Sooryanarayana Varambally

Genomics data from The Cancer Genome Atlas (TCGA) project has led to the comprehensive molecular characterization of multiple cancer types. The large sample numbers in TCGA offer an excellent opportunity to address questions associated with tumo heterogeneity. Exploration of the data by cancer researchers and clinicians is imperative to unearth novel therapeutic/diagnostic biomarkers. Various computational tools have been developed to aid researchers in carrying out specific TCGA data analyses; however there is need for resources to facilitate the study of gene expression variations and survival associations across tumors. Here, we report UALCAN, an easy to use, interactive web-portal to perform to in-depth analyses of TCGA gene expression data. UALCAN uses TCGA level 3 RNA-seq and clinical data from 31 cancer types. The portal's user-friendly features allow to perform: 1) analyze relative expression of a query gene(s) across tumor and normal samples, as well as in various tumor sub-groups based on individual cancer stages, tumor grade, race, body weight or other clinicopathologic features, 2) estimate the effect of gene expression level and clinicopathologic features on patient survival; and 3) identify the top over- and under-expressed (up and down-regulated) genes in individual cancer types. This resource serves as a platform for in silico validation of target genes and for identifying tumor sub-group specific candidate biomarkers. Thus, UALCAN web-portal could be extremely helpful in accelerating cancer research. UALCAN is publicly available at http://ualcan.path.uab.edu.

Recurrent chromosomal rearrangements have not been well characterized in common carcinomas. We used a bioinformatics approach to discover candidate oncogenic chromosomal aberrations on the basis of outlier gene expression. Two ETS transcription factors, ERG and ETV1 , were identified as outliers in prostate cancer. We identified recurrent gene fusions of the 5′ untranslated region of TMPRSS2 to ERG or ETV1 in prostate cancer tissues with outlier expression. By using fluorescence in situ hybridization, we demonstrated that 23 of 29 prostate cancer samples harbor rearrangements in ERG or ETV1 . Cell line experiments suggest that the androgen-responsive promoter elements of TMPRSS2 mediate the overexpression of ETS family members in prostate cancer. These results have implications in the development of carcinomas and the molecular diagnosis and treatment of prostate cancer.

Prostate cancer is a leading cause of cancer-related death in males and is second only to lung cancer. Although effective surgical and radiation treatments exist for clinically localized prostate cancer, metastatic prostate cancer remains essentially incurable. Here we show, through gene expression profiling, that the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in hormone-refractory, metastatic prostate cancer. Small interfering RNA (siRNA) duplexes targeted against EZH2 reduce the amounts of EZH2 protein present in prostate cells and also inhibit cell proliferation in vitro. Ectopic expression of EZH2 in prostate cells induces transcriptional repression of a specific cohort of genes. Gene silencing mediated by EZH2 requires the SET domain and is attenuated by inhibiting histone deacetylase activity. Amounts of both EZH2 messenger RNA and EZH2 protein are increased in metastatic prostate cancer; in addition, clinically localized prostate cancers that express higher concentrations of EZH2 show a poorer prognosis. Thus, dysregulated expression of EZH2 may be involved in the progression of prostate cancer, as well as being a marker that distinguishes indolent prostate cancer from those at risk of lethal progression.

Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of critical biomarkers for cancer invasion and disease aggressiveness. Although gene and protein expression have been extensively profiled in human tumours, little is known about the global metabolomic alterations that characterize neoplastic progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we profiled more than 1,126 metabolites across 262 clinical samples related to prostate cancer (42 tissues and 110 each of urine and plasma). These unbiased metabolomic profiles were able to distinguish benign prostate, clinically localized prostate cancer and metastatic disease. Sarcosine, an N-methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly increased during prostate cancer progression to metastasis and can be detected non-invasively in urine. Sarcosine levels were also increased in invasive prostate cancer cell lines relative to benign prostate epithelial cells. Knockdown of glycine-N-methyl transferase, the enzyme that generates sarcosine from glycine, attenuated prostate cancer invasion. Addition of exogenous sarcosine or knockdown of the enzyme that leads to sarcosine degradation, sarcosine dehydrogenase, induced an invasive phenotype in benign prostate epithelial cells. Androgen receptor and the ERG gene fusion product coordinately regulate components of the sarcosine pathway. Here, by profiling the metabolomic alterations of prostate cancer progression, we reveal sarcosine as a potentially important metabolic intermediary of cancer cell invasion and aggressivity.

Prostate cancer is the most frequently diagnosed cancer in American men. Screening for prostate-specific antigen (PSA) has led to earlier detection of prostate cancer, but elevated serum PSA levels may be present in non-malignant conditions such as benign prostatic hyperlasia (BPH). Characterization of gene-expression profiles that molecularly distinguish prostatic neoplasms may identify genes involved in prostate carcinogenesis, elucidate clinical biomarkers, and lead to an improved classification of prostate cancer. Using microarrays of complementary DNA, we examined gene-expression profiles of more than 50 normal and neoplastic prostate specimens and three common prostate-cancer cell lines. Signature expression profiles of normal adjacent prostate (NAP), BPH, localized prostate cancer, and metastatic, hormone-refractory prostate cancer were determined. Here we establish many associations between genes and prostate cancer. We assessed two of these genes-hepsin, a transmembrane serine protease, and pim-1, a serine/threonine kinase-at the protein level using tissue microarrays consisting of over 700 clinically stratified prostate-cancer specimens. Expression of hepsin and pim-1 proteins was significantly correlated with measures of clinical outcome. Thus, the integration of cDNA microarray, high-density tissue microarray, and linked clinical and pathology data is a powerful approach to molecular profiling of human cancer.

The Polycomb Group Protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive prostate cancer. Here we investigate the functional role of EZH2 in cancer cell invasion and breast cancer progression. EZH2 transcript and protein were consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrated that EZH2 protein levels were strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion. EZH2-mediated cell invasion required an intact SET domain and histone deacetylase activity. This study provides compelling evidence for a functional link between dysregulated cellular memory, transcriptional repression, and neoplastic transformation.

Genome-wide DNA methylation analysis of metastatic biopsies from patients with castration-resistant prostate cancer reveals marked epigenetic differences between samples with adenocarcinoma and neuroendocrine histologies. An increasingly recognized resistance mechanism to androgen receptor (AR)-directed therapy in prostate cancer involves epithelial plasticity, in which tumor cells demonstrate low to absent AR expression and often have neuroendocrine features. The etiology and molecular basis for this 'alternative' treatment-resistant cell state remain incompletely understood. Here, by analyzing whole-exome sequencing data of metastatic biopsies from patients, we observed substantial genomic overlap between castration-resistant tumors that were histologically characterized as prostate adenocarcinomas (CRPC-Adeno) and neuroendocrine prostate cancer (CRPC-NE); analysis of biopsy samples from the same individuals over time points to a model most consistent with divergent clonal evolution. Genome-wide DNA methylation analysis revealed marked epigenetic differences between CRPC-NE tumors and CRPC-Adeno, and also designated samples of CRPC-Adeno with clinical features of AR independence as CRPC-NE, suggesting that epigenetic modifiers may play a role in the induction and/or maintenance of this treatment-resistant state. This study supports the emergence of an alternative, 'AR-indifferent' cell state through divergent clonal evolution as a mechanism of treatment resistance in advanced prostate cancer.

Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and regulates the survival and metastasis of cancer cells. EZH2 is overexpressed in aggressive solid tumors by mechanisms that remain unclear. Here we show that the expression and function of EZH2 in cancer cell lines are inhibited by microRNA-101 (miR-101). Analysis of human prostate tumors revealed that miR-101 expression decreases during cancer progression, paralleling an increase in EZH2 expression. One or both of the two genomic loci encoding miR-101 were somatically lost in 37.5% of clinically localized prostate cancer cells (6 of 16) and 66.7% of metastatic disease cells (22 of 33). We propose that the genomic loss of miR-101 in cancer leads to overexpression of EZH2 and concomitant dysregulation of epigenetic pathways, resulting in cancer progression.

Chromosomal rearrangements fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG occur in approximately 50% of prostate cancers, but how the fusion products regulate prostate cancer remains unclear. Using chromatin immunoprecipitation coupled with massively parallel sequencing, we found that ERG disrupts androgen receptor (AR) signaling by inhibiting AR expression, binding to and inhibiting AR activity at gene-specific loci, and inducing repressive epigenetic programs via direct activation of the H3K27 methyltransferase EZH2, a Polycomb group protein. These findings provide a working model in which TMPRSS2-ERG plays a critical role in cancer progression by disrupting lineage-specific differentiation of the prostate and potentiating the EZH2-mediated dedifferentiation program.

Distinct translocation mechanisms and additional translocation partners for ETS genes are found in prostate cancer. This study also provides the first functional evidence that ETS gene deregulation can promote cancer cell invasion in cell lines and pre-malignant prostate lesions in a transgenic mouse model. Recently, we identified recurrent gene fusions involving the 5′ untranslated region of the androgen-regulated gene TMPRSS2 and the ETS (E26 transformation-specific) family genes ERG, ETV1 or ETV4 in most prostate cancers1,2. Whereas TMPRSS2–ERG fusions are predominant, fewer TMPRSS2–ETV1 cases have been identified than expected on the basis of the frequency of high (outlier) expression of ETV1 (refs 3–13). Here we explore the mechanism of ETV1 outlier expression in human prostate tumours and prostate cancer cell lines. We identified previously unknown 5′ fusion partners in prostate tumours with ETV1 outlier expression, including untranslated regions from a prostate-specific androgen-induced gene (SLC45A3) and an endogenous retroviral element (HERV-K_22q11.23), a prostate-specific androgen-repressed gene (C15orf21), and a strongly expressed housekeeping gene (HNRPA2B1). To study aberrant activation of ETV1, we identified two prostate cancer cell lines, LNCaP and MDA-PCa 2B, that had ETV1 outlier expression. Through distinct mechanisms, the entire ETV1 locus (7p21) is rearranged to a 1.5-megabase prostate-specific region at 14q13.3–14q21.1 in both LNCaP cells (cryptic insertion) and MDA-PCa 2B cells (balanced translocation). Because the common factor of these rearrangements is aberrant ETV1 overexpression, we recapitulated this event in vitro and in vivo, demonstrating that ETV1 overexpression in benign prostate cells and in the mouse prostate confers neoplastic phenotypes. Identification of distinct classes of ETS gene rearrangements demonstrates that dormant oncogenes can be activated in prostate cancer by juxtaposition to tissue-specific or ubiquitously active genomic loci. Subversion of active genomic regulatory elements may serve as a more generalized mechanism for carcinoma development. Furthermore, the identification of androgen-repressed and insensitive 5′ fusion partners may have implications for the anti-androgen treatment of advanced prostate cancer.

Molecular profiling of cancer at the transcript level has become routine. Large-scale analysis of proteomic alterations during cancer progression has been a more daunting task. Here, we employed high-throughput immunoblotting in order to interrogate tissue extracts derived from prostate cancer. We identified 64 proteins that were altered in prostate cancer relative to benign prostate and 156 additional proteins that were altered in metastatic disease. An integrative analysis of this compendium of proteomic alterations and transcriptomic data was performed, revealing only 48%-64% concordance between protein and transcript levels. Importantly, differential proteomic alterations between metastatic and clinically localized prostate cancer that mapped concordantly to gene transcripts served as predictors of clinical outcome in prostate cancer as well as other solid tumors.

TMPRSS2-ERG gene fusions are the predominant molecular subtype of prostate cancer. Here, we explored the role of TMPRSS2-ERG gene fusion product using in vitro and in vivo model systems. Transgenic mice expressing the ERG gene fusion product under androgen-regulation develop mouse prostatic intraepithelial neoplasia (PIN), a precursor lesion of prostate cancer. Introduction of the ERG gene fusion product into primary or immortalized benign prostate epithelial cells induced an invasion-associated transcriptional program but did not increase cellular proliferation or anchorage-independent growth. These results suggest that TMPRSS2-ERG may not be sufficient for transformation in the absence of secondary molecular lesions. Transcriptional profiling of ERG knockdown in the TMPPRSS2-ERG-positive prostate cancer cell line VCaP revealed decreased expression of genes over-expressed in prostate cancer versus PIN and genes overexpressed in ETS-positive versus -negative prostate cancers in addition to inhibiting invasion. ERG knockdown in VCaP cells also induced a transcriptional program consistent with prostate differentiation. Importantly, VCaP cells and benign prostate cells overexpressing ERG directly engage components of the plasminogen activation pathway to mediate cellular invasion, potentially representing a downstream ETS target susceptible to therapeutic intervention. Our results support previous work suggesting that TMPRSS2-ERG fusions mediate invasion, consistent with the defining histologic distinction between PIN and prostate cancer.

Abstract Understanding the biology of prostate cancer metastasis has been limited by the lack of tissue for study. We studied the clinical data, distribution of prostate cancer involvement, morphology, immunophenotypes, and gene expression from 30 rapid autopsies of men who died of hormone-refractory prostate cancer. A tissue microarray was constructed and quantitatively evaluated for expression of prostate-specific antigen, androgen receptor, chromogranin, synaptophysin, MIB-1, and α-methylacylCoA-racemase markers. Hierarchical clustering of 16 rapid autopsy tumor samples was performed to evaluate the cDNA expression pattern associated with the morphology. Comparisons were made between patients as well as within the same patient. Metastatic hormone-refractory prostate cancer has a heterogeneous morphology, immunophenotype, and genotype, demonstrating that “metastatic disease” is a group of diseases even within the same patient. An appreciation of this heterogeneity is critical to evaluating diagnostic and prognostic biomarkers as well as to designing therapeutic targets for advanced disease.

New biomarkers, such as autoantibody signatures, may improve the early detection of prostate cancer.With a phage-display library derived from prostate-cancer tissue, we developed and used phage protein microarrays to analyze serum samples from 119 patients with prostate cancer and 138 controls, with the samples equally divided into training and validation sets. A phage-peptide detector that was constructed from the training set was evaluated on an independent validation set of 128 serum samples (60 from patients with prostate cancer and 68 from controls).A 22-phage-peptide detector had 88.2 percent specificity (95 percent confidence interval, 0.78 to 0.95) and 81.6 percent sensitivity (95 percent confidence interval, 0.70 to 0.90) in discriminating between the group with prostate cancer and the control group. This panel of peptides performed better than did prostate-specific antigen (PSA) in distinguishing between the group with prostate cancer and the control group (area under the curve for the autoantibody signature, 0.93; 95 percent confidence interval, 0.88 to 0.97; area under the curve for PSA, 0.80; 95 percent confidence interval, 0.71 to 0.88). Logistic-regression analysis revealed that the phage-peptide panel provided additional discriminative power over PSA (P<0.001). Among the 22 phage peptides used as a detector, 4 were derived from in-frame, named coding sequences. The remaining phage peptides were generated from untranslated sequences.Autoantibodies against peptides derived from prostate-cancer tissue could be used as the basis for a screening test for prostate cancer.

Enhancer of zeste homolog 2 (EZH2) is a critical component of the polycomb-repressive complex 2 (PRC2), which is involved in gene silencing and histone H3 lysine 27 methylation. EZH2 has a master regulatory function in controlling such processes as stem cell differentiation, cell proliferation, early embryogenesis and X chromosome inactivation. Although benign epithelial cells express very low levels of EZH2, increased levels of EZH2 have been observed in aggressive solid tumors such as those of the prostate, breast and bladder. The mechanism by which EZH2 mediates tumor aggressiveness is unclear. Here, we demonstrate that EZH2 mediates transcriptional silencing of the tumor suppressor gene E-cadherin by trimethylation of H3 lysine 27. Histone deacetylase inhibitors can prevent EZH2-mediated repression of E-cadherin and attenuate cell invasion, suggesting a possible mechanism that may be useful for the development of therapeutic treatments. Taken together, these observations provide a novel mechanism of E-cadherin regulation and establish a functional link between dysregulation of EZH2 and repression of E-cadherin during cancer progression.

Using pair-end transcriptome sequencing, this study provides the identification of Raf pathway gene rearrangements in a small proportion of prostate and gastric cancers and in melanomas. The fusion proteins show tumorigenic potential and represent a unique activating alteration of this oncogenic pathway, which seems to be mutually exclusive from known cancer-associated Raf mutations. This suggests that therapeutic Raf inhibition can be expanded to this fusion-harboring subset of solid tumors. Although recurrent gene fusions involving erythroblastosis virus E26 transformation-specific (ETS) family transcription factors are common in prostate cancer, their products are considered 'undruggable' by conventional approaches. Recently, rare targetable gene fusions involving the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, have been identified in 1–5% of lung cancers1, suggesting that similar rare gene fusions may occur in other common epithelial cancers, including prostate cancer. Here we used paired-end transcriptome sequencing to screen ETS rearrangement–negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF (solute carrier family 45, member 3–v-raf murine sarcoma viral oncogene homolog B1) and ESRP1-RAF1 (epithelial splicing regulatory protein-1–v-raf-1 murine leukemia viral oncogene homolog-1) gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and mitogen-activated protein kinase kinase (MAP2K1) inhibitors. Screening a large cohort of patients, we found that, although rare, recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers and melanoma. Taken together, our results emphasize the key role of RAF family gene rearrangements in cancer, suggest that RAF and MEK inhibitors may be useful in a subset of gene fusion–harboring solid tumors and demonstrate that sequencing of tumor transcriptomes and genomes may lead to the identification of rare targetable fusions across cancer types.

Recurrent fusions of ETS genes are considered driving mutations in a diverse array of cancers, including Ewing's sarcoma, acute myeloid leukemia, and prostate cancer. We investigate the mechanisms by which ETS fusions mediate their effects, and find that the product of the predominant ETS gene fusion, TMPRSS2:ERG, interacts in a DNA-independent manner with the enzyme poly (ADP-ribose) polymerase 1 (PARP1) and the catalytic subunit of DNA protein kinase (DNA-PKcs). ETS gene-mediated transcription and cell invasion require PARP1 and DNA-PKcs expression and activity. Importantly, pharmacological inhibition of PARP1 inhibits ETS-positive, but not ETS-negative, prostate cancer xenograft growth. Finally, overexpression of the TMPRSS2:ERG fusion induces DNA damage, which is potentiated by PARP1 inhibition in a manner similar to that of BRCA1/2 deficiency.

A catalog of all human protein-protein interactions would provide scientists with a framework to study protein deregulation in complex diseases such as cancer. Here we demonstrate that a probabilistic analysis integrating model organism interactome data, protein domain data, genome-wide gene expression data and functional annotation data predicts nearly 40,000 protein-protein interactions in humans—a result comparable to those obtained with experimental and computational approaches in model organisms. We validated the accuracy of the predictive model on an independent test set of known interactions and also experimentally confirmed two predicted interactions relevant to human cancer, implicating uncharacterized proteins into definitive pathways. We also applied the human interactome network to cancer genomics data and identified several interaction subnetworks activated in cancer. This integrative analysis provides a comprehensive framework for exploring the human protein interaction network.

Gene fusions play a critical role in cancer progression. The mechanisms underlying their genesis and cell type specificity are not well understood. About 50% of human prostate cancers display a gene fusion involving the 5' untranslated region of TMPRSS2, an androgen-regulated gene, and the protein-coding sequences of ERG, which encodes an erythroblast transformation-specific (ETS) transcription factor. By studying human prostate cancer cells with fluorescence in situ hybridization, we show that androgen signaling induces proximity of the TMPRSS2 and ERG genomic loci, both located on chromosome 21q22.2. Subsequent exposure of the cells to gamma irradiation, which causes DNA double-strand breaks, facilitates the formation of the TMPRSS2-ERG gene fusion. These results may help explain why TMPRSS2-ERG fusions are restricted to the prostate, which is dependent on androgen signaling.

TMPRSS2-ERG gene fusions occur in 50% of prostate cancers and result in the overexpression of a chimeric fusion transcript that encodes a truncated ERG product. Previous attempts to detect truncated ERG products have been hindered by a lack of specific antibodies. Here, we characterize a rabbit anti-ERG monoclonal antibody (clone EPR 3864; Epitomics, Burlingame, CA) using immunoblot analysis on prostate cancer cell lines, synthetic TMPRSS2-ERG constructs, chromatin immunoprecipitation, and immunofluorescence. We correlated ERG protein expression with the presence of ERG gene rearrangements in prostate cancer tissues using a combined immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analysis. We independently evaluated two patient cohorts and observed ERG expression confined to prostate cancer cells and high-grade prostatic intraepithelial neoplasia associated with ERG-positive cancer, as well as vessels and lymphocytes (where ERG has a known biologic role). Image analysis of 131 cases demonstrated nearly 100% sensitivity for detecting ERG rearrangement prostate cancer, with only 2 (1.5%) of 131 cases demonstrating strong ERG protein expression without any known ERG gene fusion. The combined pathology evaluation of 207 patient tumors for ERG protein expression had 95.7% sensitivity and 96.5% specificity for determining ERG rearrangement prostate cancer. In conclusion, this study qualifies a specific anti-ERG antibody and demonstrates exquisite association between ERG gene rearrangement and truncated ERG protein product expression. Given the ease of performing IHC versus FISH, ERG protein expression may be useful for molecularly subtyping prostate cancer based on ERG rearrangement status and suggests clinical utility in prostate needle biopsy evaluation.

Abstract Recent studies suggest that NOTCH signaling can promote epithelial-mesenchymal transitions and augment signaling through AKT, an important growth and survival pathway in epithelial cells and prostate cancer in particular. Here we show that JAGGED1, a NOTCH receptor ligand, is significantly more highly expressed in metastatic prostate cancer as compared with localized prostate cancer or benign prostatic tissues, based on immunohistochemical analysis of JAGGED1 expression in human tumor samples from 154 men. Furthermore, high JAGGED1 expression in a subset of clinically localized tumors was significantly associated with recurrence, independent of other clinical parameters. These findings support a model in which dysregulation of JAGGED1 protein levels plays a role in prostate cancer progression and metastasis and suggest that JAGGED1 may be a useful marker in distinguishing indolent and aggressive prostate cancers.

Glucose availability is limiting in tumor environments. Zou and colleagues show that reduced glycolytic metabolism in T cells within tumors suppresses expression of the methyltransferase EZH2, which limits production of antitumor effector molecules and enhances T cell apoptosis. Aerobic glycolysis regulates T cell function. However, whether and how primary cancer alters T cell glycolytic metabolism and affects tumor immunity in cancer patients remains a question. Here we found that ovarian cancers imposed glucose restriction on T cells and dampened their function via maintaining high expression of microRNAs miR-101 and miR-26a, which constrained expression of the methyltransferase EZH2. EZH2 activated the Notch pathway by suppressing Notch repressors Numb and Fbxw7 via trimethylation of histone H3 at Lys27 and, consequently, stimulated T cell polyfunctional cytokine expression and promoted their survival via Bcl-2 signaling. Moreover, small hairpin RNA–mediated knockdown of human EZH2 in T cells elicited poor antitumor immunity. EZH2+CD8+ T cells were associated with improved survival in patients. Together, these data unveil a metabolic target and mechanism of cancer immune evasion.

The Polycomb Group (PcG) protein EZH2 is a critical component of a multiprotein complex that methylates Lys(27) of histone 3 (H3K27), which consequently leads to the repression of target gene expression. We have previously reported that EZH2 is overexpressed in metastatic prostate cancer and is a marker of aggressive diseases in clinically localized solid tumors. However, the global set of genes directly regulated by PcG in tumors is largely unknown, and thus how PcG mediates tumor progression remains unclear. Herein we mapped genome-wide H3K27 methylation in aggressive, disseminated human prostate cancer tissues. Integrative analysis revealed that a significant subset of these genes are also targets of PcG in embryonic stem cells, and their repression in tumors is associated with poor prognosis. By stepwise cross-validation, we developed a "Polycomb repression signature" composed of 14 direct targets of PcG in metastatic tumors. Notably, solid tumor subtypes in which this gene signature is repressed show poor clinical outcome in multiple microarray data sets of tumors including breast and prostate cancer. Taken together, our results show a fingerprint of PcG-mediated transcriptional repression in metastatic prostate cancer that is reminiscent of stem cells and associated with cancer progression. Therefore, PcG proteins play a central role in the epigenetic silencing of target genes and functionally link stem cells, metastasis, and cancer survival.

ETS gene fusions have been characterized in a majority of prostate cancers; however, the key molecular alterations in ETS-negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier expression exclusively in a subset of ETS rearrangement-negative cancers ( approximately 10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier expression can be detected noninvasively in urine, and observed that SPINK1 outlier expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier expression model and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement-negative prostate cancers.

