Kenneth J. Pienta

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.

Toward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%–60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides clinically actionable information that could impact treatment decisions for these affected individuals.

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.

Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration-resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion-negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signalling deregulated in prostate cancer, and prioritize candidates for future study.

Abstract Purpose: A method for enumerating circulating tumor cells (CTC) has received regulatory clearance. The primary objective of this prospective study was to establish the relationship between posttreatment CTC count and overall survival (OS) in castration-resistant prostate cancer (CRPC). Secondary objectives included determining the prognostic utility of CTC measurement before initiating therapy, and the relationship of CTC to prostate-specific antigen (PSA) changes and OS at these and other time points. Experimental Design: Blood was drawn from CRPC patients with progressive disease starting a new line of chemotherapy before treatment and monthly thereafter. Patients were stratified into predetermined Favorable or Unfavorable groups (<5 and ≥5 CTC/7.5mL). Results: Two hundred thirty-one of 276 enrolled patients (84%) were evaluable. Patients with Unfavorable pretreatment CTC (57%) had shorter OS (median OS, 11.5 versus 21.7 months; Cox hazard ratio, 3.3; P < 0.0001). Unfavorable posttreatment CTC counts also predicted shorter OS at 2 to 5, 6 to 8, 9 to 12, and 13 to 20 weeks (median OS, 6.7-9.5 versus 19.6-20.7 months; Cox hazard ratio, 3.6-6.5; P < 0.0001). CTC counts predicted OS better than PSA decrement algorithms at all time points; area under the receiver operator curve for CTC was 81% to 87% and 58% to 68% for 30% PSA reduction (P = 0.0218). Prognosis for patients with (a) Unfavorable baseline CTC who converted to Favorable CTC improved (6.8 to 21.3 months); (b) Favorable baseline CTC who converted to Unfavorable worsened (>26 to 9.3 months). Conclusions: CTC are the most accurate and independent predictor of OS in CRPC. These data led to Food and Drug Administration clearance of this assay for the evaluation of CRPC.

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.

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.

A 44-year old woman with recurrent solitary fibrous tumor (SFT)/hemangiopericytoma was enrolled in a clinical sequencing program including whole-exome and transcriptome sequencing. A gene fusion of the transcriptional repressor NAB2 with the transcriptional activator STAT6 was detected. Transcriptome sequencing of 27 additional SFTs identified the presence of a NAB2-STAT6 gene fusion in all tumors. Using RT-PCR and sequencing, we detected this fusion in all 51 SFTs, indicating high levels of recurrence. Expression of NAB2-STAT6 fusion proteins was confirmed in SFT, and the predicted fusion products harbor the early growth response (EGR)-binding domain of NAB2 fused to the activation domain of STAT6. Overexpression of the NAB2-STAT6 gene fusion induced proliferation in cultured cells and activated the expression of EGR-responsive genes. These studies establish NAB2-STAT6 as the defining driver mutation of SFT and provide an example of how neoplasia can be initiated by converting a transcriptional repressor of mitogenic pathways into a transcriptional activator.

Chemokines, small pro-inflammatory chemoattractant cytokines that bind to specific G-protein-coupled seven-span transmembrane receptors, are major regulators of cell trafficking and adhesion. The chemokine CXCL12 (also called stromal-derived factor-1) is an important α-chemokine that binds primarily to its cognate receptor CXCR4 and thus regulates the trafficking of normal and malignant cells. For many years, it was believed that CXCR4 was the only receptor for CXCL12. Yet, recent work has demonstrated that CXCL12 also binds to another seven-transmembrane span receptor called CXCR7. Our group and others have established critical roles for CXCR4 and CXCR7 on mediating tumor metastasis in several types of cancers, in addition to their contributions as biomarkers of tumor behavior as well as potential therapeutic targets. Here, we review the current concepts regarding the role of CXCL12 / CXCR4 / CXCR7 axis activation, which regulates the pattern of tumor growth and metastatic spread to organs expressing high levels of CXCL12 to develop secondary tumors. We also summarize recent therapeutic approaches to target these receptors and/or their ligands.