Recurrent gene fusions involving oncogenic ETS transcription factors (including ERG, ETV1, and ETV4) have been identified in a large fraction of prostate cancers. The most common fusions contain the 5' untranslated region of TMPRSS2 fused to ERG. Recently, we identified additional 5' partners in ETV1 fusions, including TMPRSS2, SLC45A3, HERV-K_22q11.23, C15ORF21, and HNRPA2B1. Here, we identify ETV5 as the fourth ETS family member involved in recurrent gene rearrangements in prostate cancer. Characterization of two cases with ETV5 outlier expression by RNA ligase-mediated rapid amplification of cDNA ends identified one case with a TMPRSS2:ETV5 fusion and one case with a SLC45A3:ETV5 fusion. We confirmed the presence of these fusions by quantitative PCR and fluorescence in situ hybridization. In vitro recapitulation of ETV5 overexpression induced invasion in RWPE cells, a benign immortalized prostatic epithelial cell line. Expression profiling and an integrative molecular concepts analysis of RWPE-ETV5 cells also revealed the induction of an invasive transcriptional program, consistent with ERG and ETV1 overexpression in RWPE cells, emphasizing the functional redundancy of ETS rearrangements. Together, our results suggest that the family of 5' partners previously identified in ETV1 gene fusions can fuse with other ETS family members, suggesting numerous rare gene fusion permutations in prostate cancer.

<h2>Summary</h2> Polycomb Repressive Complexes (PRC1 and PRC2)-mediated epigenetic regulation is critical for maintaining cellular homeostasis. Members of Polycomb Group (PcG) proteins including EZH2, a PRC2 component, are upregulated in various cancer types, implicating their role in tumorigenesis. Here, we have identified several microRNAs (miRNAs) that are repressed by EZH2. These miRNAs, in turn, regulate the expression of PRC1 proteins BMI1 and RING2. We found that ectopic overexpression of EZH2-regulated miRNAs attenuated cancer cell growth and invasiveness, and abrogated cancer stem cell properties. Importantly, expression analysis revealed an inverse correlation between miRNA and PRC protein levels in cell culture and prostate cancer tissues. Taken together, our data have uncovered a coordinate regulation of PRC1 and PRC2 activities that is mediated by miRNAs.

The heparan sulfate-degrading enzyme heparanase promotes the progression of many cancers by driving tumor cell proliferation, metastasis and angiogenesis. Heparanase accomplishes this via multiple mechanisms including its recently described effect on enhancing biogenesis of tumor exosomes. Because we recently discovered that heparanase expression is upregulated in myeloma cells that survive chemotherapy, we were prompted to investigate the impact of anti-myeloma drugs on exosome biogenesis. When myeloma cells were exposed to the commonly utilized anti-myeloma drugs bortezomib, carfilzomib or melphalan, exosome secretion by the cells was dramatically enhanced. These chemotherapy-induced exosomes (chemoexosomes) have a proteome profile distinct from cells not exposed to drug including a dramatic elevation in the level of heparanase present as exosome cargo. The chemoexosome heparanase was not found inside the chemoexosome, but was present on the exosome surface where it was capable of degrading heparan sulfate embedded within an extracellular matrix. When exposed to myeloma cells, chemoexosomes transferred their heparanase cargo to those cells, enhancing their heparan sulfate degrading activity and leading to activation of ERK signaling and an increase in shedding of the syndecan-1 proteoglycan. Exposure of chemoexosomes to macrophages enhanced their secretion of TNF-α, an important myeloma growth factor. Moreover, chemoexosomes stimulated macrophage migration and this effect was blocked by H1023, a monoclonal antibody that inhibits heparanase enzymatic activity. These data suggest that anti-myeloma therapy ignites a burst of exosomes having a high level of heparanase that remodels extracellular matrix and alters tumor and host cell behaviors that likely contribute to chemoresistance and eventual patient relapse.We find that anti-myeloma chemotherapy dramatically stimulates secretion of exosomes and alters exosome composition. Exosomes secreted during therapy contain high levels of heparanase on their surface that can degrade ECM and also can be transferred to both tumor and host cells, altering their behavior in ways that may enhance tumor survival and progression.

Multiple genetic and epigenetic events characterize tumor progression and define the identity of the tumors. Advances in high-throughput technologies, like gene expression profiling, next-generation sequencing, proteomics, and metabolomics, have enabled detailed molecular characterization of various tumors. The integration and analyses of these high-throughput data have unraveled many novel molecular aberrations and network alterations in tumors. These molecular alterations include multiple cancer-driving mutations, gene fusions, amplification, deletion, and post-translational modifications, among others. Many of these genomic events are being used in cancer diagnosis, whereas others are therapeutically targeted with small-molecule inhibitors. Multiple genes/enzymes that play a role in DNA and histone modifications are also altered in various cancers, changing the epigenomic landscape during cancer initiation and progression. Apart from protein-coding genes, studies are uncovering the critical regulatory roles played by noncoding RNAs and noncoding regions of the genome during cancer progression. Many of these genomic and epigenetic events function in tandem to drive tumor development and metastasis. Concurrent advances in genome-modulating technologies, like gene silencing and genome editing, are providing ability to understand in detail the process of cancer initiation, progression, and signaling as well as opening up avenues for therapeutic targeting. In this review, we discuss some of the recent advances in cancer genomic and epigenomic research.

GATA binding protein 3 (GATA3) is a transcriptional activator highly expressed by the luminal epithelial cells in the breast. Here we did a meta-analysis of the available breast cancer cDNA data sets on a cohort of 305 patients and found that GATA3 was one of the top genes with low expression in invasive carcinomas with poor clinical outcome. To validate its prognostic utility, we did a tissue microarray analysis on a cohort of 139 consecutive invasive carcinomas (n = 417 tissue samples) immunostained with a monoclonal antibody against GATA3. Low GATA3 expression was associated with higher histologic grade (P < 0.001), positive nodes (P = 0.002), larger tumor size (P = 0.03), negative estrogen receptor and progesterone receptor (P < 0.001 for both), and HER2-neu overexpression (P = 0.03). Patients whose tumors expressed low GATA3 had significantly shorter overall and disease-free survival when compared with those whose tumors had high GATA3 levels. The hazard ratio of metastasis or recurrence according to the GATA3 status was 0.31 (95% confidence interval, 0.13-0.74; P = 0.009). Cox multivariate analysis showed that GATA3 had independent prognostic significance above and beyond conventional variables. Our data suggest that immunohistochemical analysis of GATA3 may be the basis for a new clinically applicable test to predict tumor recurrence early in the progression of breast cancer.

The Polycomb group (PcG) protein EZH2 possesses oncogenic properties for which the underlying mechanism is unclear. We integrated in vitro cell line, in vivo tumor profiling, and genome-wide location data to nominate key targets of EZH2. One of the candidates identified was ADRB2 (Adrenergic Receptor, Beta-2), a critical mediator of beta-adrenergic signaling. EZH2 is recruited to the ADRB2 promoter and represses ADRB2 expression. ADRB2 inhibition confers cell invasion and transforms benign prostate epithelial cells, whereas ADRB2 overexpression counteracts EZH2-mediated oncogenesis. ADRB2 is underexpressed in metastatic prostate cancer, and clinically localized tumors that express lower levels of ADRB2 exhibit a poor prognosis. Taken together, we demonstrate the power of integrating multiple diverse genomic data to decipher targets of disease-related genes.

Nod1 is a member of family of intracellular proteins that mediate host recognition of bacterial peptidoglycan. To characterize immune responses mediated by Nod1, synthetic ligand compounds possessing enhanced ability to stimulate Nod1 were developed to study the function of Nod1. Stimulation of epithelial cells with Nod1 stimulatory molecules induced chemokines and other proinflammatory molecules that are important for innate immune responses and recruitment of acute inflammatory cells. Administration of Nod1 ligands into mice induced chemokines and recruitment of acute inflammatory cells, an activity that was abolished in Nod1-null mice. Microarray analysis revealed that Nod1 stimulation induces a restricted number of genes in intestinal epithelial cells compared with that induced by tumor necrosis factor (TNF) α. Nod1 stimulation did not induce TNFα, interleukin 12, and interferon γ, suggesting that the primary role of Nod1 is to induce the recruitment of immune cells. These results indicate that Nod1 functions as a pathogen recognition molecule to induce expression of molecules involved in the early stages of the innate immune response.

The proteasome controls a plethora of survival factors in all mammalian cells analyzed to date. Therefore, it is puzzling that proteasome inhibitors such as bortezomib can display a preferential toxicity toward malignant cells. In fact, proteasome inhibitors have the salient feature of promoting a dramatic induction of the proapoptotic protein NOXA in a tumor cell-restricted manner. However, the molecular determinants that control this specific regulation of NOXA are unknown. Here, we show that the induction of NOXA by bortezomib is directly dependent on the oncogene c-MYC. This requirement for c-MYC was found in a variety of tumor cell types, in marked contrast with dispensable roles of p53, HIF-1α, and E2F-1 (classical proteasomal targets that can regulate NOXA mRNA under stress). Conserved MYC-binding sites identified at the NOXA promoter were validated by ChIP and reporter assays. Down-regulation of the endogenous levels of c-MYC abrogated the induction of NOXA in proteasome-defective tumor cells. Conversely, forced expression of c-MYC enabled normal cells to accumulate NOXA and subsequently activate cell death programs in response to proteasome blockage. c-MYC is itself a proteasomal target whose levels or function are invariably up-regulated during tumor progression. Our data provide an unexpected function of c-MYC in the control of the apoptotic machinery, and reveal a long sought-after oncogenic event conferring sensitivity to proteasome inhibition.

The endothelium plays a critical role in the inflammatory process. The complement activation product, C5a, is known to have proinflammatory effects on the endothelium, but the molecular mechanisms remain unclear. We have used cDNA microarray analysis to assess gene expression in human umbilical vein endothelial cells (HUVECs) that were stimulated with human C5a in vitro. Chip analyses were confirmed by reverse transcriptase-polymerase chain reaction and by Western blot analysis. Gene activation responses were remarkably similar to gene expression patterns of HUVECs stimulated with human tumor necrosis factor-alpha or bacterial lipopolysaccharide. HUVECs stimulated with C5a showed progressive increases in gene expression for cell adhesion molecules (eg, E-selectin, ICAM-1, VCAM-1), cytokines/chemokines, and related receptors (eg, VEGFC, IL-6, IL-18R). Surprisingly, HUVECs showed little evidence for up-regulation of complement-related genes. There were transient increases in gene expression associated with broad functional activities. The three agonists used also caused down-regulation of genes that regulate angiogenesis and drug metabolism. With a single exception, C5a caused little evidence of activation of complement-related genes. These studies indicate that endothelial cells respond robustly to C5a by activation of genes related to progressive expression of cell adherence molecules, and cytokines and chemokines in a manner similar to responses induced by tumor necrosis factor-alpha and lipopolysaccharide.

Recurrent gene fusions involving E26 transformation-specific (ETS) transcription factors ERG, ETV1, ETV4, or ETV5 have been identified in 40% to 70% of prostate cancers. Here, we used a comprehensive fluorescence in situ hybridization (FISH) split probe strategy interrogating all 27 ETS family members and their five known 5' fusion partners in a cohort of 110 clinically localized prostate cancer patients. Gene rearrangements were only identified in ETS genes that were previously implicated in prostate cancer gene fusions including ERG, ETV1, and ETV4 (43%, 5%, and 5%, respectively), suggesting that a substantial fraction of prostate cancers (estimated at 30-60%) cannot be attributed to an ETS gene fusion. Among the known 5' gene fusion partners, TMPRSS2 was rearranged in 47% of cases followed by SLC45A3, HNRPA2B1, and C15ORF21 in 2%, 1%, and 1% of cases, respectively. Based on this comprehensive FISH screen, we have made four noteworthy observations. First, by screening the entire ETS transcription factor family for rearrangements, we found that a large fraction of prostate cancers (44%) cannot be ascribed to an ETS gene fusion, an observation which will stimulate research into identifying recurrent non-ETS aberrations in prostate cancers. Second, we identified SLC45A3 as a novel 5' fusion partner of ERG; previously, TMPRSS2 was the only described 5' partner of ERG. Third, we identified two prostate-specific, androgen-induced genes, FLJ35294 and CANT1, as 5' partners to ETV1 and ETV4. Fourth, we identified a ubiquitously expressed, androgen-insensitive gene, DDX5, fused in frame with ETV4, leading to the expression of a DDX5-ETV4 fusion protein.

Gene fusions involving ETS (erythroblastosis virus E26 transformation-specific) family transcription factors are found in ~50% of prostate cancers and as such can be used as a basis for the molecular subclassification of prostate cancer. Previously, we showed that marked overexpression of SPINK1 (serine peptidase inhibitor, Kazal type 1), which encodes a secreted serine protease inhibitor, defines an aggressive molecular subtype of ETS fusion-negative prostate cancers (SPINK1+/ETS⁻, ~10% of all prostate cancers). Here, we examined the potential of SPINK1 as an extracellular therapeutic target in prostate cancer. Recombinant SPINK1 protein (rSPINK1) stimulated cell proliferation in benign RWPE as well as cancerous prostate cells. Indeed, RWPE cells treated with either rSPINK1 or conditioned medium from 22RV1 prostate cancer cells (SPINK1+/ETS⁻) significantly increased cell invasion and intravasation when compared with untreated cells. In contrast, knockdown of SPINK1 in 22RV1 cells inhibited cell proliferation, cell invasion, and tumor growth in xenograft assays. 22RV1 cell proliferation, invasion, and intravasation were attenuated by a monoclonal antibody (mAb) to SPINK1 as well. We also demonstrated that SPINK1 partially mediated its neoplastic effects through interaction with the epidermal growth factor receptor (EGFR). Administration of antibodies to SPINK1 or EGFR (cetuximab) in mice bearing 22RV1 xenografts attenuated tumor growth by more than 60 and 40%, respectively, or ~75% when combined, without affecting PC3 xenograft (SPINK1⁻/ETS⁻) growth. Thus, this study suggests that SPINK1 may be a therapeutic target in a subset of patients with SPINK1+/ETS⁻ prostate cancer. Our results provide a rationale for both the development of humanized mAbs to SPINK1 and evaluation of EGFR inhibition in SPINK1+/ETS⁻ prostate cancers.

Breast cancer patients have benefited from the use of targeted therapies directed at specific molecular alterations. To identify additional opportunities for targeted therapy, we searched for genes with marked overexpression in subsets of tumors across a panel of breast cancer profiling studies comprising 3,200 microarray experiments. In addition to prioritizing ERBB2, we found AGTR1, the angiotensin II receptor type I, to be markedly overexpressed in 10–20% of breast cancer cases across multiple independent patient cohorts. Validation experiments confirmed that AGTR1 is highly overexpressed, in several cases more than 100-fold. AGTR1 overexpression was restricted to estrogen receptor-positive tumors and was mutually exclusive with ERBB2 overexpression across all samples. Ectopic overexpression of AGTR1 in primary mammary epithelial cells, combined with angiotensin II stimulation, led to a highly invasive phenotype that was attenuated by the AGTR1 antagonist losartan. Similarly, losartan reduced tumor growth by 30% in AGTR1-positive breast cancer xenografts. Taken together, these observations indicate that marked AGTR1 overexpression defines a subpopulation of ER-positive, ERBB2-negative breast cancer that may benefit from targeted therapy with AGTR1 antagonists, such as losartan.

Histone methyltransferases (HMTases), as chromatin modifiers, regulate the transcriptomic landscape in normal development as well in diseases such as cancer. Here, we molecularly order two HMTases, EZH2 and MMSET, that have established genetic links to oncogenesis. EZH2, which mediates histone H3K27 trimethylation and is associated with gene silencing, was shown to be coordinately expressed and function upstream of MMSET, which mediates H3K36 dimethylation and is associated with active transcription. We found that the EZH2-MMSET HMTase axis is coordinated by a microRNA network and that the oncogenic functions of EZH2 require MMSET activity. Together, these results suggest that the EZH2-MMSET HMTase axis coordinately functions as a master regulator of transcriptional repression, activation, and oncogenesis and may represent an attractive therapeutic target in cancer.

While miRNAs are increasingly linked to various immune responses, whether they can be targeted for regulating in vivo inflammatory processes such as endotoxin-induced Gram-negative sepsis is not known. Production of cytokines by the dendritic cells (DCs) plays a critical role in response to endotoxin, lipopolysaccharide (LPS). We profiled the miRNA and mRNA of CD11c⁺ DCs in an unbiased manner and found that at baseline, miR-142-3p was among the most highly expressed endogenous miRs while IL-6 was among the most highly expressed mRNA after LPS stimulation. Multiple computational algorithms predicted the IL-6 3' untranslated region (UTR) to be a target of miR-142-3p. Studies using luciferase reporters carrying wild-type (WT) and mutant IL-6 3'UTR confirmed IL-6 as a target for miR-142-3p. In vitro knockdown and overexpression studies demonstrated a critical and specific role for miR142-3p in regulating IL-6 production by the DCs after LPS stimulation. Importantly, treatment of only WT but not the IL-6-deficient (IL-6(⁻/⁻)) mice with locked nucleic acid (LNA)-modified phosphorothioate oligonucleotide complementary to miR 142-3p reduced endotoxin-induced mortality. These results demonstrate a critical role for miR-142-3p in regulating DC responses to LPS and provide proof of concept for targeting miRs as a novel strategy for treatment of endotoxin-induced mortality.

Squamous cell carcinoma of the head and neck (SCCHN) is a common, aggressive, treatment-resistant cancer with a high recurrence rate and mortality, but the mechanism of treatment resistance remains unclear. Here we describe a mechanism where the AAA-ATPase TRIP13 promotes treatment resistance. Overexpression of TRIP13 in non-malignant cells results in malignant transformation. High expression of TRIP13 in SCCHN leads to aggressive, treatment-resistant tumors and enhanced repair of DNA damage. Using mass spectrometry, we identify DNA-PKcs complex proteins that mediate nonhomologous end joining (NHEJ), as TRIP13-binding partners. Using repair-deficient reporter systems, we show that TRIP13 promotes NHEJ, even when homologous recombination is intact. Importantly, overexpression of TRIP13 sensitizes SCCHN to an inhibitor of DNA-PKcs. Thus, this study defines a new mechanism of treatment resistance in SCCHN and underscores the importance of targeting NHEJ to overcome treatment failure in SCCHN and potentially in other cancers that overexpress TRIP13.

Transcription factors play a key role in the development of diverse cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the gene encoding the transcription factor ERG is recurrently rearranged and plays a critical role in prostate oncogenesis. Here, we identified a series of peptides that interact specifically with the DNA binding domain of ERG. ERG inhibitory peptides (EIPs) and derived peptidomimetics bound ERG with high affinity and specificity, leading to proteolytic degradation of the ERG protein. The EIPs attenuated ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion and proliferation, and tumor growth. Thus, peptidomimetic targeting of transcription factor fusion products may provide a promising therapeutic strategy for prostate cancer as well as other malignancies.

Collagen prolyl hydroxylases (C-P4HAs) are a family of enzymes involved in collagen biogenesis.One of the isoforms of P4HA, Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), catalyzes the formation of 4-hydroxyproline that is essential for the proper three-dimensional folding of newly synthesized procollagen chains.Here, we show the overexpression of P4HA1 in aggressive prostate cancer.Immunohistochemical analysis using tissue microarray demonstrated that P4HA1 expression was correlated with prostate cancer progression.Using in vitro studies, we showed that P4HA1 plays a critical role in prostate cancer cell growth and tumor progression.Expression profiling studies using P4HA1-modulated prostate cells suggested regulation of Matrix metalloprotease 1.The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1.Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124.MiR-124 in turn is negatively regulated by transcriptional repressors EZH2 and CtBP1, both of which are overexpressed in aggressive prostate cancer.Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo.Our observations suggest that P4HA1 plays a critical role in prostate cancer progression and could serve as a viable therapeutic target.

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.

Abstract Gains in the long arm of chromosome 8 (8q) are believed to be associated with poor outcome and the development of hormone-refractory prostate cancer. Based on a meta-analysis of gene expression microarray data from multiple prostate cancer studies (D. R. Rhodes et al., Cancer Res 2002;62:4427–33), a candidate oncogene, Tumor Protein D52 (TPD52), was identified in the 8q21 amplicon. TPD52 is a coiled-coil motif-bearing protein, potentially involved in vesicle trafficking. Both mRNA and protein levels of TPD52 were highly elevated in prostate cancer tissues. Array comparative genomic hybridization and amplification analysis using single nucleotide polymorphism arrays demonstrated increased DNA copy number in the region encompassing TPD52. Fluorescence in situ hybridization on tissue microarrays confirmed TPD52 amplification in prostate cancer epithelia. Furthermore, our studies suggest that TPD52 protein levels may be regulated by androgens, consistent with the presence of androgen response elements in the upstream promoter of TPD52. In summary, these findings suggest that dysregulation of TPD52 by genomic amplification and androgen induction may play a role in prostate cancer progression.

The expression of thousands of genes can be monitored simultaneously using cDNA microarray technology. This technology is being used to understand the complexity of human disease. One significant technical concern regards potential alterations in gene expression because of the effect of tissue ischemia. This study evaluates the increase in the differential gene expression because of tissue processing time. To evaluate differential gene expression because of ischemia time, prostate samples were divided into five time points (0, 0.5, 1, 3, and 5 hours). Each time point consisted of a homogeneous mixture of 12 to 15 prostate tissue cubes (5 mm(3)). These tissues were maintained at room temperature until at the assigned time point the tissue was placed in OCT, flash frozen in liquid nitrogen, and stored at -80 degrees C until RNA extraction. RNA from each time point was hybridized against an aliquot of 0 time point RNA from the same prostate. Four prostate glands were used in parallel studies. M-A plots were graphed to compare variability between time point sample hybridizations. Statistical Analysis of Microarray software was used to identify genes overexpressed at the 1-hour time point versus the 0-hour time with statistically significance. Microarray analysis revealed only a small percentage of genes (<0.6%) from more than 9000 to demonstrate overexpression at the 1-hour time point. Among the 41 statistically significant named overexpressed genes at the 1-hour time point were early growth response 1 (EGR1), jun B proto-oncogene (jun B), jun D proto-oncogene (jun D), and activating transcription factor 3 (ATF3). Genes previously associated with prostate cancer did not have significantly altered expression with ischemia time. Increased EGR1 protein expression was confirmed by Western blot analysis. Microarray technology has opened the possibility of evaluating the expression of a multitude of genes simultaneously, however, the interpretation of this complex data needs to be assessed circumspectly using refined statistical methods. Because RNA expression represents the tissue response to insults such as ischemia, and is also sensitive to degradation, investigators need be mindful of confounding artifacts secondary to tissue processing. All attempts should be made to process tissue rapidly to ensure that the microarray gene profile accurately represents the state of the cells and confirmatory studies should be performed using alternative methods (eg, Northern blot analysis, Western blot, immunohistochemistry).

α-Methylacyl-CoA racemase (AMACR) has previously been shown to be a highly sensitive marker for colorectal and clinically localized prostate cancer (PCa). However, AMACR expression was down-regulated at the transcript and protein level in hormone-refractory metastatic PCa, suggesting a hormone-dependent expression of AMACR. To further explore the hypothesis that AMACR is hormone regulated and plays a role in PCa progression AMACR protein expression was characterized in a broad range of PCa samples treated with variable amounts and lengths of exogenous anti-androgens. Analysis included standard slides and high-density tissue microarrays. AMACR protein expression was significantly increased in localized hormone-naive PCa as compared to benign (P < 0.001). Mean AMACR expression was lower in tissue samples from patients who had received neoadjuvant hormone treatment but still higher compared to hormone-refractory metastases. The hormone-sensitive tumor cell line, LNCaP, demonstrated stronger AMACR expression by Western blot analysis than the poorly differentiated cell lines DU-145 and PC-3. AMACR protein expression in cells after exposure to anti-androgen treatment was unchanged, whereas prostate-specific antigen, known to be androgen-regulated, demonstrated decreased protein expression. Surprisingly, this data suggests that AMACR expression is not regulated by androgens. Examination of colorectal cancer, which is not hormone regulated, demonstrated high levels of AMACR expression in well to moderately differentiated tumors and weak expression in anaplastic colorectal cancers. Taken together, these data suggest that AMACR expression is not hormone-dependent but may in fact be a marker of tumor differentiation. α-Methylacyl-CoA racemase (AMACR) has previously been shown to be a highly sensitive marker for colorectal and clinically localized prostate cancer (PCa). However, AMACR expression was down-regulated at the transcript and protein level in hormone-refractory metastatic PCa, suggesting a hormone-dependent expression of AMACR. To further explore the hypothesis that AMACR is hormone regulated and plays a role in PCa progression AMACR protein expression was characterized in a broad range of PCa samples treated with variable amounts and lengths of exogenous anti-androgens. Analysis included standard slides and high-density tissue microarrays. AMACR protein expression was significantly increased in localized hormone-naive PCa as compared to benign (P < 0.001). Mean AMACR expression was lower in tissue samples from patients who had received neoadjuvant hormone treatment but still higher compared to hormone-refractory metastases. The hormone-sensitive tumor cell line, LNCaP, demonstrated stronger AMACR expression by Western blot analysis than the poorly differentiated cell lines DU-145 and PC-3. AMACR protein expression in cells after exposure to anti-androgen treatment was unchanged, whereas prostate-specific antigen, known to be androgen-regulated, demonstrated decreased protein expression. Surprisingly, this data suggests that AMACR expression is not regulated by androgens. Examination of colorectal cancer, which is not hormone regulated, demonstrated high levels of AMACR expression in well to moderately differentiated tumors and weak expression in anaplastic colorectal cancers. Taken together, these data suggest that AMACR expression is not hormone-dependent but may in fact be a marker of tumor differentiation. Prostate cancer (PCa) is the most common non-skin cancer diagnosed in men in the United States.1Dennis LK Resnick MI Analysis of recent trends in prostate cancer incidence and mortality.Prostate. 2000; 42: 247-252Crossref PubMed Scopus (82) Google Scholar One explanation for the rapid increase in the incidence of PCa diagnosis has been the advent of prostate-specific antigen (PSA) screening. PSA screening has led to earlier detection of PCa.2Catalona WJ Richie JP Ahmann FR Hudson MA Scardino PT Flanigan RC deKernion JB Ratliff TL Kavoussi LR Dalkin BL et al.Comparison of digital rectal examination and serum prostate specific antigen in the early detection of prostate cancer: results of a multicenter clinical trial of 6,630 men.J Urol. 1994; 151: 1283-1290Abstract Full Text PDF PubMed Scopus (1343) Google Scholar However, the impact of PSA screening on cancer-specific mortality is still unknown pending the results of prospective randomized screening studies.3Etzioni R Legler JM Feuer EJ Merrill RM Cronin KA Hankey BF Cancer surveillance series: interpreting trends in prostate cancer—part III: quantifying the link between population prostate-specific antigen testing and recent declines in prostate cancer mortality.J Natl Cancer Inst. 1999; 91: 1033-1039Crossref PubMed Scopus (180) Google Scholar, 4Maattanen L Auvinen A Stenman UH Rannikko S Tammela T Aro J Juusela H Hakama M European randomized study of prostate cancer screening: first-year results of the Finnish trial.Br J Cancer. 1999; 79: 1210-1214Crossref PubMed Scopus (71) Google Scholar, 5Schroder FH van der Maas P Beemsterboer P Kruger AB Hoedemaeker R Rietbergen J Kranse R Evaluation of the digital rectal examination as a screening test for prostate cancer. Rotterdam section of the European Randomized Study of Screening for Prostate Cancer.J Natl Cancer Inst. 1998; 90: 1817-1823Crossref PubMed Scopus (265) Google Scholar A major limitation of the serum PSA test is lack of PCa sensitivity and specificity especially in the intermediate range of PSA detection (4 to 10 ng/ml). Our group has concentrated on developing and validating novel PCa biomarkers using a combined expression and tissue microarray (TMA) approach.6Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar This approach by our group and others has led to the identification of hepsin, a serine protease up-regulated in PCa.6Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar, 7Magee JA Araki T Patil S Ehrig T True L Humphrey PA Catalona WJ Watson MA Milbrandt J Expression profiling reveals hepsin overexpression in prostate cancer.Cancer Res. 2001; 61: 5692-5696PubMed Google Scholar, 8Welsh JB Sapinoso LM Su AI Kern SG Wang-Rodriguez J Moskaluk CA Frierson Jr, HF Hampton GM Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer.Cancer Res. 2001; 61: 5974-5978PubMed Google Scholar, 9Stamey TA Warrington JA Caldwell MC Chen Z Fan Z Mahadevappa M McNeal JE Nolley R Zhang Z Molecular genetic profiling of Gleason grade 4/5 prostate cancers compared to benign prostatic hyperplasia.J Urol. 2001; 166: 2171-2177Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 10Luo J Duggan DJ Chen Y Sauvageot J Ewing CM Bittner ML Trent JM Isaacs WB Human prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling.Cancer Res. 2001; 61: 4683-4688PubMed Google Scholar Furthermore, our group was able to use high-density TMAs to determine associations of hepsin protein and another protein, pim-1 kinase, with clinical outcome.6Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar Using a similar approach, α-methylacyl-CoA racemase (AMACR), an enzyme that plays an important role in bile acid biosynthesis and β-oxidation of branched-chain fatty acids,11Ferdinandusse S Denis S Ijlst J Dacremont G Waterham HR Wanders RJ Subcellular localization and physiological role of alpha-methylacyl-CoA racemase.J Lipid Res. 2000; 41: 1890-1896Abstract Full Text Full Text PDF PubMed Google Scholar, 12Kotti TJ Savolainen K Helander HM Yagi A Novikov DK Kalkkinen N Conzelmann E Hiltunen JK Schmitz W In mouse alpha-methylacyl-CoA racemase, the same gene product is simultaneously located in mitochondria and peroxisomes.J Biol Chem. 2000; 275: 20887-20895Crossref PubMed Scopus (57) Google Scholar was also recently identified. AMACR was determined to be up-regulated in PCa after examination of several independent gene expression data sets, including our own.6Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar, 8Welsh JB Sapinoso LM Su AI Kern SG Wang-Rodriguez J Moskaluk CA Frierson Jr, HF Hampton GM Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer.Cancer Res. 2001; 61: 5974-5978PubMed Google Scholar, 10Luo J Duggan DJ Chen Y Sauvageot J Ewing CM Bittner ML Trent JM Isaacs WB Human prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling.Cancer Res. 2001; 61: 4683-4688PubMed Google Scholar, 13Jiang Z Woda BA Rock KL Xu Y Savas L Khan A Pihan G Cai F Babcook JS Rathanaswami P Reed SG Xu J Fanger GR P504S: a new molecular marker for the detection of prostate carcinoma.Am J Surg Pathol. 2001; 25: 1397-1404Crossref PubMed Scopus (341) Google Scholar These findings were supported by different groups on the protein level even when using different types of antibodies for immunoblot analysis and high-density TMAs.13Jiang Z Woda BA Rock KL Xu Y Savas L Khan A Pihan G Cai F Babcook JS Rathanaswami P Reed SG Xu J Fanger GR P504S: a new molecular marker for the detection of prostate carcinoma.Am J Surg Pathol. 2001; 25: 1397-1404Crossref PubMed Scopus (341) Google Scholar, 14Rubin MA Zhou M Dhanasekaran SM Varambally S Barrette TR Sanda MG Pienta KJ Ghosh D Chinnaiyan AM Alpha-methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer.JAMA. 2002; 287: 1662-1670Crossref PubMed Scopus (583) Google Scholar, 15Luo J Zha S Gage WR Dunn TA Hicks JL Bennett CJ Ewing CM Platz EA Ferdinandusse S Wanders RJ Trent JM Isaacs WB De Marzo AM Alpha-methylacyl-CoA racemase: a new molecular marker for prostate cancer.Cancer Res. 2002; 62: 2220-2226PubMed Google Scholar Interestingly, hormone-refractory metastatic PCa demonstrated lower AMACR expression than hormone-naive-localized PCa. This observation suggested that AMACR protein expression is regulated by androgens. It is extremely important to identify PCa biomarkers, which portend an aggressive clinical course, given that hormone-refractory tumors are virtually all lethal. However, currently no clinical marker is available to identify a subgroup of localized tumors that may eventually develop into lethal PCa. To examine the intriguing possibility that the PCa biomarker, AMACR, might play a role in hormone dysregulation of localized PCa, we undertook the current study. Clinical samples were taken from the Radical Prostatectomy Series and from the Rapid Autopsy Program at the University of Michigan.16Rubin MA Putzi M Mucci N Smith DC Wojno K Korenchuk S Pienta KJ Rapid (“warm”) autopsy study for procurement of metastatic prostate cancer.Clin Cancer Res. 2000; 6: 1038-1045PubMed Google Scholar Both are part of the University of Michigan Prostate Cancer Specialized Program of Research Excellence (SPORE). Primary PCa of metastatic cases as well as lymph node metastases were contributed in collaboration from the University of Ulm, Ulm, Germany. Detailed clinical expression analyses as well as TMA data were acquired and are maintained on a secure relational database17Manley S Mucci NR De Marzo AM Rubin MA Relational database structure to manage high-density tissue microarray data and images for pathology studies focusing on clinical outcome: the prostate specialized program of research excellence model.Am J Pathol. 2001; 159: 837-843Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar according to the Institutional Review Board protocol of both institutions. Tissue procurement for expression analysis on RNA level was described in detail elsewhere.6Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar For the development of TMA, samples were embedded in paraffin. The study pathologist (MAR) reviewed slides of all cases and circled areas of interest. These slides were used as a template for construction of the six TMAs used in this study. All TMAs were assembled using the manual tissue arrayer (Beecher Instruments, Silver Spring, MD). At least three tissue cores were sampled from each donor block. Histological diagnosis of the tissue cores was verified by standard hematoxylin and eosin (H&E) staining of the initial TMA slide. Standard biotin-avidin complex immunohistochemistry was performed using a polyclonal anti-AMACR antibody (kind gift of Ronald J. A. Wanders, University of Amsterdam, Amsterdam, The Netherlands). Digital images were acquired using the BLISS Imaging System (Bacus Laboratory, Lombard, IL). Staining intensity was scored as negative (score = 1), weak (score = 2), moderate (score = 3), and strong (score = 4). For exploration of the treatment effect by the means of hormonal withdrawal before radical prostatectomy, standard slides were used for regular H&E staining and consecutive sections for detection of AMACR expression. To test AMACR expression in poorly differentiated colon cancers, cases were used from a cohort of well-described colon tumors.18Hinoi T Tani M Lucas PC Caca K Dunn RL Macri E Loda M Appelman HD Cho KR Fearon ER Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon.Am J Pathol. 2001; 159: 2239-2248Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar In addition to well-differentiated colon cancers, a recently described subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance, termed “large-cell minimally differentiated carcinomas,” was used. As previously described, these poorly differentiated colon carcinomas had a high frequency of the microsatellite instability phenotype.18Hinoi T Tani M Lucas PC Caca K Dunn RL Macri E Loda M Appelman HD Cho KR Fearon ER Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon.Am J Pathol. 2001; 159: 2239-2248Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Prostate cell lines (RWPE-1, LNCaP, PC-3, and DU-145) were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 with 8% decomplemented fetal bovine serum, 0.1% glutamine, and 0.1% penicillin and streptomycin (BioWhittaker, Walkersville, MD). Cells were grown to 75% confluence and then treated for 24 and 48 hours with the anti-androgen bicalutamide (Casodex, Zeneca Pharmaceuticals, Plankstadt, Germany) at a final concentration of 20 μmol/L or with methyltrienolone (synthetic androgen) (R1881; NEN, Life Science Products, Boston, MA) at a final concentration of 1 nmol/L. Cells were harvested and lysed in Nonidet P-40 lysis buffer containing 50 mmol/L Tris-HCl, pH 7.4, 1% Nonidet P-40 (Sigma, St. Louis, MO) and complete proteinase inhibitor cocktail (Roche, Indianapolis, IN). Fifteen μg of protein extracts were mixed with sodium dodecyl sulfate sample buffer and electrophoresed onto a 10% sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. After transferring, the membranes (Amersham Pharmacia Biotech, Piscataway, NJ) were incubated for 1 hour in blocking buffer (Tris-buffered saline with 0.1% Tween and 5% nonfat dry milk). The AMACR antibody (kind gift of Dr. Wanders) was applied at 1:10,000 diluted blocking buffer overnight at 4°C. After three washes with TBS-T buffer, the membrane was incubated with horseradish peroxidase-linked donkey anti-rabbit IgG antibody (Amersham Pharmacia Biotech) at 1:5000 for 1 hour at room temperature. The signals were visualized with the ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ). For β-tubulin blots, membranes were stripped with Western Re-Probe buffer (Geno-tech, St. Louis, MO) and blocked in Tris-buffered saline with 0.1% Tween with 5% nonfat dry milk, and incubated with rabbit anti-β-tubulin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500 for 2 hours. For PSA expression the membranes were reprobed in the described manner with PSA antibody (rabbit polyclonal; DAKO Corporation, Carpinteria, CA) at 1:1000 dilution and further processed. Primary analysis of the cDNA expression data were done with the Genepix software. Cluster analysis with the program Cluster and generation of figures with TreeView was performed as described earlier.6Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar AMACR protein expression was statistically evaluated using the mean score result for each prostate tissue type (ie, benign prostate, naive localized or advanced PCa, hormone-treated and hormone-refractory PCa). To test for significant differences in AMACR protein expression between all tissue types, we performed a one-way analysis of variance test. To determine differences between all pairs a post hoc analysis using the Scheffé method was applied as described earlier.14Rubin MA Zhou M Dhanasekaran SM Varambally S Barrette TR Sanda MG Pienta KJ Ghosh D Chinnaiyan AM Alpha-methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer.JAMA. 2002; 287: 1662-1670Crossref PubMed Scopus (583) Google Scholar For comparison of naive primaries to their corresponding lymph node metastases with respect to AMACR protein expression, a nonparametric analysis (Mann-Whitney test) was performed. To compare AMACR expression intensity to the scored hormonal effect of the pretreated localized PCa cases the Mantel-Haenszel chi-square test was applied. AMACR expression scores are presented in a graphical format using error bars with 95% confidence intervals (CIs). P values <0.05 were considered statistically significant. Hierarchical clustering of 76 prostate tissues including benign prostate, benign prostatic hyperplasia, localized PCa, and metastatic PCa and filtering for only those genes with a 1.5-fold expression difference or greater, clustered the samples into histologically distinct groups as previously described.6Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar As demonstrated by a TreeView presentation of this data (Figure 1), AMACR was one of several genes that demonstrated overexpression at the cDNA level of PCa samples with respect to benign pooled prostate tissue. Interestingly, although some of the hormone-refractory cases demonstrated overexpression, as depicted by the red shading, the highest level of overexpression by cDNA analysis was in the clinically localized PCa cases. To further investigate the role of AMACR protein expression in samples with variable differentiation and exposure to anti-androgen treatment, several TMAs with a wide range of PCa were constructed: a total of 119 benign prostate samples, 365 primary hormone-naive PCa samples, 37 naive PCa lymph node metastases, and 41 hormone-refractory metastatic PCa samples were evaluated. An additional 49 hormone-treated primary PCas (including 22 on standard slides) were examined for histological changes associated with anti-androgen treatment and AMACR protein expression. In our hands the percentage of stained cancer cells per sample was 95% in the cases studied. This was independent from the scored intensity of AMACR staining. The mean AMACR protein expression levels for each tissue category is presented in Figure 2A. Benign prostate, naive primary PCa, hormone-treated primary cancer, and hormone-refractory metastatic tissue had a mean staining intensity of 1.28 [standard error (SE), 0.038; 95% CI, 1.20 to 1.35], 3.11 (SE, 0.046; CI, 3.02 to 3.20), 2.86 (SE, 0.15; CI, 2.56 to 3.15), and 2.52 (SE, 0.15; CI, 2.22 to 2.28), respectively. One-way analysis of variance analysis revealed a P value of <0.0001. To specifically examine the difference between different tissue types, a post hoc pair-wise comparison was performed. Clinically localized PCa demonstrated a significantly stronger AMACR protein expression as compared to benign prostate tissue (post hoc analysis using Scheffé method; mean difference = 1.83; P < 0.0001; CI, 1.53 to 2.13). As expected from previous work, a significant decrease in AMACR protein expression was observed in the metastatic hormone-refractory PCa samples with respect to clinically localized PCa (0.59; P = 0.002; CI, 0.15 to 1.03). Hormone-treated primaries had a mean AMACR expression of 2.86, which was between the expression levels of naive primaries (3.11) and hormone-refractory cases (2.52) (post hoc analysis using Scheffé method; P = 0.51; CI, −0.66 to 0.16; and P = 0.56; CI, −0.23 to 0.91). Interestingly there was no significant difference in AMACR expression in the 37 naive primary prostate samples and lymph node metastases derived from the same patient (Mann-Whitney test, P = 0.8). In other words, matched primaries and lymph node metastases showed a similar AMACR expression pattern (Figure 2B). Examples of in situ AMACR protein expression in a naive primary PCa and a naive lymph node metastasis are presented in Figure 2. Because there seems to be a wider variability in AMACR protein expression in the hormone-treated PCa samples as compared to naive cases (Figure 2), we further investigated this observation to correlate the hormone treatment effect with AMACR protein expression. We examined a subset of 22 PCa cases in which the patients received variable amounts and types of anti-androgen treatment before surgery. These cases were evaluated blindly with respect to treatment protocol for histological evidence of hormone treatment (H&E slide) and AMACR protein expression. The hormonal effect visible on the H&E slides was classified from 1 to 4 with 1 representing “no effect” and 4 showing a “very strong effect.” Thirteen cases demonstrated either no or moderate hormonal effect and nine cases had a very strong hormonal effect. Statistical analysis revealed a significant difference between these two groups with respect to AMACR expression intensity (Figure 3, Mantel-Haenszel chi-square, P = 0.009). Figure 3 presents an example of a PCa case treated before surgery with anti-androgens, which has a strong hormonal effect appreciated on H&E and decreased AMACR protein expression (Figure 3A). In these cases, no association was detected between the type of hormone treatment (ie, monotherapy or complete hormonal withdrawal for hormone deprivation) or duration of treatment and AMACR expression. For further exploration of the hormonal effect on AMACR expression, primary cell culture experiments and Western blot analysis were performed. As demonstrated in Figure 3B, LNCaP cells, derived from a metastatic lesion but considered hormone responsive, showed a higher baseline AMACR expression as compared to PC-3 and DU-145 cells, which are both hormone-independent cell lines derived from metastatic lesions. A benign cell line, RWPE-1,19Bello D Webber MM Kleinman HK Wartinger DD Rhim JS Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18.Carcinogenesis. 1997; 18: 1215-1223Crossref PubMed Scopus (331) Google Scholar showed near absent AMACR expression, which is consistent with our in situ protein expression data. To simulate an anti-androgen treatment, we used the hormone-responsive cell line, LNCaP, and treated the cells with bicalutamide in a final concentration of 20 μmol/L for a time period of 24 and 48 hours. Interestingly, AMACR expression in cell lysates of LNCaP cells did not change at either time point when exposed to anti-androgen therapy. Under the same conditions, PSA, a gene known to be regulated by the androgen receptor, showed decreased protein expression. In addition, when LNCaP cells were exposed to a synthetic androgen R1881, no increase in AMACR expression was observed (Figure 3B). Therefore, these cell culture experiments provide evidence that AMACR expression may not be directly regulated by the androgen pathway. However, another explanation for these observations was that AMACR overexpression occurred in PCa, but as these tumors became poorly differentiated, as in the hormone-refractory PCa, AMACR expression was down-regulated either directly or indirectly because of the process of dedifferentiation. To elucidate this potential correlation we examined colon cancer samples for AMACR expression. As we have recently identified,20Zhou M Chinnaiyan A Kleer C Lucas P Rubin MA Alpha-methyl-CoA racemase: a novel tumor marker over expressed in several human cancers and their precursor lesions.Am J Surg Pathol. 2002; 26: 926-931Crossref PubMed Scopus (259) Google Scholar AMACR protein expression is also observed in some other tumor types, with the highest overall expression in colorectal cancers. Colorectal cancers are not known to be regulated by androgens and therefore represent a good control to test this hypothesis. Twenty well-differentiated and nine anaplastic colon cancer samples were chosen. As previously described, the poorly differentiated tumors have distinct molecular alterations distinguishing them from the common well to moderately differentiated colorectal tumors.18Hinoi T Tani M Lucas PC Caca K Dunn RL Macri E Loda M Appelman HD Cho KR Fearon ER Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon.Am J Pathol. 2001; 159: 2239-2248Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Figure 4 demonstrates strong AMACR protein expression in a moderately differentiated colon cancer. This tumor still forms well-defined glandular structures. The surrounding benign colonic tissue does not express AMACR. The anaplastic colon cancers demonstrated weak AMACR protein expression (Figure 4). Moderate to strong AMACR expression was seen in 18 of 20 differentiated cases but in only 2 of 9 anaplastic colonic cancers (chi-square, P < 0.001). Seven of nine of the anaplastic colon cancers had weak to moderate expression. For comparative purposes examples of metastatic hormone-refractory PCa from the TMA experiments are shown in Figure 4. Both of these cases demonstrate weak AMACR protein expression. High-throughput technologies such as cDNA microarrays and TMA have opened a gateway for research. The combination of a tool for discovery with one for verification allows surveying target genes in a very efficient manner. By exploring gene expression data it could be shown that AMACR is consistently overexpressed in PCa.14Rubin MA Zhou M Dhanasekaran SM Varambally S Barrette TR Sanda MG Pienta KJ Ghosh D Chinnaiyan AM Alpha-methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer.JAMA. 2002; 287: 1662-1670Crossref PubMed Scopus (583) Google Scholar These findings are supported by AMACR expression data described recently in at least two more independent studies using cDNA microarrays.8Welsh JB Sapinoso LM Su AI Kern SG Wang-Rodriguez J Moskaluk CA Frierson Jr, HF Hampton GM Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer.Cancer Res. 2001; 61: 5974-5978PubMed Google Scholar, 10Luo J Duggan DJ Chen Y Sauvageot J Ewing CM Bittner ML Trent JM Isaacs WB Human prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling.Cancer Res. 2001; 61: 4683-4688PubMed Google Scholar Even combined analysis across the three studies revealed statistically significant differential expression of AMACR between benign prostate and PCa.14Rubin MA Zhou M Dhanasekaran SM Varambally S Barrette TR Sanda MG Pienta KJ Ghosh D Chinnaiyan AM Alpha-methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer.JAMA. 2002; 287: 1662-1670Crossref PubMed Scopus (583) Google Scholar This study focuses on the AMACR expression in different groups of PCa including the aspect of neoadjuvant hormonal withdrawal in localized disease. The most interesting finding in this context was that AMACR expression is decreased in hormone-refractory metastatic tissue samples compared to localized PCa. From the clinical point of view, this observation is very important because localized PCa can be controlled well by different means such as surgery or radiotherapy or even watchful waiting.21Krisch EB Koprowski CD Deciding on radiation therapy for prostate cancer: the physician's perspective.Semin Urol Oncol. 2000; 18: 214-225PubMed Google Scholar, 22Han M Partin AW Pound CR Epstein JI Walsh PC Long-term biochemical disease-free and cancer-specific survival following anatomic radical retropubic prostatectomy. The 15-year Johns Hopkins experience.Urol Clin N Am. 2001; 28: 555-565Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar On the other hand, there is no consensus as how to treat metastatic disease successfully to positively impact life expectancy.23Altwein JE Therapeutic options in locally defined or advanced prostate cancer.Eur Urol. 1999; 35: 9-16Crossref PubMed Scopus (2) Google Scholar Therefore, identifying PCa biomarkers that are up-regulated in localized PCa but decrease again in far advanced hormone-refractory PCa could help to understand the mechanism involved with the ultimate goal of identifying new treatments. A surprising finding is that AMACR expression seemed to be hormone-independent in the setting of our cell culture experiments. PSA, a gene known to be regulated by androgens, demonstrated hormone-related alterations in expression under the same conditions. These findings may provide evidence that AMACR is not regulated by the androgen pathway. Still, because we see a decreased AMACR expression in hormone-refractory tissue, it might be postulated that AMACR could play a role as a biomarker for hormone resistance. Considering the fact that hormone treatment in the mean of hormonal withdrawal did not affect AMACR expression in the cell culture, may lead to the conclusion that some othermechanism than the androgen pathway is responsible for AMACR down-regulation in the integrity of cancer tissue. These findings are considered to be very important and require further investigation. An alternative hypothesis would suggest that AMACR is overexpressed in the development of cancer, perhaps playing an important role in providing energy for the neoplastic cells. However as the tumors become dedifferentiated, they no longer require these sources of energy. Poorly differentiated tumors may take over other pathways to accomplish this same activity of branched fatty acid oxidation. Recent work failed to identify an association with the proliferative rate of the tumor cells and AMACR expression14Rubin MA Zhou M Dhanasekaran SM Varambally S Barrette TR Sanda MG Pienta KJ Ghosh D Chinnaiyan AM Alpha-methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer.JAMA. 2002; 287: 1662-1670Crossref PubMed Scopus (583) Google Scholar or with the Gleason score and AMACR expression.13Jiang Z Woda BA Rock KL Xu Y Savas L Khan A Pihan G Cai F Babcook JS Rathanaswami P Reed SG Xu J Fanger GR P504S: a new molecular marker for the detection of prostate carcinoma.Am J Surg Pathol. 2001; 25: 1397-1404Crossref PubMed Scopus (341) Google Scholar, 14Rubin MA Zhou M Dhanasekaran SM Varambally S Barrette TR Sanda MG Pienta KJ Ghosh D Chinnaiyan AM Alpha-methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer.JAMA. 2002; 287: 1662-1670Crossref PubMed Scopus (583) Google Scholar Examination of other tumors demonstrated that colon cancer has the highest AMACR expression (unpublished observations). As colorectal cancers are not known to be hormonally regulated, the fact that dedifferentiation and decreased AMACR expression went hand in hand, further supports the hypothesis that dedifferentiation leads to decreased AMACR expression in the hormone-refractory metastatic PCa. Hormone treatment is also a front line therapy in metastatic PCa but is known to loose efficacy, selecting out hormone-insensitive clones. Anticipating the selection of potentially more dedifferentiated cells because of hormone treatment, an observed strong treatment effect may be consistent with decreased AMACR expression because of the selection of more dedifferentiated cells. The AMACR gene product is an enzyme, that plays an important role in bile acid biosynthesis and β-oxidation of branched-chain fatty acids.12Kotti TJ Savolainen K Helander HM Yagi A Novikov DK Kalkkinen N Conzelmann E Hiltunen JK Schmitz W In mouse alpha-methylacyl-CoA racemase, the same gene product is simultaneously located in mitochondria and peroxisomes.J Biol Chem. 2000; 275: 20887-20895Crossref PubMed Scopus (57) Google Scholar, 24Ferdinandusse S Overmars H Denis S Waterham HR Wanders RJ Vreken P Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency.J Lipid Res. 2001; 42: 137-141PubMed Google Scholar The link of AMACR expression and neoplasia, however, has only recently been made. AMACR overexpression appears to occur in tumors that have been linked to high-fat diet such as PCa and colorectal cancer.25Bartsch H Nair J Owen RW Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers.Carcinogenesis. 1999; 20: 2209-2218Crossref PubMed Scopus (406) Google Scholar The relationship between fatty acid consumption and cancer is a controversial subject in the development of PCa and colorectal cancer.26Moyad MA Fat reduction to prevent prostate cancer: waiting for more evidence?.Curr Opin Urol. 2001; 11: 457-461Crossref PubMed Scopus (7) Google Scholar, 27Willett WC Diet and cancer.Oncologist. 2000; 5: 393-404Crossref PubMed Scopus (207) Google Scholar An essential role for AMACR in the oxidation of bile acid intermediates has been demonstrated. AMACR encodes an enzyme that catalyzes the racemization of α-methyl-branched carboxylic coenzyme A thioesters and is localized in peroxisomes and mitochondria.28Schmitz W Albers C Fingerhut R Conzelmann E Purification and characterization of an alpha-methylacyl-CoA racemase from human liver.Eur J Biochem. 1995; 231: 815-822Crossref PubMed Scopus (122) Google Scholar As AMACR is involved in the metabolism of lipids it may be speculated that this could lead to alterations in the oxidant balance of a cell. These changes might be associated with DNA damage, malignant transformation, and other parameters of cell disturbance. It is still not clear if AMACR is directly involved in carcinogenesis via a degradation pathway of branched chain fatty acids or if it represents an epiphenomenon, because it is overexpressed in some human tumors. These hypotheses need to be further elucidated. We thank Dr. Ronald J. A. Wanders (University of Amsterdam) for the anti-AMACR antibody; Robin Kunkel with assistance in preparing figures; Dr. Henry D. Appelman (Pathology, University of Michigan) for pathology support; Prof. Dr. Peter Möller, Chairman Department of Pathology, Ulm, Germany, for providing tissue samples; and Dr. Kenneth J. Pienta, Associate Professor, University of Michigan, for his support through the Rapid Autopsy Program and the SPORE affiliation.

Distinguishing aggressive prostate cancer from indolent disease represents an important clinical challenge, as current therapy requires over treating men with prostate cancer to prevent the progression of a few cases. Expression of the metastasis-associated protein 1 (MTA1) has previously been found to be associated with progression to the metastatic state in various cancers. Analyzing DNA microarray data, we found MTA1 to be selectively overexpressed in metastatic prostate cancer compared with clinically localized prostate cancer and benign prostate tissue. These results were validated by demonstrating overexpression of MTA1 in metastatic prostate cancer by immunoblot analysis. MTA1 protein expression was evaluated by immunohistochemistry in a broad spectrum of prostate tumors with tissue microarrays containing 1940 tissue cores from 300 cases. Metastatic prostate cancer demonstrated significantly higher mean MTA1 protein expression intensity (score = 3.4/4) and percentage of tissue cores staining positive for MTA1 (83%) compared with clinically localized prostate cancer (score = 2.8/4, 63% positive cores) or benign prostate tissue (score = 1.5/4, 25% positive cores) with a mean difference of 0.54 and 1.84, respectively (P < 0.00001 for both). Paradoxically, for localized disease, higher MTA1 protein expression was associated with lower rates of prostate specific antigen recurrence after radical prostatectomy for localized disease. In summary, this study identified an association of MTA1 expression and prostate cancer progression.

The aim of the current study was to evaluate the expression of ADAM15 disintegrin (ADAM15) in a broad spectrum of human tumors. The transcript for ADAM15 was found to be highly upregulated in a variety of tumor cDNA expression arrays. ADAM15 protein expression was examined in tissue microarrays (TMAs) consisting of 638 tissue cores. TMA analysis revealed that ADAM15 protein was significantly increased in multiple types of adenocarcinoma, specifically in prostate and breast cancer specimens. Statistical association was observed with disease progression within clinical parameters of predictive outcome for both prostate and breast cancers, pertaining to Gleason sum and angioinvasion, respectively. In this report, we also present data from a cDNA microarray of prostate cancer (PCa), where we compared transfected LNCaP cells that overexpress ADAM15 to vector control cells. In these experiments, we found that ADAM15 expression was associated with the induction of specific proteases and protease inhibitors, particularly tissue inhibitor of metalloproteinase 2, as validated in a separate PCa TMA. These results suggest that ADAM15 is generally overexpressed in adenocarcinoma and is highly associated with metastatic progression of prostate and breast cancers.

Prostate cancer is the most common type of tumor found in American men and is the second leading cause of cancer death in males. To identify biomarkers that distinguish prostate cancer from normal, we compared multiple gene expression profiling studies. Through meta-analysis of expression array data from multiple prostate cancer studies, we identified GOLM1 (Golgi membrane protein 1, Golm 1) as consistently up-regulated in clinically localized prostate cancer. This observation was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and validated at the protein level by immunoblot assay and immunohistochemistry. Prostate epithelial cells were identified as the cellular source of GOLM1 expression using laser capture microdissection. Immunohistochemical staining localized the GOLM1 signal to the subapical cytoplasmic region, typical of a Golgi distribution. Surprisingly, GOLM1 immunoreactivity was detected in the supernatants of prostate cell lines and in the urine of patients with prostate cancer. The mechanism by which intact GOLM1 might be released from cells has not yet been elucidated. GOLM1 transcript levels were measured in urine sediments using quantitative PCR on a cohort of patients presenting for biopsy or radical prostatectomy. We found that urinary GOLM1 mRNA levels were a significant predictor of prostate cancer. Further, GOLM1 outperformed serum prostate-specific antigen (PSA) in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.622 for GOLM1 (P = .0009) versus 0.495 for serum PSA (P = .902). Our data indicating the up-regulation of GOLM1 expression and its appearance in patients' urine suggest GOLM1 as a potential novel biomarker for clinically localized prostate cancer.

Rap1GAP is a critical tumor suppressor gene that is downregulated in multiple aggressive cancers, such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP downregulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 upregulation, and the concomitant downregulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of histone 3 at lysine 27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression.

Abstract Using an integrative genomics approach called amplification breakpoint ranking and assembly analysis, we nominated KRAS as a gene fusion with the ubiquitin-conjugating enzyme UBE2L3 in the DU145 cell line, originally derived from prostate cancer metastasis to the brain. Interestingly, analysis of tissues revealed that 2 of 62 metastatic prostate cancers harbored aberrations at the KRAS locus. In DU145 cells, UBE2L3-KRAS produces a fusion protein, a specific knockdown of which attenuates cell invasion and xenograft growth. Ectopic expression of the UBE2L3-KRAS fusion protein exhibits transforming activity in NIH 3T3 fibroblasts and RWPE prostate epithelial cells in vitro and in vivo. In NIH 3T3 cells, UBE2L3-KRAS attenuates MEK/ERK signaling, commonly engaged by oncogenic mutant KRAS, and instead signals via AKT and p38 mitogen-activated protein kinase (MAPK) pathways. This is the first report of a gene fusion involving the Ras family, suggesting that this aberration may drive metastatic progression in a rare subset of prostate cancers. Significance: This is the first description of an oncogenic gene fusion of KRAS, one of the most studied proto-oncogenes. KRAS rearrangement may represent the driving mutation in a rare subset of metastatic prostate cancers, emphasizing the importance of RAS-RAF-MAPK signaling in this disease. Cancer Discovery; 1(1); 35–43. © 2011 AACR. Read the Commentary on this article by Edgren et al., p. 12 This article is highlighted in the In This Issue feature, p. 4

The neuronal repellent SLIT2 is repressed in a number of cancer types primarily through promoter hypermethylation. SLIT2, however, has not been studied in prostate cancer. Through genome-wide location analysis we identified SLIT2 as a target of polycomb group (PcG) protein EZH2. The EZH2-containing polycomb repressive complexes bound to the SLIT2 promoter inhibiting its expression. SLIT2 was downregulated in a majority of metastatic prostate tumors, showing a negative correlation with EZH2. This repressed expression could be restored by methylation inhibitors or EZH2-suppressing compounds. In addition, a low level of SLIT2 expression was associated with aggressive prostate, breast and lung cancers. Functional assays showed that SLIT2 inhibited prostate cancer cell proliferation and invasion. Thus, this study showed for the first time the epigenetic silencing of SLIT2 in prostate tumors, and supported SLIT2 as a potential biomarker for aggressive solid tumors. Importantly, PcG-mediated repression may serve as a precursor for the silencing of SLIT2 by DNA methylation in cancer.

Transforming growth factor-β (TGF-β) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-β, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-β signaling. BUB1 interacted with TGFBRI in the presence of TGF-β and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-β-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-β signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-β signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-β signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-β pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion.

MicroRNA-101, a tumor suppressor microRNA (miR), is often downregulated in cancer and is known to target multiple oncogenes. Some of the genes that are negatively regulated by miR-101 expression include histone methyltransferase EZH2 (enhancer of zeste homolog 2), COX2 (cyclooxygenase-2), POMP (proteasome maturation protein), CERS6, STMN1, MCL-1 and ROCK2, among others. In the present study, we show that miR-101 targets transcriptional coactivator SUB1 homolog (Saccharomyces cerevisiae)/PC4 (positive cofactor 4) and regulates its expression. SUB1 is known to have diverse role in vital cell processes such as DNA replication, repair and heterochromatinization. SUB1 is known to modulate transcription and acts as a mediator between the upstream activators and general transcription machinery. Expression profiling in several cancers revealed SUB1 overexpression, suggesting a potential role in tumorigenesis. However, detailed regulation and function of SUB1 has not been elucidated. In this study, we show elevated expression of SUB1 in aggressive prostate cancer. Knockdown of SUB1 in prostate cancer cells resulted in reduced cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Gene expression analyses coupled with chromatin immunoprecipitation revealed that SUB1 binds to the promoter regions of several oncogenes such as PLK1 (Polo-like kinase 1), C-MYC, serine-threonine kinase BUB1B and regulates their expression. Additionally, we observed SUB1 downregulated CDKN1B expression. PLK1 knockdown or use of PLK1 inhibitor can mitigate oncogenic function of SUB1 in benign prostate cancer cells. Thus, our study suggests that miR-101 loss results in increased SUB1 expression and subsequent activation of known oncogenes driving prostate cancer progression and metastasis. This study therefore demonstrates functional role of SUB1 in prostate cancer, and identifies its regulation and potential downstream therapeutic targets of SUB1 in prostate cancer.

Metastatic urothelial carcinoma is generally incurable with current systemic therapies. Chromatin modifiers are frequently mutated in bladder cancer, with ARID1A-inactivating mutations present in about 20% of tumors. EZH2, a histone methyltransferase, acts as an oncogene that functionally opposes ARID1A. In addition, PI3K signaling is activated in more than 20% of bladder cancers. Using a combination of in vitro and in vivo data, including patient-derived xenografts, we show that ARID1A-mutant tumors were more sensitive to EZH2 inhibition than ARID1A WT tumors. Mechanistic studies revealed that (a) ARID1A deficiency results in a dependency on PI3K/AKT/mTOR signaling via upregulation of a noncanonical PI3K regulatory subunit, PIK3R3, and downregulation of MAPK signaling and (b) EZH2 inhibitor sensitivity is due to upregulation of PIK3IP1, a protein inhibitor of PI3K signaling. We show that PIK3IP1 inhibited PI3K signaling by inducing proteasomal degradation of PIK3R3. Furthermore, ARID1A-deficient bladder cancer was sensitive to combination therapies with EZH2 and PI3K inhibitors in a synergistic manner. Thus, our studies suggest that bladder cancers with ARID1A mutations can be treated with inhibitors of EZH2 and/or PI3K and revealed mechanistic insights into the role of noncanonical PI3K constituents in bladder cancer biology.

Therapeutic interventions that counter emerging targets in diabetes eye diseases are lacking. We hypothesize that a combination therapy targeting inflammation and hyperglycemia can prevent diabetic eye diseases. Here, we report a multipronged approach to prevent diabetic cataracts and retinopathy by combining orally bioavailable curcumin-laden double-headed (two molecules of gambogic acid conjugated to terminal carboxyl groups of poly(d,l-lactide-co-glycolide)) nanoparticles and injectable basal insulin. The combination treatment led to a significant delay in the progression of diabetic cataracts and retinopathy, improving liver function and peripheral glucose homeostasis. We found a concurrent reduction in lens aggregate protein, AGEs, and increased mitochondrial ATP production. Importantly, inhibition of Piezo1 protected against hyperglycemia-induced retinal vascular damage suggesting possible involvement of Piezo1 in the regulation of retinal phototransduction. Histologic evaluation of murine small intestines revealed that chronic administration of curcumin-laden double-headed nanoparticles was well tolerated, circumventing the fear of nanoparticle toxicity. These findings establish the potential of anti-inflammatory and anti-hyperglycemic combination therapy for the prevention of diabetic cataracts and retinopathy.

The differentiation and maturation of dendritic cells (DCs) is governed by various signals in the microenvironment. Monocytes and DCs circulate in peripheral blood, which contains high levels of natural antibodies (NAbs). NAbs are germ-line-encoded and occur in the absence of deliberate immunization or microbial aggression. To assess the importance of NAbs in the milieu on DC development, we examined the status of DCs in patients with X-linked agammaglobulinemia, a disease characterized by paucity of B cells and circulating antibodies. We demonstrate that the in vitro differentiation of DCs is severely impaired in these patients, at least in part because of low levels of circulating NAbs. We identified NAbs reactive with the CD40 molecule as an important component that participates in the development of DCs. CD40-reactive NAbs restored normal phenotypes of DCs in patients. The maturation process induced by CD40-reactive NAbs was accompanied by an increased IL-10 and decreased IL-12 production. The transcription factor analysis revealed distinct signaling pathways operated by CD40-reactive NAbs compared to those by CD40 ligand. These results suggest that B cells promote bystander DC development through NAbs and the interaction between NAbs and DCs may play a role in steady-state migration of DCs.

TRAIL/APO-2L induces apoptosis in a variety of transformed cells and has potential as an anti-cancer therapeutic. The physiologic role of TRAIL is presumably more complex than merely activating caspase-mediated cell death. To shed light into TRAIL-mediated signaling, we used DNA microarrays to profile gene expression mediated by TRAIL in breast carcinoma cells. Primary response genes induced by TRAIL included a number of known NF-κB-dependent genes such as cIAP2, A20, and E-selectin. Remarkably, global transcriptome analysis revealed that TRAIL also induced a cohort of genes related to the interferon-signaling pathway. Assessing interferon-induced gene expression suggested various points of interaction with the TRAIL signaling pathway. Interestingly, while we observed interferon-mediated up-regulation of TRAIL, we also demonstrated a concomitant TRAIL-mediated induction of interferon-β. Combining TRAIL and interferon in vitro, synergistically induced apoptosis and caspase activation in breast cancer cells. Together, these data indicate multiple levels of molecular cross-talk between the two diverse cytokines with anti-tumor properties. TRAIL/APO-2L induces apoptosis in a variety of transformed cells and has potential as an anti-cancer therapeutic. The physiologic role of TRAIL is presumably more complex than merely activating caspase-mediated cell death. To shed light into TRAIL-mediated signaling, we used DNA microarrays to profile gene expression mediated by TRAIL in breast carcinoma cells. Primary response genes induced by TRAIL included a number of known NF-κB-dependent genes such as cIAP2, A20, and E-selectin. Remarkably, global transcriptome analysis revealed that TRAIL also induced a cohort of genes related to the interferon-signaling pathway. Assessing interferon-induced gene expression suggested various points of interaction with the TRAIL signaling pathway. Interestingly, while we observed interferon-mediated up-regulation of TRAIL, we also demonstrated a concomitant TRAIL-mediated induction of interferon-β. Combining TRAIL and interferon in vitro, synergistically induced apoptosis and caspase activation in breast cancer cells. Together, these data indicate multiple levels of molecular cross-talk between the two diverse cytokines with anti-tumor properties. tumor necrosis factor death receptor interleukin signal transducers and activators of transcription interferon interferon-stimulating gene factor-3 γ phosphate-buffered saline reverse transcriptase glyceraldehyde-3-phosphate dehydrogenase cycloheximide seven in absentia homolog-2 Ras suppressor-1 cell-division cycle-6 TRAIL/APO-2L is a member of the tumor necrosis factor (TNF)1 cytokine family, which are type II transmembrane proteins that share homology in their extracellular domains (1Schulze-Osthoff K. Ferrari D. Los M. Wesselborg S. Peter M.E. Eur. J. Biochem. 1998; 254: 439-459Crossref PubMed Scopus (864) Google Scholar, 2Golstein P. Curr. Biol. 1997; 7: R750-753Crossref PubMed Google Scholar, 3Pitti R.M. Marsters S.A. Ruppert S. Donahue C.J. Moore A. Ashkenazi A. J. Biol. Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1655) Google Scholar). A subset of these ligands is known to activate the cell death program including TNF, Fas ligand, DR3 ligand (TWEAK), and TRAIL. Of these, TRAIL has garnered the most interest therapeutically, as several studies have demonstrated in vivo tumoricidal activity in animal models without significant toxicity (4Chinnaiyan A.M. Prasad U. Shankar S. Hamstra D.A. Shanaiah M. Chenevert T.L. Ross B.D. Rehemtulla A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1754-1759Crossref PubMed Scopus (488) Google Scholar, 5Walczak H. Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A. Le T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2236) Google Scholar, 6Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Invest. 1999; 104: 155-162Crossref PubMed Scopus (2008) Google Scholar). Death ligands induce apoptosis by activating their cognate receptors on the surface of cells. These death receptors are members of the TNF receptor superfamily and include TNFRI, Fas (CD95/APO-1), DR-3 (death receptor-3), DR-4, DR-5, and DR-6 (1Schulze-Osthoff K. Ferrari D. Los M. Wesselborg S. Peter M.E. Eur. J. Biochem. 1998; 254: 439-459Crossref PubMed Scopus (864) Google Scholar, 7Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5169) Google Scholar). TRAIL specifically binds and induces apoptosis via DR-4 and DR-5 (8Walczak H. Degli-Esposti M.A. Johnson R.S. Smolak P.J. Waugh J.Y. Boiani N. Timour M.S. Gerhart M.J. Schooley K.A. Smith C.A. Goodwin R.G. Rauch C.T. EMBO J. 1997; 16: 5386-5397Crossref PubMed Scopus (1020) Google Scholar, 9Pan G. Ni J. Wei Y.F. Yu G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1383) Google Scholar, 10Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1562) Google Scholar). Once activated, the death receptors, in general, bind the cytoplasmic adaptor molecule FADD, which functions as a molecular bridge to caspase-8, a protease at the apex of the cell death cascade (11Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 12Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar). DR-4 and DR-5 have been proposed to indirectly bind FADD via the GTP-binding adaptor molecule, DAP3 (13Miyazaki T. Reed J.C. Nat. Immunol. 2001; 2: 493-500Crossref PubMed Scopus (86) Google Scholar). By inducing proximity between pro-caspase-8 molecules, which possess weak protease activity, death receptor clustering facilitates trans-proteolysis and consequent activation of the cell death machinery (14Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (773) Google Scholar, 15Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). TNF family ligands also have important nonapoptotic functions. For example, many death ligands (especially TNFα) are known to activate transcription through NF-κB and AP-1, leading to induction of inflammatory and immunomodulatory genes (16Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1131) Google Scholar). Activation of these nonapoptotic pathways involves a host of signaling mediators including TRADD, TRAF2, RIP, NIK, and others. Even FADD, a molecule required for endogenous activation of capsase-8, has functions outside of regulating cell suicide, including proposed roles in survival and proliferation (17Newton K. Harris A.W. Strasser A. EMBO J. 2000; 19: 931-941Crossref PubMed Scopus (129) Google Scholar, 18Newton K. Kurts C. Harris A.W. Strasser A. Curr. Biol. 2001; 11: 273-276Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Strasser A. Newton K. Int. J. Biochem. Cell Biol. 1999; 31: 533-537Crossref PubMed Scopus (82) Google Scholar, 20Walsh C.M. Wen B.G. Chinnaiyan A.M. O'Rourke K. Dixit V.M. Hedrick S.M. Immunity. 1998; 8: 439-449Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 21Zornig M. Hueber A.O. Evan G. Curr. Biol. 1998; 8: 467-470Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). FADD-deficient mice die in utero, suggesting that FADD has an important role in developmental pathways (22Zhang J. Cado D. Chen A. Kabra N.H. Winoto A. Nature. 1998; 392: 296-300Crossref PubMed Scopus (640) Google Scholar, 23Yeh W.C. Pompa J.L. McCurrach M.E. Shu H.B. Elia A.J. Shahinian A. Ng M. Wakeham A. Khoo W. Mitchell K. El-Deiry W.S. Lowe S.W. Goeddel D.V. Mak T.W. Science. 1998; 279: 1954-1958Crossref PubMed Scopus (804) Google Scholar). However, relatively little is known about possible nonapoptotic functions induced by TRAIL. To shed light into death ligand signaling and function, we studied gene expression profiles elicited by these cytokines. By using a functional genomics approach, we identified novel primary response genes induced by TRAIL and related death ligands. A dominant negative version of FADD (FADD-DN), inhibited much of the gene activation mediated by death ligands, further emphasizing the role of FADD in nonapoptotic signaling induced by death receptors. To our surprise, a cohort of genes induced by TRAIL at a late time point (≥16 h), is classically associated with the interferon signaling pathway, including ISGF3γ, STAT-1, PKR, IL-6, and IFNγR2, among others. Several genes identified by microarray were validated at the transcript level by Northern blot analysis and at the protein level by immunoblotting. While TRAIL transcript and protein were induced by interferons, we also demonstrated TRAIL-mediated up-regulation of IFNβ transcript and protein. Functionally, TRAIL and interferons had a synergistic effect in the induction of apoptosis in breast cancer cells. As our study demonstrates that TRAIL can induce interferon pathways, presumably via up-regulation of IFNβ, exposure of cancer cells to interferons may “prime” cells for apoptosis by TRAIL. The diverse gene expression profiles activated by the TRAIL signaling pathway may provide clues to diverse physiologic roles of TRAIL in vivo. MCF7 breast cancer cells, stably transfected with pCDNA3 and FADD-DN constructs (24Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar) were grown in RPMI medium containing 10% fetal bovine serum and 0.5 mg/ml geneticin (Invitrogen) at 37 °C in a humidified atmosphere of 10% CO2. SUM149 breast cancer cells (25van Golen K.L. Wu Z.F. Qiao X.T. Bao L. Merajver S.D. Neoplasia. 2000; 2: 418-425Crossref PubMed Scopus (143) Google Scholar) were grown in Ham's F12 medium containing 5% fetal bovine serum and supplemented with 5 μg/ml insulin (Sigma) and 1 μg/ml hydrocortisone (Sigma) at 37 °C in a humidified atmosphere of 10% CO2. Treatment with various reagents was carried out on subconfluent plates (∼4–6 million cells per 100-mm tissue culture plate) with fresh medium at the following concentrations: 1000 ng/ml TRAIL (4Chinnaiyan A.M. Prasad U. Shankar S. Hamstra D.A. Shanaiah M. Chenevert T.L. Ross B.D. Rehemtulla A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1754-1759Crossref PubMed Scopus (488) Google Scholar), 100 ng/ml TNFα (specific activity ≥ 2 × 107 units/mg; ED50 ≤0.05 ng/ml) (Atlanta Biologicals), 100 ng/ml αFasAb (Alexis), 100 ng/ml TWEAK (TNF relatedweak inducer of apoptosis) (ED50 ≤ 10 ng/ml) (Atlanta Biologicals), 10 μg/ml cycloheximide (Sigma), 5 μm zVADfmk (Enzyme Systems Products), 1000 units/ml IFN-α (PBL Biomedical Labs), 1000 units/ml IFN-β (BIOSOURCE Int.), or 1000 units/ml IFN-γ (R&D Systems). Cells were harvested by trypsinization, washed once with Dulbecco's phosphate-buffered saline, and fixed in 70% ice-cold ethanol at −20 °C for 10 min. Cells were then washed twice with PBS and stained at room temperature for 10 min in Dulbecco's phosphate-buffered saline containing 100 μg/ml propidium iodide (Sigma), 10 μg/ml RNase A (Promega), and 0.01% Triton X-100. After an additional PBS wash, the cells were visualized by fluorescence microscopy. Propidium iodide stained the condensed fragmented chromatin of apoptotic nuclei intensely as opposed to diffuse nuclear staining associated with normal cells (24Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar). Three independent experiments were performed for each data point with over 250 cells counted per sample. Percent apoptosis was calculated as a mean ± S.D. DNA microarray analysis of gene expression was done essentially as described by the Brown Lab (available at www.microarrays.org) and in Refs. 26Dhanasekaran M.S. Barrette T.R. Ghosh D. Shah R. Varambally S. Kurachi K. Pienta K. Rubin M.A. Chinnaiyan A.M. Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar and 27Chinnaiyan A.M. Huber-Lang M. Kumar-Sinha C. Barrette T.R. Shankar-Sinha S. Sarma V.J. Padgaonkar V.A. Ward P.A. Am. J. Pathol. 2001; 159: 1199-1209Abstract Full Text Full Text PDF PubMed Google Scholar. The sequence-verified cDNA clones on the human cDNA microarray are listed in the Supplementary Material and are available from the Research Genetics web site (www.resgen.com). Purified PCR products, generated using the clone inserts as template, were spotted onto poly-l-lysine-coated microscope slides using an Omnigrid robotic arrayer (GeneMachines) equipped with quill-type pins (Majer Scientific). Based on the latest Unigene build-136, our 10,000 human cDNA microarray covers ∼5996 unique, known, named genes and 2674 ESTs. Of these, 495 genes are present in duplicates, which serve as internal controls. Protocols for printing and post-processing of arrays are available in the public domain (www.microarrays.org). Approximately 4–6 million cells per 100-mm tissue culture plate were washed with PBS, homogenized in Trizol (Invitrogen), and total RNA was isolated according to the manufacturer's protocol. An extra phenol-chloroform extraction was performed to improve the quality of RNA. RNA integrity was judged by denaturing formaldehyde-agarose gel electrophoresis. Total RNA was quantified and stored in aliquots at −80 °C until use. Fifteen micrograms of total RNA was routinely used as template for cDNA generation using reverse transcriptase (RT) (Invitrogen). Inclusion of amino allyl-dUTP (Sigma) in the RT reaction allowed for subsequent fluorescent labeling of cDNA using monofunctional NHS ester dyes (Amersham Bioscience, Inc.) as described at www.microarrays.org. In each experiment, fluorescent cDNA probes were prepared from an experimental RNA sample (coupled to monofunctional Cy5 NHS-ester) and an appropriate reference RNA sample (coupled to monofunctional Cy3 NHS-ester). The labeled probes were then hybridized to 10,000 human cDNA microarrays at 65 °C overnight as described previously (www.microarrays.org). Fluorescent images of hybridized microarrays were obtained using a GenePix 4000A microarray scanner (www.axon.com, Axon Instruments). Primary analysis was done using the GenePix software package (Axon Instruments). Cy5 to Cy3 ratios are determined for the individual genes along with various other quality control parameters (e.g. intensity over local background). Furthermore, bad spots or areas of the array with obvious defects were manually flagged. Spots with small diameters (<50 microns) and spots with low signal strengths <350 fluorescence intensity units over local background in the more intense channel were discarded. Flagged spots were not included in subsequent analyses. These files were then imported into a Microsoft Access data base. Prior to clustering, the normalized median of ratio values of the genes were log base 2 transformed and filtered for the presence across arrays and selected for expression levels and patterns depending on the experimental set as stated in the figure legends. Average linkage hierarchical clustering of an uncentered Pearson correlation similarity matrix was applied using the program Cluster (28Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13268) Google Scholar), and the results were visualized with the program TreeView (28Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13268) Google Scholar). Thirty micrograms of total RNA was resolved by denaturing formaldehyde-agarose gel and transferred onto Hybond membrane (Amersham Bioscience, Inc.) by a capillary transfer set up. Hybridizations were performed as previously described (29Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7266) Google Scholar). Briefly, prehybridization was performed for 1 h at 65 °C in a solution containing 1% bovine serum albumin (fraction V), 8% SDS, 0.5m phosphate buffer, pH 7.0, and 1 mm EDTA, pH 8.0. Hybridization was performed in prehybridization buffer for 16 h at 65 °C after adding the denatured probe at 2–3 × 106 cpm/ml concentration. Blots were washed with 2 × SSC, 0.1% SDS at room temperature three times over a period of 30 min. Subsequently, the blots were washed twice in 0.2 × SSC, 0.1% SDS at 65 °C for 60 min. Signal was visualized and quantitated by PhosphorImager. Data was culled using PhosphorImager software. The signal intensities were normalized against GAPDH signal intensities. For relative fold estimation, the ratio of the intensity of the respective transcript in the test sample over the transcript intensity in the reference sample was determined. One microgram of total RNA from experimental samples was reverse transcribed with Superscript reverse transcriptase (Invitrogen), using oligo(dT) and random hexanucleotide primer for first-strand cDNA synthesis. PCRs were performed directly on 1 μl of first-strand cDNA using 500 nmol each of gene-specific primer (IFNβ: forward primer, 5′-tctcctccaaattgctctcctgtt-3′, reverse primer, 5′-cagaaggaggacgccgca-3′; IFNγ: forward primer, 5′-tttgggttctcttggctgttact-3′, reverse, primer 5′-acagaaaaataatgcagagccaaa-3′; and GAPDH: forward primer, 5′-cggagtcaacggatttggtcgtat-3′, and reverse primer, 5′-agccttctccatggtggtgaagac-3′) in a 100-μl reaction volume comprised of 50 mm KCl, 10 mm Tris-HCl, pH 8.3, 1.5 mm MgCl2, 200 μm of each deoxynucleotide triphosphate. PCR was carried out for initial denaturation at 94 °C for 5 min, followed by 25, 42, and 45 cycles (for GAPDH, IFNβ, and IFNγ, respectively) of denaturation (94 °C for 1 min), annealing (55 °C for GAPDH and IFNβ, 59 °C for IFNγ, for 1 min), and extension (72 °C for 1 min) with 5 units ofTaq polymerase (Invitrogen). This was followed by a final extension step of 72 °C for 10 min. The products were analyzed on 2% agarose gels stained with ethidium bromide and visualized with ultraviolet light. Whole cell lysates prepared from MCF7 cells treated with a combination of zVADfmk, IFN-β, and TRAIL were separated on SDS-PAGE. The proteins were blotted onto nitrocellulose membrane (Amersham Bioscience, Inc.) and blocked overnight in Tris-buffered saline containing 5% nonfat milk and 0.1% Tween 20 at 4 °C. The membranes were then incubated for 2 h with antibodies directed against caspase-7 (Cell Signaling Technology), caspase-8 (Cell Signaling Technology), STAT-1 (Cell Signaling Technology), phospho (Tyr-701)-specific STAT-1, BID (Cell Signaling Technology), poly(ADP-ribose) polymerase (Cell Signaling Technology), TRAIL (PharMingen), β-tubulin (NeoMarkers), or actin (Sigma). Membranes were washed with Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and incubated with HRP-conjugated secondary antibody (Amersham Bioscience, Inc.) for 2 h at room temperature. After washing the membrane with TBS-T, signals were visualized by the ECL chemiluminescence system (Amersham Bioscience, Inc.). Cells were lysed in Nonidet P-40 lysis buffer (IP buffer) containing 50 mmTris-HCl, pH 7.4, 1% Nonidet P-40 (Sigma), complete proteinase inhibitor mixture tablet (Roche Molecular Biochemicals) and incubated with 1.5 μg of mouse monoclonal antibody against human IFN-β (PBL Biomedicals) in a total volume of 400 μl of IP buffer and rotated end-over-end at 4 °C for 90 min. Protein G-Sepharose beads (Amersham Bioscience, Inc.) equilibrated in IP buffer were added and the reactions were incubated for a further 60 min. IP reactions were washed three times with 1 ml of ice-cold IP buffer, resuspended in 40 μl of SDS-PAGE sample buffer, boiled for 5 min, and 10 μl of sample was run on 4–20% SDS-PAGE gradient gels (Gradipore). The separated proteins were transferred onto PVDF membrane (Amersham Bioscience, Inc.). The membrane was incubated for 1 h in blocking buffer (Tris-buffered saline containing 0.1% Tween (TBS-T) and 5% nonfat dry milk). Biotinylated anti-human IFN-β (R&D systems) was applied at 1:10000 in TBS-T with 1% bovine serum albumin and incubated for 1 h at room temperature. After washing three times with TBS-T buffer, the membrane was incubated with streptavidin-horseradish peroxidase (Invitrogen) at 1:5000 in TBST with 1% bovine serum albumin for 1 h at room temperature. ECL was performed as described in the previous section. MCF7 cells were split and grown overnight in 24-well tissue culture plates (Costar) at a density of 62,500 cells per well. Preincubation with 10 μg/ml cycloheximide (Sigma) for 30 min was followed by incubation in Dulbecco's modified Eagle's medium minus methionine and cysteine for 15 min. The cells were then incubated in the above medium containing 250 μCi/ml Tran35S-label (l-methionine:l-cysteine 70:30), specific activity >1,000 Ci/mm (10 mCi/ml) (ICN Radiochemicals) at both 4 and 24 h at 37 °C in 10% CO2. Cells were harvested in 100 μl of lysis buffer (1% Nonidet P-40, 0.02m Tris, pH 7.4, and complete, EDTA-free protease inhibitor mixture tablet). Lysates were spotted on 2.4-cm glass microfiber (GF/C) filter (Whatman) and air dried. Labeled proteins were precipitated by soaking the filters in 20% trichloroacetic acid (JT Baker Inc.) for 4 min with gentle shaking. The filters were then washed in 70% alcohol, dried, and counted in Econo-SafeTM (Research Products Int. Corp) in a scintillation counter. 96-Well plates (Nunc Immunoplate Maxisorb) were coated with 100 ng/ml anti-IFN-γ mAb (PBL Biomedical Laboratories) and incubated overnight at 4 °C. The plates were washed with PBS containing 0.05% Tween 20 (Fisher Biotech) and nonspecific binding sites were blocked with BlockerTMBlotto (Pierce) in PBS for 1 h at room temperature. All subsequent incubations were conducted at room temperature with shaking. The plates were washed and incubated for 1 h with different dilutions of purified IFN-γ (Standard) and the total cell lysates (diluted 1:2) in dilution medium (PBS with 10% Blotto, 0.1% bovine serum albumin (Sigma), and 0.05% Tween 20. Plates were washed and incubated with the detection antibody, biotinylated rabbit polyclonal α-IFN-γ Ab (1:500 in dilution medium) for 1 h. After washing, horseradish peroxidase-conjugated streptavidin (Jackson Immuno Research Laboratories, 1:20,000 in dilution medium) was added and plates were incubated for 30 min. After a final wash, 3,3′,5,5′-tetramethylbenzidine (Genzyme Diagnostics) was added, and plates were incubated in the dark for 15 min. The reaction was terminated using 1.5 n H2SO4. The plates were then read at dual wavelengths (465 and 590 nm) on a Bio-Tek microplate reader (Bio-Tek Instruments) and IFN-γ concentrations were estimated using a standard curve based on dilutions of recombinant IFNγ (R&D Biosystems). SUM149 cells were grown at a density of 2.5 × 104 cells per well overnight in 12-well tissue culture plates. Cells were lysed in buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, and 1% Triton X-100. The assay was carried out according to the manufacturer's protocol (Enzyme Systems Products). Briefly, 50 μl of lysate was mixed with 50 μl of 2 × reaction buffer (200 mmHEPES, pH 7.5, 40% glycerol, 10 mm dithiothreitol, 1 mm EDTA) and incubated at 30 °C for 30 min. Lysates were transferred to dark walled 96-well plates (BD) and caspase 8 flurometric substrate Ac-IETD-AFC (Enzyme Systems Products) was added at a 1 mm concentration. Fluorescence was recorded at 400 nm excitation and 530 nm emission using a Packard Fluorometer. Values obtained from three independent experiments were averaged and fluorescence intensity was plotted as arbitrary units. To study genes induced by activation of cell death receptors we used a 9984 element (10,000) cDNA microarray consisting of ∼5996 known, named genes and 2674 ESTs (Unigene Build 136) (26Dhanasekaran M.S. Barrette T.R. Ghosh D. Shah R. Varambally S. Kurachi K. Pienta K. Rubin M.A. Chinnaiyan A.M. Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar). Fluorescently labeled (Cy5) cDNA was prepared from total RNA from each experimental sample. A specific reference sample was paired with each experimental sample and labeled with a second distinguishable fluorescent dye (Cy3). Fig.1 is an Eisen matrix representation that provides an overview of the 35 different experimental samples analyzed and includes: various control hybridizations (Fig. 1, columns 5, 7–10, and 21 and 22), MCF7 and SUM149 cells treated with TRAIL (Fig. 1, columns 1–4, 6, 11–15, and20) or with other death inducing agents including TNF, αFasAb, or TWEAK (Fig. 1, columns 16–19), MCF7-FADD-DN cells treated with various death ligands (Fig. 1, columns 23–26), and cells treated with interferon-α, -β, or -γ (Fig. 1, columns 27–35). A hierarchical clustering algorithm was employed to group genes based on similarity in gene expression across all the samples tested (28Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13268) Google Scholar). Coordinated patterns of gene expression mediated by these cytokines were evident and selected clusters will be dissected in detail. The entire dataset presented in Fig. 1 is available in the Supplementary Material. To focus on primary response genes induced by TRAIL in breast carcinoma cells, we used cycloheximide (CHX) to block protein synthesis, thereby inhibiting secondary and tertiary responses. CHX alone does not induce significant apoptosis in either MCF7 or SUM149 cells, in the time frame assessed (data not shown). As extended treatment with TRAIL causes induction of apoptosis in these cell lines (TRAIL, at 1000 ng/ml, significantly increases apoptosis within 3 and 5 h in SUM149 and MCF7 cells, respectively), we used the broad-spectrum caspase inhibitor z-VADfmk (zVAD) to block cell death, allowing for the study of gene expression alterations independent of apoptosis. While CHX and zVAD alter the expression of a limited set of genes, these changes were outside of the death ligand-specific response clusters (Fig. 1,columns 7–10 and 21 and 22). Furthermore, zVAD and CHX mediated effects were negated by using these agents in the specific reference samples used for microarray analysis. Analysis of gene expression in breast cancer cells treated with TRAIL revealed, primarily, “up-regulation” of specific gene products. While selected genes were down-regulated (as evident in Fig. 1), they are not presented here and are included in the Supplementary Material. Our analysis of a time course of TRAIL-treated MCF7 cells revealed three distinct clusters of up-regulated genes. In the “early” primary response cluster, genes were up-regulated between 1 and 4 h and declined to normal levels thereafter (Fig.2, A, top panel, andB). The “intermediate” cluster was comprised of genes that were up-regulated at the 4-h time point and remained elevated (Fig. 2, A, middle panel, and C). Finally, the “late” cluster contained genes that were up-regulated at 16 h and beyond (Fig. 2, A, bottom panel, and D). We next considered each cluster in some detail. The early cluster of TRAIL-induced genes (Fig. 2B) includes an activator of immediate early genes HCFC-1 (30Wysocka J. Reilly P.T. Herr W. Mol. Cell. Biol. 2001; 21: 3820-3829Crossref PubMed Scopus (158) Google Scholar), an example of an immediate early gene INSIG-1 (31Bortoff K.D. Zhu C.C. Hrywna Y. Messina J.L. Endocrine. 1997; 7: 199-207Crossref PubMed Google Scholar), and transcriptional repressors, such as seven in absentia homolog-2 (SIAH-2) (32Bogdan S. Senkel S. Esser F. Ryffel G.U. Pogge v Strandmann E. Mech. Dev. 2001; 103: 61-69Crossref PubMed Scopus (21) Google Scholar), Ras suppressor-1 (RSU-1) (33Masuelli L. Cutler M.L. Mol. Cell. Biol. 1996; 16: 5466-5476Crossref PubMed Scopus (46) Google Scholar), and CDC-6 (34Takayama M. Taira T. Iguchi-Ariga S.M. Ariga H. FEBS Lett. 2000; 477: 43-48Crossref PubMed Scopus (16) Google Scholar). Other notable genes in the early primary response cluster are the endothelial adhesion molecule, E-selectin, known to be regulated by NF-κB (35Hurwitz A.A. Lyman W.D. Guida M.P. Calderon T.M. Berman J.W. J. Exp. Med. 1992; 176: 1631-1636Crossref PubMed Scopus (102) Google Scholar, 36Harashima S. Horiuchi T. Hatta N. Morita C. Higuchi M. Sawabe T. Tsukamoto H. Tahira T. Hayashi K. Fujita S. Niho Y. J. Immunol. 2001; 166: 130-136Crossref PubMed Scopus (111) Google Scholar), BNIP-3, a pro-apoptotic BCl-2 family mitochondrial protein (37Vande Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. Mol. Cell. Biol. 2000; 20: 5454-5468Crossref PubMed Scopus (542) Google Scholar), and the CXC chemokine GRO1α, which possesses antiapoptotic activity (38Dunican A.L. Leuenroth S.J. Ayala A. Simms H.H. Shock. 2000; 13: 244-250Crossref PubMed Scopus (40) Google Scholar). The apoptotic threshold of cells may be regulated by intracellular Ca2+ levels (39Pinton P. 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The critical clinical question in prostate cancer research is: How do we develop means of distinguishing aggressive disease from indolent disease? Using a combination of proteomic and expression array data, we identified a set of 36 genes with concordant dysregulation of protein products that could be evaluated in situ by quantitative immunohistochemistry. Another five prostate cancer biomarkers were included using linear discriminant analysis, we determined that the optimal model used to predict prostate cancer progression consisted of 12 proteins. Using a separate patient population, transcriptional levels of the 12 genes encoding for these proteins predicted prostate-specific antigen failure in 79 men following surgery for clinically localized prostate cancer (P = .0015). This study demonstrates that cross-platform models can lead to predictive models with the possible advantage of being more robust through this selection process.

The Polycomb group protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive and metastatic breast cancer. Here we report that EZH2 decreased the expression of five RAD51 paralog proteins involved in homologous recombination (HR) repair of DNA doublestrand breaks (RAD51B/RAD51L1, RAD51C/RAD51L2, RAD51D/RAD51L3, XRCC2, and XRCC3), but did not affect the levels of DMC1, a gene that only functions in meiosis. EZH2 overexpression impaired the formation of RAD51 repair foci at sites of DNA breaks. Overexpression of EZH2 resulted in decreased cell survival and clonogenic capacity following DNA damage induced independently by etoposide and ionizing radiation. We suggest that EZH2 may contribute to breast tumorigenesis by specific downregulation of RAD51-like proteins and by impairment of HR repair. We provide mechanistic insights into the function of EZH2 in mammalian cells and uncover a link between EZH2, a regulator of homeotic gene expression, and HR DNA repair. Our study paves the way for exploring the blockade of EZH2 overexpression as a novel approach for the prevention and treatment of breast cancer.

Abstract There is considerable evidence that the presence of cancer can elicit a humoral immune response to specific proteins in the host, and these resulting autoantibodies may have potential as noninvasive biomarkers. To characterize the autoantibody repertoire present in the sera of patients with lung adenocarcinoma, we developed a high-density peptide microarray derived from biopanning a lung cancer phage display library. Using a 2,304-element microarray, we interrogated a total of 250 sera from Michigan lung cancer patients and noncancer controls to develop an “autoantibody profile” of lung adenocarcinoma. A set of 22 discriminating peptides derived from a training set of 125 serum samples from lung adenocarcinoma patients and control subjects was found to predict cancer status with 85% sensitivity and 86% specificity in an independent test set of 125 sera. Sequencing of the immunoreactive phage-peptide clones identified candidate humoral immune response targets in lung adenocarcinoma, including ubiquilin 1, a protein that regulates the degradation of several ubiquitin-dependent proteasome substrates. An independent validation set of 122 serum samples from Pittsburgh was examined using two overlapping clones of ubiquilin 1 that showed 0.79 and 0.74 of the area under the receiver operating characteristics curve, respectively. Significantly increased levels of both ubiquilin 1 mRNA and protein, as well as reduced levels of the phosphorylated form of this protein, were detected in lung tumors. Immunofluorescence using anti–ubiquilin 1 antibodies confirmed intracellular expression within tumors cells. These studies indicate that autoantibody profiles, as well as individual candidates, may be useful for the noninvasive detection of lung adenocarcinoma. [Cancer Res 2007;67(7):3461–7]

Localized prostate cancer (CaP) can be cured using several strategies. However, the need to identify active substances in advanced tumor stages is tremendous, as the outcome in such cases is still disappointing. One approach is to deliver human tumor antigen-targeted therapy, which is recognized by T cells or antibodies. We used data mining of the Cancer Immunome Database (CID), which comprises potential immunologic targets identified by serological screening of expression libraries. Candidate antigens were screened by DNA microarrays. Genes were then validated at the protein level by tissue microarrays, representing various stages of CaP disease. Of 43 targets identified by CID, 10 showed an overexpression on the complementary DNA array in CaP metastases. The RHAMM (CD168) gene, earlier identified by our group as an immunogenic antigen in acute and chronic leukemia, also showed highly significant overexpression in CaP metastases compared with localized disease and benign prostatic hyperplasia. At the protein level, RHAMM was highest in metastatic tissue samples and significantly higher in neoplastic localized disease compared with benign tissue. High RHAMM expression was associated with clinical parameters known to be linked to better clinical outcome. Patients with high RHAMM expression in the primaries had a significantly lower risk of biochemical failure. The number of viable cells in cell cultures was reduced in blocking experiments using hormone-sensitive and hormone-insensitive metastatic CaP cell lines. Acknowledging the proven immunogenic effects of RHAMM in leukemia, this antigen is intriguing as a therapeutic target in far-advanced CaP.

With a flood of cancer genome sequences expected soon, distinguishing 'driver' from 'passenger' mutations will be an important task. Wang et al. describe a bioinformatic method for identifying cancer-associated fusions and apply it to discover a recurrent rearrangement in lung cancer. Cancer genomes contain many aberrant gene fusions—a few that drive disease and many more that are nonspecific passengers. We developed an algorithm (the concept signature or 'ConSig' score) that nominates biologically important fusions from high-throughput data by assessing their association with 'molecular concepts' characteristic of cancer genes, including molecular interactions, pathways and functional annotations. Copy number data supported candidate fusions and suggested a breakpoint principle for intragenic copy number aberrations in fusion partners. By analyzing lung cancer transcriptome sequencing and genomic data, we identified a novel R3HDM2-NFE2 fusion in the H1792 cell line. Lung tissue microarrays revealed 2 of 76 lung cancer patients with genomic rearrangement at the NFE2 locus, suggesting recurrence. Knockdown of NFE2 decreased proliferation and invasion of H1792 cells. Together, these results present a systematic analysis of gene fusions in cancer and describe key characteristics that assist in new fusion discovery.

Transcriptional repressors and corepressors play a critical role in cellular homeostasis and are frequently altered in cancer. C-terminal binding protein 1 (CtBP1), a transcriptional corepressor that regulates the expression of tumor suppressors and genes involved in cell death, is known to play a role in multiple cancers. In this study, we observed the overexpression and mislocalization of CtBP1 in metastatic prostate cancer and demonstrated the functional significance of CtBP1 in prostate cancer progression. Transient and stable knockdown of CtBP1 in prostate cancer cells inhibited their proliferation and invasion. Expression profiling studies of prostate cancer cell lines revealed that multiple tumor suppressor genes are repressed by CtBP1. Furthermore, our studies indicate a role for CtBP1 in conferring radiation resistance to prostate cancer cell lines. In vivo studies using chicken chorioallantoic membrane assay, xenograft studies, and murine metastasis models suggested a role for CtBP1 in prostate tumor growth and metastasis. Taken together, our studies demonstrated that dysregulated expression of CtBP1 plays an important role in prostate cancer progression and may serve as a viable therapeutic target.

Histone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep caused selective apoptosis in alloantigen-activated T cells mediating host tissue injury. This effect was associated with the ability of DZNep to selectively reduce trimethylation of histone H3 lysine 27, deplete the histone methyltransferase Ezh2 specific to trimethylation of histone H3 lysine 27, and activate proapoptotic gene Bim repressed by Ezh2 in antigenic-activated T cells. In contrast, DZNep did not affect the survival of alloantigen-unresponsive T cells in vivo and naive T cells stimulated by IL-2 or IL-7 in vitro. Importantly, inhibition of histone methylation by DZNep treatment in vivo preserved the antileukemia activity of donor T cells and did not impair the recovery of hematopoiesis and lymphocytes, leading to significantly improved survival of recipients after allogeneic BM transplantation. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat ongoing GVHD and other T cell-mediated inflammatory disorders in a broad context.

We have recently reported that treatment with gemcitabine, a potent chemotherapeutic agent and radiation sensitizer, stimulates phosphorylation of the epidermal growth factor receptor (EGFR). Because phosphorylation of EGFR is known to precede receptor degradation, we hypothesized that gemcitabine treatment may also result in EGFR degradation. In two human head and neck cancer cell lines, UMSCC-1 and UMSCC-6, we demonstrated an approximately 80% decrease in total EGFR levels at 72 h after a 2-h treatment with 1 μ M gemcitabine. Neither cisplatin nor 5-fluorouracil, which are used to treat head and neck cancer, caused EGFR degradation. EGFR downregulation did not occur at the level of transcription, as assessed by reverse transcription-polymerase chain reaction (RT–PCR), but instead occurred via phosphorylation and ubiquitination of the receptor along a proteosome/lysosome-mediated pathway. Inhibition of EGFR degradation, by either pretreatment with the EGFR tyrosine kinase inhibitor gefitinib or by exposure to the proteosome/lysosome inhibitor MG132, significantly reduced gemcitabine-induced cell death. These results suggest that EGFR degradation may be a novel mechanism for gemcitabine-mediated cell death. These findings also indicate that caution should be exercised when combining gemcitabine with agents that may prevent EGFR degradation, such as EGFR tyrosine kinase inhibitors administered in a suboptimal sequence or proteosome inhibitors.

The cysteine-rich protein CCN6 [or Wnt-1-induced signaling protein 3 (WISP3)] exerts tumor-suppressive effects in aggressive inflammatory breast cancer. Loss of CCN6 is associated with poorly differentiated phenotypes and increased invasion. Here, we show that reduction of CCN6 expression occurs in 60% of invasive breast carcinomas and is associated with axillary lymph node metastases. Furthermore, low CCN6 expression in invasive carcinoma tissue samples correlates with reduced expression of E-cadherin. In vitro, RNA interference knockdown of CCN6 in two benign human mammary epithelial cell lines (HME and MCF10A) decreased expression of E-cadherin protein and mRNA and reduced activity of the E-cadherin promoter; this reduction was dependent on intact E-box elements. CCN6 knockdown in HME cells resulted in up-regulation of the E-cadherin transcriptional repressors Snail and ZEB1 and enhanced their recruitment and binding to the E-cadherin promoter as analyzed by chromatin immunoprecipitation assays. Small interfering RNA-mediated knockdown of ZEB1 or Snail blocked the down-regulation of E-cadherin caused by CCN6 inhibition. These data show, for the first time, that CCN6 expression is reduced or lost in a substantial number of invasive breast carcinomas and that CCN6 modulates transcriptional repressors of E-cadherin. Together, our results lead to a new hypothesis that Snail and ZEB1 are downstream of CCN6 and play a critical role in CCN6-mediated regulation of E-cadherin in breast cancer. The cysteine-rich protein CCN6 [or Wnt-1-induced signaling protein 3 (WISP3)] exerts tumor-suppressive effects in aggressive inflammatory breast cancer. Loss of CCN6 is associated with poorly differentiated phenotypes and increased invasion. Here, we show that reduction of CCN6 expression occurs in 60% of invasive breast carcinomas and is associated with axillary lymph node metastases. Furthermore, low CCN6 expression in invasive carcinoma tissue samples correlates with reduced expression of E-cadherin. In vitro, RNA interference knockdown of CCN6 in two benign human mammary epithelial cell lines (HME and MCF10A) decreased expression of E-cadherin protein and mRNA and reduced activity of the E-cadherin promoter; this reduction was dependent on intact E-box elements. CCN6 knockdown in HME cells resulted in up-regulation of the E-cadherin transcriptional repressors Snail and ZEB1 and enhanced their recruitment and binding to the E-cadherin promoter as analyzed by chromatin immunoprecipitation assays. Small interfering RNA-mediated knockdown of ZEB1 or Snail blocked the down-regulation of E-cadherin caused by CCN6 inhibition. These data show, for the first time, that CCN6 expression is reduced or lost in a substantial number of invasive breast carcinomas and that CCN6 modulates transcriptional repressors of E-cadherin. Together, our results lead to a new hypothesis that Snail and ZEB1 are downstream of CCN6 and play a critical role in CCN6-mediated regulation of E-cadherin in breast cancer. Afflicting one of eight women, breast cancer is the second leading cause of cancer-related deaths in women in the United States.1Jemal A Murray T Samuels A Ghafoor A Ward E Thun MJ Cancer statistics, 2003.CA Cancer J Clin. 2003; 53: 5-26Crossref PubMed Scopus (3355) Google Scholar Despite advances in the early detection and treatment of breast cancer, mortality for those 20% of patients with recurrences and/or metastases is nearly 100%.2Battaglia C Salani G Consolandi C Bernardi LR De Bellis G Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II [In Process Citation].Biotechniques. 2000; 29: 78-81PubMed Google Scholar Discovering and characterizing key genes and pathways that define breast cancers with metastatic ability will help identify molecular markers that predict prognosis before metastasis develops and that may represent appropriate therapeutic and/or preventative targets. Once cancer develops, the acquisition of invasive capabilities is important for the progression of disease. Tumor cell migration and metastasis is a highly coordinated process. It requires not only alteration of cell adhesion to extracellular matrix proteins, but also the disruption of cell-cell junctions and changes in cell morphology, which is termed epithelial-mesenchymal transition (EMT).3Batlle E Sancho E Franci C Dominguez D Monfar M Baulida J Garcia De Herreros A The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.Nat Cell Biol. 2000; 2: 84-89Crossref PubMed Scopus (2131) Google Scholar, 4Cano A Perez-Moreno MA Rodrigo I Locascio A Blanco MJ del Barrio MG Portillo F Nieto MA The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.Nat Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2839) Google Scholar, 5Hajra KM Chen DY Fearon ER The SLUG zinc-finger protein represses E-cadherin in breast cancer.Cancer Res. 2002; 62: 1613-1618PubMed Google Scholar During this complex and dynamic process, epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility. The initiation of EMT in epithelial-derived cancer types is marked by E-cadherin repression.6Yook JI Li XY Ota I Fearon ER Weiss SJ Wnt-dependent regulation of the E-cadherin repressor snail.J Biol Chem. 2005; 280: 11740-11748Crossref PubMed Scopus (349) Google Scholar, 7Zhou BP Deng J Xia W Xu J Li YM Gunduz M Hung MC Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition.Nat Cell Biol. 2004; 6: 931-940Crossref PubMed Scopus (1285) Google Scholar The key events that lead to the down-regulation of E-cadherin and initiation of EMT in breast cancer remain elusive. CCN6 [Wnt-1 induced signaling protein (WISP3)] is a cysteine-rich protein down-regulated in the most lethal form of locally advanced breast cancer, inflammatory breast cancer, and in a group of advanced-stage noninflammatory breast cancer tumors.8van Golen KL Davies S Wu ZF Wang Y Bucana CD Root H Chandrasekharappa S Strawderman M Ethier SP Merajver SD A novel putative low-affinity insulin-like growth factor-binding protein, LIBC (lost in inflammatory breast cancer), and RhoC GTPase correlate with the inflammatory breast cancer phenotype.Clin Cancer Res. 1999; 5: 2511-2519PubMed Google Scholar CCN6 re-expression restores differentiated epithelial phenotypes in the breast. Consistently, accumulated evidence shows that CCN6 inhibits tumor cell motility and invasion in vitro and inhibits tumor growth in vivo.9Kleer CG Zhang Y Pan Q Merajver SD WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer.Neoplasia. 2004; 6: 179-185Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 10Kleer CG Zhang Y Pan Q Gallagher G Wu M Wu ZF Merajver SD WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer.Breast Cancer Res. 2004; 6: R110-R115Crossref Google Scholar, 11Kleer CG Zhang Y Pan Q van Golen KL Wu ZF Livant D Merajver SD WISP3 is a novel tumor suppressor gene of inflammatory breast cancer.Oncogene. 2002; 21: 3172-3180Crossref PubMed Scopus (124) Google Scholar, 12Zhang Y Pan Q Zhong H Merajver SD Kleer CG Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells.Breast Cancer Res. 2005; 7: R1080-R1089Crossref PubMed Scopus (54) Google Scholar CCN6 is also shown to inhibit tumor-induced angiogenesis.10Kleer CG Zhang Y Pan Q Gallagher G Wu M Wu ZF Merajver SD WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer.Breast Cancer Res. 2004; 6: R110-R115Crossref Google Scholar, 11Kleer CG Zhang Y Pan Q van Golen KL Wu ZF Livant D Merajver SD WISP3 is a novel tumor suppressor gene of inflammatory breast cancer.Oncogene. 2002; 21: 3172-3180Crossref PubMed Scopus (124) Google Scholar Recently, we demonstrated that CCN6 inhibition causes EMT of breast epithelial cells.12Zhang Y Pan Q Zhong H Merajver SD Kleer CG Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells.Breast Cancer Res. 2005; 7: R1080-R1089Crossref PubMed Scopus (54) Google Scholar This function of CCN6 correlates with its effect on cell motility and invasion. The current study describes the first mechanistic evidence that CCN6 regulates E-cadherin levels and that the transcriptional repressors Snail and ZEB1 are required for this function, and demonstrates that CCN6 expression in human breast tissues correlates with lymph node metastasis and E-cadherin levels. For tissue microarray (TMA) construction, 116 invasive breast carcinoma tissues obtained with Institutional Review Board approval were used. The TMA was constructed using triplicate tissue samples as previously described.13Kleer CG Cao Q Varambally S Shen R Ota I Tomlins SA Ghosh D Sewalt RG Otte AP Hayes DF Sabel MS Livant D Weiss SJ Rubin MA Chinnaiyan AM EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells.Proc Natl Acad Sci USA. 2003; 100: 11606-11611Crossref PubMed Scopus (1319) Google Scholar Clinical and pathological variables were determined following well established criteria. All invasive carcinomas were graded according to the method described by Elston and Ellis.14Elston EW Ellis IO Method for grading breast cancer.J Clin Pathol. 1993; 46: 189-190Crossref PubMed Scopus (145) Google Scholar Standard biotin-avidin complex immunohistochemistry was performed on 4-μm-thick paraffin-embedded tissue sections of the TMA, using a primary anti-CCN6 antibody (dilution 1:150; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a monoclonal anti-E-cadherin antibody (1:400; Zymed, Carlsbad, CA).9Kleer CG Zhang Y Pan Q Merajver SD WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer.Neoplasia. 2004; 6: 179-185Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 13Kleer CG Cao Q Varambally S Shen R Ota I Tomlins SA Ghosh D Sewalt RG Otte AP Hayes DF Sabel MS Livant D Weiss SJ Rubin MA Chinnaiyan AM EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells.Proc Natl Acad Sci USA. 2003; 100: 11606-11611Crossref PubMed Scopus (1319) Google Scholar, 15Kowalski PJ Rubin MA Kleer CG E-cadherin expression in primary carcinomas of the breast and its distant metastases.Breast Cancer Res. 2003; 5: R217-R222Crossref PubMed Scopus (316) Google Scholar CCN6 protein expression was scored using a standard, pathologist-based four-tiered scoring system as 1, negative; 2, weak; 3, moderate; and 4, strong.13Kleer CG Cao Q Varambally S Shen R Ota I Tomlins SA Ghosh D Sewalt RG Otte AP Hayes DF Sabel MS Livant D Weiss SJ Rubin MA Chinnaiyan AM EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells.Proc Natl Acad Sci USA. 2003; 100: 11606-11611Crossref PubMed Scopus (1319) Google Scholar, 16Kleer CG Griffith KA Sabel MS Gallagher G van Golen KL Wu ZF Merajver SD RhoC-GTPase is a novel tissue biomarker associated with biologically aggressive carcinomas of the breast.Breast Cancer Res Treat. 2005; 93: 101-110Crossref PubMed Scopus (105) Google Scholar Negative and weak staining were considered low CCN6, and moderate and strong were considered high CCN6 based on our studies on the biology of CCN6.9Kleer CG Zhang Y Pan Q Merajver SD WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer.Neoplasia. 2004; 6: 179-185Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 10Kleer CG Zhang Y Pan Q Gallagher G Wu M Wu ZF Merajver SD WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer.Breast Cancer Res. 2004; 6: R110-R115Crossref Google Scholar, 11Kleer CG Zhang Y Pan Q van Golen KL Wu ZF Livant D Merajver SD WISP3 is a novel tumor suppressor gene of inflammatory breast cancer.Oncogene. 2002; 21: 3172-3180Crossref PubMed Scopus (124) Google Scholar To analyze the expression of E-cadherin on tissue samples, we used a quantitative image analysis system (ACIS; ChromaVision Medical Systems, Inc., San Juan Capistrano, CA), which allows for a continuous rather than categorical scoring, and it is well suited to analyzed small changes in protein expression.17Witkiewicz AK Varambally S Shen R Mehra R Sabel MS Ghosh D Chinnaiyan AM Rubin MA Kleer CG Alpha-methylacyl-CoA racemase protein expression is associated with the degree of differentiation in breast cancer using quantitative image analysis.Cancer Epidemiol Biomarkers Prev. 2005; 14: 1418-1423Crossref PubMed Scopus (50) Google Scholar, 18Camp RL Dolled-Filhart M King BL Rimm DL Quantitative analysis of breast cancer tissue microarrays shows that both high and normal levels of HER2 expression are associated with poor outcome.Cancer Res. 2003; 63: 1445-1448PubMed Google Scholar Using this system, E-cadherin staining intensity was evaluated on a scale of 73 to 139 (mean, 100.5). A Wilcoxon exact test was used to compare the distributions of E-cadherin intensity according to CCN6 protein levels, and a P value <0.05 was considered statistically significant. Paraffin embedded tissue sections of six xenografts derived from SUM149 breast cancer (CCN6-deficient) cells overexpressing CCN6 (four tumors) or vector controls (two tumors) were studied. We have previously found that xenografts derived from SUM149/CCN6 cells developed more slowly, were significantly smaller, and were better differentiated than SUM149/vector (CCN6-deficient) tumors.11Kleer CG Zhang Y Pan Q van Golen KL Wu ZF Livant D Merajver SD WISP3 is a novel tumor suppressor gene of inflammatory breast cancer.Oncogene. 2002; 21: 3172-3180Crossref PubMed Scopus (124) Google Scholar We analyzed CCN6 and E-cadherin expression on these xenografts using anti-CCN6 and anti-E-cadherin antibodies as described above. HME cells were cultured in Ham's F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (Cambrex, Walkersville, MD), 1 μg/ml hydrocortisone, 5 μg/ml insulin, 10 ng/ml epidermal growth factor, and 100 ng/ml cholera toxin at 37°C under 10% CO2. MDA-MB-231 and HEK293 cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. MCF10A (American Type Culture Collection) cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 (Invitrogen) supplemented with 10% fetal bovine serum, 1 μg/ml hydrocortisone, 5 μg/ml insulin, and 10 μg/ml epidermal growth factor.12Zhang Y Pan Q Zhong H Merajver SD Kleer CG Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells.Breast Cancer Res. 2005; 7: R1080-R1089Crossref PubMed Scopus (54) Google Scholar HME CCN6 knockdown stable cell lines were generated by small interfering RNA (siRNA-CCN6) and short hairpin RNA (shRNA-CCN6), respectively. The empty vectors were used as controls. The construction of HME siRNA-CCN6 has been described previously.12Zhang Y Pan Q Zhong H Merajver SD Kleer CG Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells.Breast Cancer Res. 2005; 7: R1080-R1089Crossref PubMed Scopus (54) Google Scholar shRNA-CCN6 plasmid was purchased from Sigma (St. Louis, MO), based on a lentiviral vector (pLKO.1-puro). The shRNA-CCN6 was packaged at University of Michigan Vector Core, and the virus-containing supernatant was diluted 1:1 with fresh medium and used to infect HME cells. Selection was initiated in 10 μg/ml puromycin (Sigma) 48 hours after infection of HME cells. Stable transfectants were established after 3 weeks. The shRNA-CCN6 target sequence was as follows: 5′-CCGGCCATTAGATACAACACCTGAACTCGAGTTCAGGTGTTGTA-3′. Snail and ZEB1 mRNA were blocked by using siRNA SMARTpool (Dharmacon, Lafayette, CO) following the manufacturer's instructions. Briefly, HME shRNA-CCN6 or siRNA-CCN6 stable cells were grown to 70 to 80% confluence for 24 hours and were then treated with 100 nmol/L siRNA-Snail and 100 nmol/L siRNA-Zeb1, respectively. siCONTROL nontargeting siRNA pool (100 nmol/L; Dharmacon) was used as negative control. DharmaFECT Transfection reagent (Dharmacon) was used following the protocol described by the manufacturer. The medium was changed 24 hours after transfection, and the cells were incubated in fresh medium for an additional 48 to 72 hours. The following pairs (sense/antisense) of SMARTpool siRNA were used: siRNA-Snail, 5′-ACUCAGAUGUCAAGAAUAUU-3′/5′-PUACUUCUUGACAUCUGAGUUU-3′, 5′-GCAAAUACUGCAACAAGGAUU-3′/5′-PUCCUUGUUGCAGUAUUUGCUU-3′, 5′-GCUCGGACCUUCUCCCGAAUU-3′/5′-PUUCGGGAGAAGGUCCGAGCCUU-3′, and 5′-GCUUGGGCCAAGUGCCCAAUU-3′/5′-PUUGGGCACUUGGCCCAAGCUU-3′; and siRNA-Zeb1, 5′-GAACCACCCUUGAAAGUGAUU-3′/5′-PUCACUUUCAAGGGUGGUUCUU-3′, 5′-GAAGCAGGAUGUACAGUAAUU-3′/5′-PUUACUGUACAUCCUGCUUCUU-3′, 5′- AAACUGAACCUGUGGAUUAUU-3′/5′-PUAAUCCACAGGUUCAGUUUUU-3′, and 5′-GAUAGCACUUGUCUUCUGUUU-3′/5′-PACAGAAGACAAGUGCUAUCUU-3′. Samples for analysis by immunoblot were prepared as described previously.9Kleer CG Zhang Y Pan Q Merajver SD WISP3 (CCN6) is a secreted tumor-suppressor protein that modulates IGF signaling in inflammatory breast cancer.Neoplasia. 2004; 6: 179-185Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 10Kleer CG Zhang Y Pan Q Gallagher G Wu M Wu ZF Merajver SD WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer.Breast Cancer Res. 2004; 6: R110-R115Crossref Google Scholar, 11Kleer CG Zhang Y Pan Q van Golen KL Wu ZF Livant D Merajver SD WISP3 is a novel tumor suppressor gene of inflammatory breast cancer.Oncogene. 2002; 21: 3172-3180Crossref PubMed Scopus (124) Google Scholar In brief, cell lysates were prepared in lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 1% Nonidet P-40, and a mixture of protease inhibitors (Roche, Indianapolis, IN). The proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. After blocking with 5% nonfat milk in Tris-buffered saline/Tween 20 at room temperature for 1 hour, the membranes were incubated with the following antibodies: anti-CCN6 (Santa Cruz Biotechnology) at 1:1000 dilution, anti-Snail (rabbit polyclonal; Santa Cruz Biotechnology) at 1:1000 dilution, anti-Zeb1 (Santa Cruz Biotechnology) at 1:500 dilution, anti-E-cadherin (BD Transduction Laboratories, San Jose, CA) at 1:2500, and anti-β-actin (Sigma) at1:10.000. After washing in Tris-buffered saline/Tween 20, the blot was incubated with horseradish peroxidase-conjugated secondary antibodies at 1:2000 (Amersham Bioscience, Piscataway, NJ), and the antigen-antibody complexes were visualized by ECL system (Amersham Bioscience). Total RNA was isolated from cells with a TriZol kit (Life Technologies, Inc., Gaithersburg, MD). cDNA was synthesized using a reverse transcription system (Promega, Madison, WI) and 1 μg of total RNA as template. For real-time PCR, relative gene expression was determined using SYB Green PCR Master Mix kit (Applied Biosystems, Foster City, CA) in an Applied Biosystems 7300 Real Time PCR system following the manufacturer's protocol. Amplification was performed for 40 cycles of 3 minutes at 95°C, 60 seconds at 60°C, and 3 minutes at 72°C. The following pairs of primers (forward/reverse, 250 nmol/L of each) were used: SNAIL, 5′-GCGAGCTGCAGGACTCTAAT-3′/5′-CCRCTGTCCTCATCTGACA-3′; SLUG, 5′-TTCGGACCCACACATTACCT-3/5′-TTGGAGCAGTTTTTGCACTG- 3′; TWIST1, 5′-GGAGTCCGCAGTCTTACGAG-3′/5′-TGGAGGACCTGGTAGAGGAA-3′; TWIST2, 5′-AGCAAGAAGTCGAGCTAAGA-3′/5′-CAGCTTGAGCGTCTGGATCT-3′; smad-interacting protein-1, 5′-AATGGCAACAGCAACAAGTG-3′/5′-CCCCGTCAGCACATAACTTT-3′; ZEB1, 5′-GCACAACCAAGTGCAGAAGA-3′/5′-CATTTGCAGATTGAGGCTGA-3′; E-CADHERIN, 5′-CGACCAACCCAAGAATCTA-3′/5′-AGGCTGTGCCTTCCTACAGA-3′; and β-ACTIN, 5′-TCCCTGGAGAAGAGCTACGA-3′/5′-AGCACTGTGTTGGCGTACAG-3′. The quantity of DNA in each sample was calculated by interpolating its threshold cycle value versus a standard curve of threshold cycle values obtained from serially diluted cDNA from a mixture of all of the samples. The calculated quantity of the target gene for each sample was then divided by the average calculated quantity of β-actin corresponding to each sample to give a relative expression of the target gene for each sample. HME, MCF10A, and HEK293 cell transfections were performed in six-well plates using FuGENE6 Transfection Reagent (Roche) following the protocol described by the manufacturer. HEK293 and MCF10A cells were cotransfected with 1 g of reporter gene Ecad(−108)-Luc (kind gift of Dr. Eric Fearon) and 0.5 g of pSilencer2.1-U6-CCN6-siRNA5Hajra KM Chen DY Fearon ER The SLUG zinc-finger protein represses E-cadherin in breast cancer.Cancer Res. 2002; 62: 1613-1618PubMed Google Scholar or pSilencer2.1-U6 vector. HME siRNA-vector and HME siRNA-CCN6 stable clones were transfected 1.0 g of reporter gene Ecad(−108)-Luc only. Luciferase assays were performed 24 hours after transfection using a Dual Luciferase Assay System (Promega) and normalized by measuring Renilla activities (cotransfected with 4 ng of pSV-40). E-cadherin promoter activities were presented as relative light units to that obtained from pGL3-transfected cells. Triplicate samples were run in all of the experiments, which were repeated at least three times. To examine the transcriptional regulation of E-cadherin in CCN6 knockdown clones, we used the wild-type [Ecad(−108)-Luc] and E-box-mutated E-cadherin reporter gene constructs [Ecad(−108)-AMut, Ecad(−108)-CMut, and Ecad(−108)-ABCMut; kind gift of Dr. Eric Fearon].6Yook JI Li XY Ota I Fearon ER Weiss SJ Wnt-dependent regulation of the E-cadherin repressor snail.J Biol Chem. 2005; 280: 11740-11748Crossref PubMed Scopus (349) Google Scholar, 19Hajra KM Ji X Fearon ER Extinction of E-cadherin expression in breast cancer via a dominant repression pathway acting on proximal promoter elements.Oncogene. 1999; 18: 7274-7279Crossref PubMed Scopus (94) Google Scholar HME siRNA-vector and HME siRNA-CCN6 stable clones were transiently transfected with either Ecad(−108)-Luc or Ecad(−108)-Ebox mutants as above along with the pRL-null vector. Cells were lysed 48 hours later, and luciferase assays were performed using the dual luciferase assay system (Promega). Each experiment was performed in triplicate. Cells were grown on chamber slides (Nalgen Nunc International, Naperville, IL) and fixed with 3.7% formaldehyde in PBS (pH 7.4) for 10 minutes at room temperature and then permeabilized with PBS containing 0.5% Triton X-100 for 10 minutes at room temperature. The cells were blocked with 5% bovine serum albumin in PBS for 1 hour at room temperature. After washing the slides with PBS, a mixture of mouse monoclonal anti-E-cadherin antibody at 1:500 dilution (BD Transduction Laboratories) and rabbit polyclonal anti-Snail antibody at 1:200 dilution (Abcam, Cambridge, UK) was added and incubated overnight at 4°C. After washing the slides, a mixture of secondary donkey anti-rabbit Alexa 488 and donkey anti-mouse Alexa 555 (Molecular Probes, Eugene, OR) was applied at 1:1500 dilution and incubated in the dark for 1 hour. Washing the slides with PBS, anti-fade with 4′6-diamidino-2-phenylindole was applied to stain nuclei and covered by a glass coverslip. Confocal images were taken with a Zeiss LSM510 META imaging system using UV Argon and Helium Neon 1 light source. Chromatin immunoprecipitation analysis was performed using the ChIP-IT Enzymatic kit (Active Motif, Carlsbad, CA) following the manufacturer's protocol. Briefly, HME shRNA-CCN6 stable clones and control HME shRNA-vector were grown to 70 to 80% confluence on 150-mm plates. The cells were fixed with 1.0% formaldehyde, and the nuclei were released using lysis buffer. The chromatin was enzymatically sheared into small uniform fragments by incubation at 37°C for 10 minutes, and the protein/DNA complexes were then immunoprecipitated overnight at 4°C using anti-Snail (rabbit polyclonal; Abcam), anti-Zeb1 (Santa Cruz Biotechnology), and control IgG (normal Rabbit IgG) antibodies, respectively, with protein G magnetic beads. The beads were then collected by magnetic pull-down. The cross-linked protein/DNA complexes were eluted from G beads at 94°C for 15 minutes and then treated with proteinase K at 37°C for 1 hour to reverse protein/DNA complex. Stop buffer was subsequently added to inhibit proteinase K function. The resulting DNA was subjected directly to PCR analysis for 36 cycles using the following conditions: 5 minutes at 94°C, 30 seconds at 94°C, 30 seconds at 55°C, 20 seconds at 72°C, and 7 minutes at 72°C. The primers (400 nmol/L) for E-cadherin promoter were synthesized as follows: forward, 5′-TAGAGGGTCACCGCGTCTAT-3′ (−170∼−151); reverse, 5′-TCACAGGTGCTTTGCAGTTC-3′ (+10∼+39). The PCR product covers the A, B, and C E-box elements in the proximal E-cadherin: E-box A, 5′-CAGGTG-3′ (−79∼−74); E-box B, 5′-CACCTG-3′ (−59∼−54); and E-box C, 5′-CACCTG-3′ (+21∼+26). Using high-density TMAs, we evaluated the expression of CCN6 and E-cadherin proteins on 116 unselected invasive carcinoma tissue samples to characterize its expression in situ by immunohistochemistry. Clinical and pathological characteristics of the patients can be found in Table 1. CCN6 protein expression was observed primarily in the cytoplasm of breast cancer cells and less frequently in the nucleus (Figure 1). Occasional stromal cells also expressed CCN6 protein. Invasive carcinomas expressing high levels of CCN6 (scores 3 to 4, CCN6+) and those that expressed low levels (scores 1 to 2, CCN6−) were readily apparent (Figure 1). We noted that of the 102 invasive carcinomas with available tumor for immunohistochemistry, CCN6 expression was lost or reduced (scores 1 to 2) in 62 (60.7%) tumors. Furthermore, invasive carcinomas with low CCN6 had increased incidence of lymph node metastasis when compared with tumors with high CCN6 (χ2 test, P = 0.04). CCN6 expression was not associated with other tumor characteristics, including histological type, grade, size, hormonal receptor status, or HER-2/neu overexpression (Table 2).Table 1Clinical and Pathological Characteristics of the Patients Included in This StudyParameterValueNo. of patients116Median age [years (range)]52 (30–80)Histopathological grade [n (%)] 124 (24.49) 258 (59.18) 316 (16.33)Histopathological type [n (%)] Ductal85 (73.28) Lobular19 (16.38) Mixed ductal and lobular11 (9.48) Tubular1 (0.86)Median tumor size [cm (range)]1.7 (0.3–8.0)Lymph nodes [n (%)] Negative34 (30.91) Positive76 (69.09)ER status [n (%)] Negative14 (14.00) Positive86 (86.00)PR status [n (%)] Negative22 (22.00) Positive78 (78.00)Her-2/neu status [n (%)] Not overexpressed57 (66.28) Overexpressed29 (33.72)CCN6 [n (%)] Normal40 (39.2) Reduced62 (60.8) Open table in a new tab Table 2Analysis of CCN6 Expression According to Clinical and Pathological Characteristics of the Patient CohortCCN6LowHighParametern%n%P value*P value computed using χ2 test.Histopathological grade0.18 11529.4616.7 22956.92363.9 3713.7719.4Tumor size0.37 <2 cm3559.32468.6 ≥2 cm2440.71121.4Lymph nodes0.04 Negative2746.6824.2 Positive3153.42575.8ER status0.21 Negative59.4719.4 Positive4890.62980.6PR status0.08 Negative815.11130.6 Positive4584.92569.4HER-2/neu status0.37 Not overexpressed3068.21858.1 Overexpressed1431.81341.9* P value computed using χ2 test. Open table in a new tab As expected, E-cadherin expression had a crisp membranous staining pattern. The intensity of the staining was measured using an image analysis system in a continuous scale, allowing for high reproducibility and precise scoring of protein expression.17Witkiewicz AK Varambally S Shen R Mehra R Sabel MS Ghosh D Chinnaiyan AM Rubin MA Kleer CG Alpha-methylacyl-CoA racemase protein expression is associated with the degree of differentiation in breast cancer using quantitative image analysis.Cancer Epidemiol Biomarkers Prev. 2005; 14: 1418-1423Crossref PubMed Scopus (50) Google Scholar There was a strong association between CCN6 and E-cadherin proteins. Invasive carcinomas with low CCN6 expression also had reduced or absent E-cadherin protein at the cell membrane. Invasive carcinomas with low CCN6 had a mean E-cadherin expression of 98, whereas the invasive carcinomas with high CCN6 had a mean E-cadherin expression of 105.3 (t-test, P = 0.02; Figure 1). Collectively, these data show that CCN6 down-regulation is a frequent event in invasive breast carcinomas and that it is associated with lymph node metastasis, and they highlight the strength of the association between CCN6 and E-cadherin proteins in human breast cancer. Immunoblot analysis of a panel of benign breast cells and breast cancer cells showed that CCN6 expression is highest in benign mammary epithelial cells (HME and MCF10A). Supporting our findings in breast tissue samples, CCN6 protein is decreased in breast cancer cell lines. When compared with benign breast cells, the well differentiated, noninvasive MCF-7 breast cancer cells have slightly decreased levels of CCN6 protein, whereas CCN6 is greatly decreased in the invasive and metastasizing MDA-MB-231 cell line and is almost absent in the poorly differentiated SUM149 inflammatory breast cancer cell line (Figure 2). We have stably knocked down CCN6 in HME cells using siRNA and shRNA (Figure 2B). Stable CCN6 knockdown in HME cells resulted in phenotypic features of EMT and

Prostate cancer remains the most common malignancy among men in United States, and there is no remedy currently available for the advanced stage hormone-refractory cancer. This is partly due to the incomplete understanding of androgen-regulated proteins and their encoded functions. Whole-cell proteomes of androgen-starved and androgen-treated LNCaP cells were analyzed by semi-quantitative MudPIT ESI- ion trap MS/MS and quantitative iTRAQ MALDI- TOF MS/MS platforms, with identification of more than 1300 high-confidence proteins. An enrichment-based pathway mapping of the androgen-regulated proteomic data sets revealed a significant dysregulation of aminoacyl tRNA synthetases, indicating an increase in protein biosynthesis- a hallmark during prostate cancer progression. This observation is supported by immunoblot and transcript data from LNCaP cells, and prostate cancer tissue. Thus, data derived from multiple proteomics platforms and transcript data coupled with informatics analysis provides a deeper insight into the functional consequences of androgen action in prostate cancer.

Recurrent gene fusions involving ETS family genes are a distinguishing feature of human prostate cancers, with TMPRSS2-ERG fusions representing the most common subtype. The TMPRSS2-ERG fusion transcript and its splice variants are well characterized in prostate cancers; however, not much is known about the levels and regulation of wild-type ERG. By employing an integrative approach, we show that the TMPRSS2-ERG gene fusion product binds to the ERG locus and drives the overexpression of wild-type ERG in prostate cancers. Knockdown of TMPRSS2-ERG in VCaP cells resulted in the downregulation of wild-type ERG transcription, whereas stable overexpression of TMPRSS2-ERG in the gene fusion-negative PC3 cells was associated with the upregulation of wild-type ERG transcript. Further, androgen signaling-mediated upregulation of TMPRSS2-ERG resulted in the concomitant upregulation of wild-type ERG transcription in VCaP cells. The loss of wild-type ERG expression was associated with a decrease in the invasive potential of VCaP cells. Importantly, 38% of clinically localized prostate cancers and 27% of metastatic prostate cancers harboring the TMPRSS2-ERG gene fusions exhibited overexpression of wild-type ERG. Taken together, these results provide novel insights into the regulation of ERG in human prostate cancers.

Our goal was to investigate de novo purine biosynthetic gene PAICS expression and evaluate its role in prostate cancer progression.Next-generation sequencing, qRTPCR and immunoblot analysis revealed an elevated expression of a de novo purine biosynthetic gene, Phosphoribosylaminoimidazole Carboxylase, Phosphoribosylaminoimidazole Succinocarboxamide Synthetase (PAICS) in a progressive manner in prostate cancer. Functional analyses were performed using prostate cancer cell lines- DU145, PC3, LnCaP, and VCaP. The oncogenic properties of PAICS were studied both by transient and stable knockdown strategies, in vivo chicken chorioallantoic membrane (CAM) and murine xenograft models. Effect of BET bromodomain inhibitor JQ1 on the expression level of PAICS was also studied.Molecular staging of prostate cancer is important factor in effective diagnosis, prognosis and therapy. In this study, we identified a de novo purine biosynthetic gene; PAICS is overexpressed in PCa and its expression correlated with disease aggressiveness. Through several in vitro and in vivo functional studies, we show that PAICS is necessary for proliferation and invasion in prostate cancer cells. We identified JQ1, a BET bromodomain inhibitor previously implicated in regulating MYC expression and demonstrated role in prostate cancer, abrogates PAICS expression in several prostate cancer cells. Furthermore, we observe loss of MYC occupancy on PAICS promoter in presence of JQ1.Here, we report that evaluation of PAICS in prostate cancer progression and its role in prostate cancer cell proliferation and invasion and suggest it as a valid therapeutic target. We suggest JQ1, a BET-domain inhibitor, as possible therapeutic option in targeting PAICS in prostate cancer. Prostate 77:10-21, 2017. © 2016 Wiley Periodicals, Inc.

Kennedy disease, a degenerative disorder caused by an expanded glutamine tract, is mediated by misfolding of the mutant androgen receptor (AR) protein, a process that may disrupt proteasome function. We hypothesized that this might lead to endoplasmic reticulum (ER) stress and induction of the unfolded protein response (UPR), a complex physiologic pathway that regulates cell survival. To test this hypothesis, we used aminoterminal fragments of wild type (AR16Q) or mutant (AR112Q) AR that triggered glutamine length-dependent cell death and activated an ER stress-inducible promoter. To evaluate the role of the UPR, we examined the contributions of three proximal sensors of ER stress: activating transcription factor 6 (ATF6), inositol requiring 1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK). AR112Q toxicity was significantly increased by a dominant negative ATF6 mutant and significantly decreased by a constitutively active ATF6 mutant, indicating that ATF6 promoted cell survival. In contrast, co-transfection with three separate IRE1α dominant negative mutants failed to alter glutamine length-dependent toxicity, suggesting that this arm of the UPR did not significantly affect AR112Q induced cell death. Activation of PERK, an ER transmembrane protein that functions as the third proximal UPR sensor, promoted glutamine length-dependent toxicity. Although nuclear localization sequence- and nuclear export sequence-targeted proteins both activated the UPR, this pathway more potently influenced toxicity when proteins were targeted to the cytoplasm. Taken together, our data demonstrate that the UPR is activated in cells expressing long glutamine tracts and that this pathway modulates polyglutamine toxicity.

The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5′-phosphorylated, 2′,5′-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2′,5′-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2.

To optimally integrate epidermal growth factor receptor (EGFR) inhibitors into the clinical treatment of head and neck cancer, two important questions must be answered: (a) does EGFR inhibition add to the effects of radiochemotherapy, and (b) if so, which method of inhibiting EGFR is superior (an EGFR antibody versus a small molecule tyrosine kinase inhibitor)? We designed an in vivo study to address these questions.Nude mice with UMSCC-1 head and neck cancer xenografts received either single, double, or triple agent therapy with an EGFR inhibitor (either cetuximab or gefitinib), gemcitabine, and/or radiation for 3 weeks. Tumor volumes and animal weights were measured for up to 15 weeks. Immunoblotting and immunofluorescent staining were done on tumors treated with either cetuximab or gefitinib alone.The addition of an EGFR inhibitor significantly delayed the tumor volume doubling time, from a median of 40 days with radiochemotherapy (gemcitabine and radiation) alone, to 106 days with cetuximab and 66 days with gefitinib (both P < 0.005). Cetuximab resulted in significantly less weight loss than gefitinib. Immunoblot analysis and immunofluorescent staining of tumors show that although levels of phosphorylated AKT and extracellular signal-regulated kinase were decreased similarly in response to cetuximab or gefitinib, cetuximab caused prolonged suppression of pEGFR, pSTAT3, and Bcl(XL) compared with gefitinib.EGFR inhibition, particularly with cetuximab, improves the effectiveness of radiochemotherapy in this model of head and neck cancer. The correlation of response with prolonged suppression of EGFR, STAT3, and Bcl(XL) offers the possibility that these may be candidate biomarkers for response.

Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in the metabolism of fatty acids and is an important tissue biomarker in the prostate to distinguish normal glands from prostate cancer. Here, for the first time, we evaluated the expression of AMACR protein in normal breast, ductal carcinoma in situ, and invasive carcinomas. By immunofluorescence and immunohistochemistry, AMACR was seen in cytoplasmic granules consistent with a mitochondrial and peroxisomal localization. AMACR expression was determined by immunohistochemistry on 160 invasive carcinomas with long follow-up, using a high-density tissue microarray, and evaluated by two methods: standard pathology review and quantitative image analysis. AMACR was overexpressed in 42 of 160 (26%) invasive carcinomas, and it was associated with a decrease in tumor differentiation, a feature of aggressive breast cancer. Quantitative analysis allowed for better discrimination and more accurate evaluation of low-intensity staining. In conclusion, AMACR protein is expressed in normal breast and its expression seems to increase in invasive carcinomas. We observed stronger AMACR protein expression in high-grade carcinomas when compared with low-grade ones. Quantitative image analysis is a novel way to accurately and reproducibly evaluate immunohistochemistry in breast tissue samples using high-density tissue microarrays.

Molecular inhibition of the ErbB signaling pathway represents a promising cancer treatment strategy. Preclinical studies suggest that enhancement of antitumor activity can be achieved by maximizing ErbB signaling inhibition. Using cDNA microarrays, we identified histone deacetylase (HDAC) inhibitors as having strong potential to enhance the effects of anti-ErbB agents. Studies using a 20,000 element (20K) cDNA microarray demonstrate decreased transcript expression of ErbB1 (epidermal growth factor receptor) and ErbB2 in DU145 (prostate) and ErbB2 in SKBr3 (breast) cancer cell lines. Additional changes in the DU145 gene expression profile with potential interaction to ErbB signaling include down-regulation of caveolin-1 and hypoxia inducible factor 1-alpha (HIF1-alpha), and up-regulation of gelsolin, p19(INK4D) and Nur77. Findings were validated using real time RT-PCR and Western blot analysis. Enhanced proliferative inhibition, apoptosis induction and signaling inhibition were demonstrated when combining HDAC inhibition with ErbB blockade. These results suggest that used cooperatively, anti-ErbB agents and HDAC inhibitors may offer a promising strategy of dual targeted therapy. Additionally, microarray data suggest that the beneficial interaction of these agents may not derive solely from modulation of ErbB expression, but may result from effects on other oncogenic processes including angiogenesis, invasion and cell cycle kinetics.

Long noncoding RNAs (lncRNAs) are RNA molecules with over 200 nucleotides that do not code for proteins, but are known to be widely expressed and have key roles in gene regulation and cellular functions. They are also found to be involved in the onset and development of various cancers, including prostate cancer (PCa). Since PCa are commonly driven by androgen regulated signaling, mainly stimulated pathways, identification and determining the influence of lncRNAs in androgen response is useful and necessary. LncRNAs regulated by the androgen receptor (AR) can serve as potential biomarkers for PCa. In the present study, gene expression data analysis were performed to distinguish lncRNAs related to the androgen response pathway.We used publicly available RNA-sequencing and ChIP-seq data to identify lncRNAs that are associated with the androgen response pathway. Using Universal Correlation Coefficient (UCC) and Pearson Correlation Coefficient (PCC) analyses, we found 15 lncRNAs that have (a) highly correlated expression with androgen response genes in PCa and are (b) differentially expressed in the setting of treatment with an androgen agonist as well as antagonist compared to controls. Using publicly available ChIP-seq data, we investigated the role of androgen/AR axis in regulating expression of these lncRNAs. We observed AR binding in the promoter regions of 5 lncRNAs (MIR99AHG, DUBR, DRAIC, PVT1, and COLCA1), showing the direct influence of AR on their expression and highlighting their association with the androgen response pathway.By utilizing publicly available multiomics data and by employing in silico methods, we identified five candidate lncRNAs that are involved in the androgen response pathway. These lncRNAs should be investigated as potential biomarkers for PCa.

No AccessJournal of UrologyUrological survey1 May 2006Recurrent Fusion of TMPRSS2 and ETS Transcription Factor Genes in Prostate Cancer S.A. Tomlins, D.R. Rhodes, S. Perner, S.M. Dhanasekaran, R. Mehra, X.-W. Sun, S. Varambally, X. Cao, J. Tchinda, R. Kuefer, C. Lee, J.E. Montie, R.B. Shah, K.J. Pienta, M.A. Rubin, and A.M. Chinnaiyan S.A. TomlinsS.A. Tomlins Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , D.R. RhodesD.R. Rhodes Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , S. PernerS. Perner Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , S.M. DhanasekaranS.M. Dhanasekaran Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , R. MehraR. Mehra Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , X.-W. SunX.-W. Sun Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , S. VaramballyS. Varambally Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , X. CaoX. Cao Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , J. TchindaJ. Tchinda Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , R. KueferR. Kuefer Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , C. LeeC. Lee Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , J.E. MontieJ.E. Montie Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , R.B. ShahR.B. Shah Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , K.J. PientaK.J. Pienta Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , M.A. RubinM.A. Rubin Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author , and A.M. ChinnaiyanA.M. Chinnaiyan Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(06)00096-6AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "Recurrent Fusion of TMPRSS2 and ETS Transcription Factor Genes in Prostate Cancer." The Journal of Urology, 175(5), p. 1707 © 2006 by American Urological AssociationFiguresReferencesRelatedDetails Volume 175Issue 5May 2006Page: 1707 Advertisement Copyright & Permissions© 2006 by American Urological AssociationMetricsAuthor Information S.A. Tomlins Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author D.R. Rhodes Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author S. Perner Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author S.M. Dhanasekaran Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author R. Mehra Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author X.-W. Sun Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author S. Varambally Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author X. Cao Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author J. Tchinda Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author R. Kuefer Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author C. Lee Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author J.E. Montie Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author R.B. Shah Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author K.J. Pienta Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author M.A. Rubin Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author A.M. Chinnaiyan Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan Bioinformatics Program, University of Michigan Medical School, Ann Arbor, Michigan Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Michigan Urology Center, University of Michigan Medical School, Ann Arbor, Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan Department of Pathology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts Department of Pathology, Harvard Medical School, Boston, Massachusetts Department of Urology, Faculty of Medicine, University of Ulm, Ulm, Germany More articles by this author Expand All Advertisement PDF downloadLoading ...

Centrosome amplification (CA), the presence of centrosomes that are abnormally numerous or enlarged, is a well-established driver of tumor initiation and progression associated with poor prognosis across a diversity of malignancies. Pancreatic ductal adenocarcinoma (PDAC) carries one of the most dismal prognoses of all cancer types. A majority of these tumors are characterized by numerical and structural centrosomal aberrations, but it is unknown how CA contributes to the disease and patient outcomes. In this study, we sought to determine whether CA was associated with worse clinical outcomes, poor prognostic indicators, markers of epithelial-mesenchymal transition (EMT), and ethnicity in PDAC. We also evaluated whether CA could precipitate more aggressive phenotypes in a panel of cultured PDAC cell lines. Using publicly available microarray data, we found that increased expression of genes whose dysregulation promotes CA was associated with worse overall survival and increased EMT marker expression in PDAC. Quantitative analysis of centrosomal profiles in PDAC cell lines and tissue sections uncovered varying levels of CA, and the expression of CA markers was associated with the expression of EMT markers. We induced CA in PDAC cells and found that CA empowered them with enhanced invasive and migratory capabilities. In addition, we discovered that PDACs from African American (AA) patients exhibited a greater extent of both numerical and structural CA than PDACs from European American (EA) patients. Taken together, these findings suggest that CA may fuel a more aggressive disease course in PDAC patients.

Centrosome aberrations (CA) and abnormal mitoses are considered beacons of malignancy. Cancer cell doubling times in patient tumors are longer than in cultures, but differences in CA between tumors and cultured cells are uncharacterized. We compare mitoses and CA in patient tumors, xenografts, and tumor cell lines. We find that mitoses are rare in patient tumors compared with xenografts and cell lines. Contrastingly, CA is more extensive in patient tumors and xenografts (~35-50% cells) than cell lines (~5-15%), although CA declines in patient-derived tumor cells over time. Intratumoral hypoxia may explain elevated CA in vivo because exposure of cultured cells to hypoxia or mimicking hypoxia pharmacologically or genetically increases CA, and HIF-1α and hypoxic gene signature expression correlate with CA and centrosomal gene signature expression in breast tumors. These results highlight the importance of utilizing low-passage-number patient-derived cell lines in studying CA to more faithfully recapitulate in vivo cellular phenotypes.

Abstract Cancer is a complex disease effecting different organs and a major cause of death and a burden on the society. Multiple molecular alteration occur during cancer initiation and disease progression and metastasis. Recent advances in technology led to generation of large amount of molecular data including transcriptome. These large datasets can be used to analyze and identify sub-class specific cancer biomarkers and targets. However, there is a need for the development user friendly tools for its analysis and dissemination of the large scale cancer molecular data. Earlier, we had developed and upgraded web based comprehensive proteogenomic platform UALCAN (ualcan.path.uab.edu), which allows users to analyze and integrate the disparate data to better understand the gene, proteins, and pathways perturbed in cancer and make discoveries. UALCAN web platform enables intuitive analysis and discovery by cancer research community. In the current study, we describe the development of UALCAN Mobile application (App) that will provide cancer transcriptomic data obtained from The Cancer Genome Atlas (TCGA) project, to evaluate protein-coding gene expression based on various stratification including stage, grade, race, gender and molecular-subtypes across over 30 types of cancers. The UALCAN Mobile app aims at providing large cancer dataset on the go. In order to find causative gene expression changes, identify biomarkers and therapeutic targets, UALCAN Mobile will assist as the data will be easily mobile and transportable with this App. Here, we describe the development of UALCAN Mobile and its utility to the cancer research community. UALCAN Mobile App can be found at both iOS/Apple and Android play store and is free to download and use. Citation Format: Sooryanarayana Varambally, Darshan Shimoga Chandrashekar, Gopi Chand Puli, Santhosh Kumar Karthikeyan, Upender Manne, Chad J. Creighton, Sidarth Kumar. UALCAN Mobile, An app for cancer gene expression data analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2270.

Abstract Purpose: In the United States, colorectal cancer (CRC) is the second leading cause of cancer mortality. Metastatic CRC (mCRC) refractory to traditional chemotherapy is managed with regorafenib, a multiple-kinase inhibitor. However, patients show only a modest improvement in overall survival but experience high drug toxicity, adverse side effects, and poor tolerability. Thus, to reduce regorafenib-induced toxicity and to enhance antitumor activity, we combined it with a hybrid/dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition in a single agent. With syngeneic mice, the efficacy and impact on immunomodulation of this drug combination was assessed. Methods: To assess effects of the agents, C57BL/6 immunocompetent mice were injected with MC38 murine CRC cells and treated with regorafenib (6mg/kg body wt) or JAK/HDACi (30mg/kg body wt), and their combination every third day for 21 days. Upon completion of the experiment, the tumors were harvested and processed for RNA and protein expression profiles. The effect of drug treatment on immune response was analyzed by nCounter Gene Expression assays (NanoString Technologies). Cytokines were measured in serum using the MesoScale Discovery mouse V-Plex Proinflammatory Panel kit. For pharmacokinetic studies, plasma samples were assayed from C57BL/6 treated with drugs after 1, 7, 21, and 48 hrs of treatment. Results: The combination treatment significantly reduced tumor growth (volume and weight),relative to the vehicle control or single treatments. Gene expression showed higher CD45 abundance score in the combination treatment. This is an exciting observation, as CD45 has phosphatase activity and dephosphorylates key targets of the drugs used in the study. Additionally, after treatment with the combination, there was higher expression of Gzmb and Gzme and lower expression of Cx3cr1, indicating enhanced immuno modulation relative to regorafenib alone. We confirmed these findings by immunoprofiling of tumors. Higher CD45 staining, noted in tumors of mice treated with the combination, corroborated the gene expression results. Pronounced CD8 T lymphocytes infiltration was observed in combination, compared to regorafenib alone. Reduction of proinflammatory cytokine, TNF-α was noted in the combination group. This is an interesting observation as CD45 negatively regulates TNF, and combination treated mice had highest number of CD45 positive cells. Pharmacokinetic studies showed that the bioavailability of regorafenib was elevated after combination treatment relative to single-agent treatment. Conclusions: Relative to the single agents, the combination of regorafenib with JAK/HDACi was more effective, with an elevated antitumor immune response and with sustained inhibition of tumor growth. A clinical trial to evaluate this combination for the treatment of mCRCs is warranted. Citation Format: Prachi Bajpai, Farrukh Afaq, Sameer Al Diffalha, Sumit Agarwal, Hyung Gyoon Kim, Dennis Otali, Sooryanarayana Varambally, Ashish Manne, Ravi Paluri, Moh’d Khushman, Upender Manne. Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 585.

Natural polyreactive IgM autoantibodies, encoded by unmutated germline Ig V genes, represent a major fraction of the normal circulating IgM repertoire. We have previously shown that therapeutic preparation of pooled IgM exerts immunomodulatory effects as assessed by in vitro and in vivo studies. Here, we show that the IgM preparation induces cell death in lymphoblastoid cell lines and in human peripheral blood mononuclear cells. The IgM‐induced cell death involved classical features of apoptosis such as nuclear fragmentation and activation of caspases. Treatment of leukemic cells with IgM resulted in the cleavage of poly‐(A)DP ribose polymerase, a substrate of caspase, and in a reduction in mitochondrial transmembrane potential during the early period of apoptosis induction. Natural IgM‐induced apoptosis was inhibited by soluble Fas molecules and affinity‐purified Fas antibodies from pooled IgM preparation induced apoptosis in lymphoblastoid cells, suggesting the involvement of the Fas receptor. Our results suggest a role for normal IgM in controlling cell death and proliferation, and imply a possible therapeutic role for IgM in autoimmune and lymphoproliferative disorders.

In men with advanced penile squamous cell carcinoma receiving first-line chemotherapy, visceral metastases (VM) and Eastern Cooperative Oncology Group performance status ≥1 are poor prognostic factors for overall survival (OS). We hypothesized that tumor gene expression profiling may enhance prognostic stratification and identify potential therapeutic targets. In this retrospective study, RNA extracted from macrodissected tumors underwent profiling for the expression of 738 genes using NanoString. Univariate and multivariate analyses assessed the association of genes, VM, and performance status with OS. Tumors were available from 25 men who received first-line cisplatin-based chemotherapy. In univariate analysis, upregulated MAML2 (p = 0.004), KITLG (p ≤ 0.0001), and JAK1 (p = 0.029) genes were associated with poor OS, and upregulated FANCA was associated with better OS (p = 0.024). In stepwise multivariate analyses, VM (hazard ratio = 12.75, p = 0.0001) and MAML2 (hazard ratio = 10.411, p = 0.003) were associated with poor OS. The presence of none, one, and both of these poor risk factors was associated with significantly different median OS of 18.4 mo, 7.2 mo, and 2.1 mo, respectively. Unsupervised clustering demonstrated two major molecular subtypes with trend for different survivals (p = 0.052). Validation of results is necessary. Patient summary The expression of the MAML2 gene in penile cancers from men receiving first-line cisplatin-based chemotherapy predicted overall survival independent of clinical factors.

(Cancer Cell 31, 532–548; April 10, 2017) During the course of revising the manuscript and providing higher-magnification images, the authors inadvertently selected an incorrect image for Figure 4E (DU145 cells, lower right panel). This was a mistake in enlarging these images, and the initial submission did not contain this error. The correction to the image in Figure 4E does not affect the conclusions of the paper. This error has now been corrected here and in the article online. In the Supplemental Information, the images shown in Figure S3G were incorrect. These images were mistakenly duplicated from Figure S4B during preparation of the figures. Figure S3G has now been corrected here and in the Supplemental Information online. The text, the figure legends, and the conclusions of this article are not affected by this Correction. The authors apologize for any inconvenience that these errors in final figure revision/preparation may have caused.Figure 4. Retroinverso EIPs Specifically Bind to and Destabilize ERG (Original)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure S3G. Cell-Permeable Peptides Block ERG-Mediated Cell Invasion (Corrected)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure S3G. Cell-Permeable Peptides Block ERG-Mediated Cell Invasion (Original)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Development of Peptidomimetic Inhibitors of the ERG Gene Fusion Product in Prostate CancerWang et al.Cancer CellMarch 23, 2017In BriefWang et al. identify peptides that interact with the DNA binding domain of ERG, thereby attenuating protein-protein interactions and chromatin recruitment of ERG and inducing ERG degradation. These peptides reduce proliferation, invasion, and tumor growth of prostate cancer cells with the TMPRSS2:ERG gene fusion. Full-Text PDF Open Archive

Abstract Because survival of patients with metastatic colorectal cancer remain poor, there is an urgent need to identify potential novel druggable targets that are associated with colorectal cancer progression. One such target, basic leucine zipper and W2 domains 2 (BZW2), is involved in regulation of protein translation, and its overexpression is associated with human malignancy. Thus, we investigated the expression and regulation of BZW2, assessed its role in activation of WNT/β-catenin signaling, identified its downstream molecules, and demonstrated its involvement in metastasis of colorectal cancer. In human colorectal cancers, high mRNA and protein expression levels of BZW2 were associated with tumor progression. BZW2-knockdown reduced malignant phenotypes, including cell proliferation, invasion, and spheroid and colony formation. BZW2-knockdown also reduced tumor growth and metastasis; conversely, transfection of BZW2 into BZW2 low-expressing colorectal cancer cells promoted malignant features, including tumor growth and metastasis. BZW2 expression was coordinately regulated by microRNA-98, c-Myc, and histone methyltransferase enhancer of zeste homolog 2 (EZH2). RNA sequencing analyses of colorectal cancer cells modulated for BZW2 identified P4HA1 and the long noncoding RNAs, MALAT1 and NEAT1, as its downstream targets. Further, BZW2 activated the Wnt/β-catenin signaling pathway in colorectal cancers expressing wild-type β-catenin. In sum, our study suggests the possibility of targeting BZW2 expression by inhibiting EZH2 and/or c-Myc. Implications: FDA-approved small-molecule inhibitors of EZH2 can indirectly target BZW2 and because BZW2 functions as an oncogene, these inhibitors could serve as therapeutic agents for colorectal cancer.

Abstract Ataxia telangiectasia mutated (ATM) kinase plays a crucial role in the cellular response to DNA damage and in radiation resistance. Although much effort has focused on the relationship between ATM and other nuclear signal transducers, little is known about interactions between ATM and mitogenic signaling pathways. In this study, we show a novel relationship between ATM kinase and extracellular signal-regulated kinase 1/2 (ERK1/2), a key mitogenic stimulator. Activation of ATM by radiation down-regulates phospho-ERK1/2 and its downstream signaling via increased expression of mitogen-activated protein kinase phosphatase MKP-1 in both cell culture and tumor models. This dephosphorylation of ERK1/2 is independent of epidermal growth factor receptor (EGFR) activity and is associated with radioresistance. These findings show a new function for ATM in the control of mitogenic pathways affecting cell signaling and emphasize the key role of ATM in coordinating the cellular response to DNA damage. (Cancer Res 2006; 66(24): 11554-9)

Active surveillance (AS) is a widely adopted strategy to monitor men with low-risk, localized prostate cancer (PCa). Current AS inclusion criteria may misclassify as many as one in four patients. The advent of multiparametric magnetic resonance imaging (mpMRI) and novel PCa biomarkers may offer improved risk stratification. We performed a review of recently published literature to characterize emerging evidence in support of these novel modalities.An English literature search was conducted on PubMed for available original investigations on localized PCa, AS, imaging, and biomarkers published within the past 3 years. Our Boolean criteria included the following terms: PCa, AS, imaging, biomarker, genetic, genomic, prospective, retrospective, and comparative. The bibliographies and diagnostic modalities of the identified studies were used to expand our search.Our review identified 222 original studies. Our expanded search yielded 244 studies. Among these, 70 met our inclusion criteria. Evidence suggests mpMRI offers improved detection of clinically significant PCa, and MRI-fusion technology enhances the sensitivity of surveillance biopsies. Multiple studies demonstrate the promise of commercially available screening assays for prediction of AS failure, and several novel biomarkers show promise in this setting.In the era of AS for men with low-risk PCa, improved strategies for proper stratification are needed. mpMRI has dramatically enhanced the detection of clinically significant PCa. The advent of novel biomarkers for prediction of aggressive disease and AS failure has shown some initial promise, but further validation is warranted.

Molecular alterations in the prostate cancer proteome mediate the functional and phenotypic transformation from clinically localised to metastatic cancer, a transition that drives patient's mortality and challenges therapeutic intervention. A first approximation of differential proteomic alterations stratified by disease stage has yielded repertoires of potential diagnostic and prognostic markers, multiplex signatures of predictive value, and yield fundamental insight into molecular commonalities in cancer progression. Deciphering these causative proteomic alterations from the molecular noise will continue to mature our understanding of tumour biology and drive new computational and integrative approaches to model a system's view that accommodates the heterogeneity of prostate cancer progression.

In the paper published by Jentzmik et al. [1], the authors address the importance of urine-derived sarcosine based on public interest in our report [2], which describes elevated levels of the metabolite in urine of biopsy-proven prostate cancer (PCa) patients. We found especially elevated sarcosine levels in tumor specimens from patients with metastatic PCa, compared with organ-confined tumors. In their paper [1], the authors have examined urine supernatants collected after digital rectal examination (DRE) from 139 patients with prostate-specific antigen (PSA) levels between 2 and 20 ng/ml. These patients included 106 patients with PCa and 33 individuals with no evidence of malignancy (NEM), as assessed by biopsy. A total of 99 patients in this cohort had PSA levels between 0 and 10 ng/ml. Jentzmik et al. [1] report that ratio of free to total PSA (%fPSA) performed significantly better than sarcosine in delineating PCa from NEM, with an area under the curve (AUC) of 0.81 versus 0.63 (p = 0.012), whereas PSA performed similarly to sarcosine (AUC: 0.64 vs 0.63; p = 0.933). When analysis was restricted to patients with a total PSA range of 0–10 ng/ml, %fPSA performed better than sarcosine and PSA (AUC: 0.79 vs 0.67 vs 0.59). Based on this information, Jentzmik et al conclude that measurement of sarcosine in urine after DRE is hardly suitable to improve the diagnostic performance in comparison with routine markers like %fPSA. Our studies, reported in Nature [2], did not aim to establish sarcosine as a clinical test in urine to detect PCa. Rather, we reported results of a proof-of-concept study in which unbiased metabolomic profiling was used to delineate tissue-derived biomarkers for PCa aggressiveness and prognosis, of which sarcosine also could be detected in urine and, additionally, had a role in PCa progression. The intent of the initial study was to demonstrate the power of metabolomics in the identification of potential biomarkers of cancer. In this study [1], using post-DRE urine-derived sediments and supernatants, sarcosine levels were observed to be significantly elevated in biopsy-positive patients versus negative controls, indicative of its potential to serve as a biomarker. Notably, using an independent set of 40 urine sediments (n = 20; each from biopsy-positive patients and negative individuals) (unpubl. data), we have recently confirmed our earlier findings on elevated levels of sarcosine in biopsy-positive PCa patients (Fig. 1). It is recognized that the use of urinary sarcosine in clinical implementation is in its early stages, and its diagnostic value can be ascertained only after a series of comprehensive blinded validation studies. Fig. 1 Box plot of sarcosine levels based on isotope-dilution gas chromatography–mass spectrometry analysis showing median sarcosine-to-alanine levels in urine sediments from an independent group of biopsy-positive and -negative individuals. The AUCs of 0.67 and 0.59 that Jentzmik et al. [1] report for sarcosine and PSA, respectively, in patients having PSA levels between 0 and 10 ng/ml are similar to the ones reported in the Nature paper [2] from an independent set of 110 samples (ie, 0.69 and 0.53, respectively). These studies comparing sarcosine to PSA are independent validations of the initially reported observation. Furthermore, in our report [2], we used caution when describing the biomarker potential of urine-derived sarcosine, as is evident by the following statement: “The overall receiver operating characteristic curves for sarcosine indicate that its predictive value is modest, with an area under the curve (AUC) of 0.71 for urine sediments and 0.67 for supernatants (Supplementary Fig. 14b, c).” In summary, we maintain that sarcosine, when multiplexed with other metabolomic markers and perhaps with other diagnostic modalities, will have the potential to increase the accuracy of detecting PCa in future. We agree that %fPSA could be a valuable parameter to include when developing such multiplex clinical tests. Moreover, tumor and urinary sarcosine may prove to be highly predictive of prognosis, which would be very helpful for clinical decision making.

Abstract Background Recent microarray and sequencing studies of prostate cancer showed multiple molecular alterations during cancer progression. It is critical to evaluate these molecular changes to identify new biomarkers and targets. We performed analysis of glycine‐ N ‐acyltransferase like 1 (GLYATL1) expression in various stages of prostate cancer in this study and evaluated the regulation of GLYATL1 by androgen. Method We performed in silico analysis of cancer gene expression profiling and transcriptome sequencing to evaluate GLYATL1 expression in prostate cancer. Furthermore, we performed immunohistochemistry using specific GLYATL1 antibody using high‐density prostate cancer tissue microarray containing primary and metastatic prostate cancer. We also tested the regulation of GLYATL1 expression by androgen and ETS transcription factor ETV1. In addition, we performed RNA‐sequencing of GLYATL1 modulated prostate cancer cells to evaluate the gene expression and changes in molecular pathways. Results Our in silico analysis of cancer gene expression profiling and transcriptome sequencing we revealed an overexpression of GLYATL1 in primary prostate cancer. Confirming these findings by immunohistochemistry, we show that GLYATL1 is overexpressed in primary prostate cancer compared with metastatic prostate cancer and benign prostatic tissue. Low‐grade cancers had higher GLYATL1 expression compared to high‐grade prostate tumors. Our studies showed that GLYATL1 is upregulated upon androgen treatment in LNCaP prostate cancer cells which harbors ETV1 gene rearrangement. Furthermore, ETV1 knockdown in LNCaP cells showed downregulation of GLYATL1 suggesting potential regulation of GLYATL1 by ETS transcription factor ETV1. Transcriptome sequencing using the GLYATL1 knockdown prostate cancer cell lines LNCaP showed regulation of multiple metabolic pathways. Conclusions In summary, our study characterizes the expression of GLYATL1 in prostate cancer and explores the regulation of its regulation in prostate cancer showing role for androgen and ETS transcription factor ETV1. Future studies are needed to decipher the biological significance of these findings.

Abstract BACKGROUND Androgens play a crucial role in prostate cancer, hence the androgenic pathway has become an important target of therapeutic intervention. Previously we discovered that gene fusions between the 5′‐untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases. The resulting aberrant overexpression of ETS genes drives tumor progression. METHODS Here, we evaluated the expression levels of 5α‐reductase isoenzymes in prostate cancer cell lines and tissues. We tested the effect of dutasteride, a 5α‐reductase inhibitor, in TMPRSS2–ERG fusion‐positive VCaP cell proliferation and cell invasion. We also evaluated the effect of dutasteride on the TMPRSS2–ERG fusion gene expression. Finally, we tested dutasteride alone or in combination with an anti‐androgen in VCaP cell xenografts tumor model. RESULTS Our data showed that 5α‐reductase SRD5A1 and SRD5A3 isoenzymes that are responsible for the conversion of testosterone to DHT, are highly expressed in metastatic prostate cancer compared to benign and localized prostate cancer. Dutasteride treatment attenuated VCaP cell proliferation and invasion. VCaP cells pre‐treated with dutasteride showed a reduction in ERG and PSA expression. In vivo studies demonstrated that dutasteride in combination with the anti‐androgen bicalutamide significantly decreased tumor burden in VCaP cell xenograft model. CONCLUSIONS Our findings suggest that dutasteride can inhibit ERG fusion‐positive cell growth and in combination with anti‐androgen, significantly reduce the tumor burden. Our study suggests that anti‐androgens used in combination with dutasteride could synergistically augment the therapeutic efficacy in the treatment of ETS‐positive prostate cancer. Prostate 72:1542–1549, 2012. © 2012 Wiley Periodicals, Inc.

Triple-negative breast cancer (TNBC) is a molecularly complex and heterogeneous breast cancer subtype with distinct biological features and clinical behavior. Although TNBC is associated with an increased risk of metastasis and recurrence, the molecular mechanisms underlying TNBC metastasis remain unclear. We performed whole-exome sequencing (WES) analysis of primary TNBC and paired recurrent tumors to investigate the genetic profile of TNBC.Genomic DNA extracted from 35 formalin-fixed paraffin-embedded tissue samples from 26 TNBC patients was subjected to WES. Of these, 15 were primary tumors that did not have recurrence, and 11 were primary tumors that had recurrence (nine paired primary and recurrent tumors). Tumors were analyzed for single-nucleotide variants and insertions/deletions.The tumor mutational burden (TMB) was 7.6 variants/megabase in primary tumors that recurred (n = 9); 8.2 variants/megabase in corresponding recurrent tumors (n = 9); and 7.3 variants/megabase in primary tumors that did not recur (n = 15). MUC3A was the most frequently mutated gene in all groups. Mutations in MAP3K1 and MUC16 were more common in our dataset. No alterations in PI3KCA were detected in our dataset.We found similar mutational profiles between primary and paired recurrent tumors, suggesting that genomic features may be retained during local recurrence.

C1orf74, also known as URCL4, has been reported to have higher expression and be associated with poor prognosis in lung adenocarcinoma patients, and its role in regulation of the EGFR/AKT/mTORC1 pathway has been recently elucidated. In the current study, we used publicly available data and experimental validation of C1orf74 gene expression and its association with prognosis in cervical cancer patients. qRT-PCR was performed using RNA from cervical cancer cell lines and twenty-five cervical cancer patients. Data from TNMplot revealed that mRNA expression of the C1orf74 gene in primary tumor tissues, as well as metastatic tissues from cervical cancer patients, was significantly higher compared to normal cervical tissues. HPV-positive tumors had higher expression of this gene compared to HPV-negative tumors. qPCR analysis also demonstrated higher expression of C1orf74 in HPV-positive cervical cancer cell lines and most cervical cancer patients. The promoter methylation levels of the C1orf74 gene in cervical cancer tissues were lower compared to normal cervical tissues (p &lt; 0.05). Collectively, our study indicates that higher expression of the C1orf74 gene caused by hypomethylation of its promoter is associated with poor overall survival in cervical cancer patients. Thus, C1orf74 is a novel prognostic marker in cervical cancer.

Runt-related transcription factor-3 (RUNX3) is an apoptotic factor correlated with tumorigenesis and cancer progression. Enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, has been shown to mediate silencing of RUNX3. We investigated RUNX3 and EZH2 expression in diffuse large B-cell lymphoma (DLBCL). A chart review was conducted and tissue-microarray (TMA) was constructed using archived tissue from 83 DLBCL cases. RUNX3 and EZH2 protein expression was correlated with immunophenotypic subtypes and survival. Loss of RUNX3 was observed in 20 cases; EZH2 expression was observed in 59 cases. RUNX3-negative tumors had significantly lower overall and recurrence-free survival (log-rank test, p < 0.0001 for each). No correlation was found between RUNX3 and EZH2 staining (r = 0.14; p = 0.2). Results suggest a role for the RUNX3 gene in the pathogenesis of DLBCL. Loss of RUNX3 expression strongly correlated with adverse prognosis, independent of subtype. Further studies are warranted to elucidate the biology and prognostic utility of RUNX3 in DLBCL.

Why does a first pregnancy after age 35 increase the risk of breast cancer, and what can be done to combat this?

The mobile phones (MP) are low power radio devices which work on electromagnetic fields (EMFs), in the frequency range of 900-1800 MHz. Exposure to MPEMFs may affect brain physiology and lead to various health hazards including brain tumors. Earlier studies with positron emission tomography (PET) have found alterations in cerebral blood flow (CBF) after acute exposure to MPEMFs. It is widely accepted that DNA double-strand breaks (DSBs) and their misrepair in stem cells are critical events in the multistage origination of various leukemia and tumors, including brain tumors such as gliomas. Both significant misbalance in DSB repair and severe stress response have been triggered by MPEMFs and EMFs from cell towers. It has been shown that stem cells are most sensitive to microwave exposure and react to more frequencies than do differentiated cells. This may be important for cancer risk assessment and indicates that stem cells are the most relevant cellular model for validating safe mobile communication signals. Recently developed technology for recording the human bio-electromagnetic (BEM) field using Electron photonic Imaging (EPI) or Gas Discharge Visualisation (GDV) technique provides useful information about the human BEM. Studies have recorded acute effects of Mobile Phone Electromagnetic Fields (MPEMFs) using EPI and found quantifiable effects on human BEM field. Present manuscript reviews evidences of altered brain physiology and stem cell functioning due to mobile phone/cell tower radiations, its association with increased cancer risk and explores early diagnostic value of EPI imaging in detecting EMF induced changes on human BEM.

Nature 457, 910–914 (2009); doi:10.1038/nature07762 In Fig. 4b of this Article, a typographical error was made in reporting sarcosine levels in the DU145 cell line represented. The y axis values should be in the scale of 0–50 pmoles per 106 cells, rather than 0–500 pmoles per 106 cells. This error has been verified and does not affect the conclusion of the paper.

Proteomic profiling of human disease has seen much early activity with the accessibility of the newest generation of high-throughput platforms and technologies. Nevertheless, the nature of the dynamic physiologic milieu and high dimensionality of the data has complicated major diagnostic and prognostic breakthroughs. Our recent article in Cancer Cell delineates an integrative model for culling a molecular signature of metastatic progression in prostate cancer from proteomic and transcriptomic analyses and shows its facility as a predictor of prognosis. The study leveraged direct proteomic analysis of tumor tissue extracts, differential feature selection characterizing the proteomic alterations of prostate cancer subclasses, and integration with public and study-derived genomic data to construct a multiplex gene signature representing progression of indolent cancer to aggressive disease. This further predicted clinical outcome in a variety of solid tumors. This review describes the context of the work, the framework for the analysis itself, and a look forward to the promise of this systems approach to human disease.

Abstract Understanding the molecular mechanism of SARS-CoV-2 infection (the cause of COVID-19) is a scientific priority for 2020. Various research groups are working toward development of vaccines and drugs, and many have published genomic and transcriptomic data related to this viral infection. The power inherent in publicly available data can be demonstrated via comparative transcriptome analyses. In the current study, we collected high-throughput gene expression data related to human lung epithelial cells infected with SARS-CoV-2 or other respiratory viruses (SARS, H1N1, rhinovirus, avian influenza, and Dhori) and compared the effect of these viruses on the human transcriptome. The analyses identified fifteen genes specifically expressed in cells transfected with SARS-CoV-2; these included CSF2 (colony-stimulating factor 2) and S100A8 and S100A9 (calcium-binding proteins), all of which are involved in lung/respiratory disorders. The analyses showed that genes involved in the Type1 interferon signaling pathway and the apoptosis process are commonly altered by infection of SARS-CoV-2 and influenza viruses. Furthermore, results of protein-protein interaction analyses were consistent with a functional role of CSF2 in COVID-19 disease. In conclusion, our analysis has revealed cellular genes associated with SARS-CoV-2 infection of the human lung epithelium; these are potential therapeutic targets.

Nature Communications 5 Article number: 4527 (2014); Published 31 July 2014; Updated 24 February 2016 In Fig. 4e,f of this Article, the plotted tumour volumes are incorrect due to an error in the formula used to convert length and width measurements into volume. The formula used was ((length*width)^2)*(22/7)*(1/6), and this should have read (length*(width^2))*(22/7)*(1/6).

&lt;p&gt;Supplementary figure legends 1-8, and supplementary tables 1-5&lt;/p&gt;