Androgen receptor (AR) is a ligand-dependent transcription factor that plays a key role in prostate cancer. Little is known about the nature of AR cis-regulatory sites in the human genome. We have mapped the AR binding regions on two chromosomes in human prostate cancer cells by combining chromatin immunoprecipitation (ChIP) with tiled oligonucleotide microarrays. We find that the majority of AR binding regions contain noncanonical AR-responsive elements (AREs). Importantly, we identify a noncanonical ARE as a cis-regulatory target of AR action in TMPRSS2, a gene fused to ETS transcription factors in the majority of prostate cancers. In addition, through the presence of enriched DNA-binding motifs, we find other transcription factors including GATA2 and Oct1 that cooperate in mediating the androgen response. These collaborating factors, together with AR, form a regulatory hierarchy that governs androgen-dependent gene expression and prostate cancer growth and offer potential new opportunities for therapeutic intervention.

We have designed MI-219 as a potent, highly selective and orally active small-molecule inhibitor of the MDM2-p53 interaction. MI-219 binds to human MDM2 with a K(i) value of 5 nM and is 10,000-fold selective for MDM2 over MDMX. It disrupts the MDM2-p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumor cells. MI-219 stimulates rapid but transient p53 activation in established tumor xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis, and complete tumor growth inhibition. MI-219 activates p53 in normal tissues with minimal p53 accumulation and is not toxic to animals. MI-219 warrants clinical investigation as a new agent for cancer treatment.

As the incidence of prostate cancer in the United States exceeds 330,000 in 1997, increasingly more men are faced with treatment choices for which there is no clear approach. At every stage of disease, these treatment choices may involve clinically equivalent modalities that differ in side effects and impact upon quality of life (QOL). Comprehensive, yet efficient, questionnaires are needed to measure QOL in patients with prostate cancer.Developed as a disease-specific adjunct to the Functional Assessment of Cancer Therapy (FACT) measurement system, a 12-item prostate cancer subscale (PCS) was developed and tested in three independent samples: a subscale development sample (n = 43), validity sample 1 (n = 34), and validity sample 2 (n = 96). The 12 items ask about symptoms and problems specific to prostate cancer. These questions are added to the general (FACT-G) instrument, thereby comprising a 47-item questionnaire.Internal consistency of the PCS ranged from 0.65 to 0.69, with coefficients for FACT-G subscales and aggregated scores ranging from 0.61 to 0.90. Concurrent validity was confirmed by the ability to discriminate patients by disease stage, performance status, and baseline prostate-specific antigen (PSA) level. Sensitivity to change in performance status and PSA score over a 2-month period suggested that some subscales of the FACT-Prostate (P) (including the PCS) are sensitive to meaningful clinical change.Our findings support use of the FACT-P as a meaningful component of QOL evaluation in men undergoing therapy for 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.

Intermediate or surrogate endpoints for survival can shorten time lines for drug approval. We aimed to assess circulating tumour cell (CTC) count as a prognostic factor for survival in patients with progressive, metastatic, castration-resistant prostate cancer receiving first-line chemotherapy.We identified patients with progressive metastatic castration-resistant prostate cancer starting first-line chemotherapy in the IMMC38 trial. CTCs were isolated by immunomagnetic capture from blood samples at baseline and after treatment. Baseline variables, including CTC count, titre of prostate-specific antigen (PSA), and concentration of lactate dehydrogenase (LDH), and post-treatment variables (change in CTCs and PSA) were tested for association with survival with Cox proportional hazards models. Concordance probability estimates were used to gauge discriminatory strength of the informative factors in identifying patients at low-risk and high-risk of survival.Variables associated with high risk of death were high LDH concentration (hazard ratio 6.44, 95% CI 4.24-9.79), high CTC count (1.58, 1.41-1.77), and high PSA titre (1.26, 1.10-1.45), low albumin (0.10, 0.03-0.39), and low haemoglobin (0.72, 0.64-0.81) at baseline. At 4 weeks, 8 weeks, and 12 weeks after treatment, changes in CTC number were strongly associated with risk, whereas changes in PSA titre were weakly or not associated (p>0.04). The most predictive factors for survival were LDH concentration and CTC counts (concordance probability estimate 0.72-0.75).CTC number, analysed as a continuous variable, can be used to monitor disease status and might be useful as an intermediate endpoint of survival in clinical trials. Prospective recording of CTC number as an intermediate endpoint of survival in randomised clinical trials is warranted.

The mutations present in advanced cancers can be identified by integrative high-throughput sequencing to enable biomarker-driven clinical trials and, ultimately, treatment.

CCL2 and interleukin (IL)-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b(+) cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X(L) and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyperactivation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages toward the CD206(+) M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14(+)/CD206(+) double-positive cell population compared with normal tissues. Altogether, these findings suggest a role for CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation toward tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy.

Abstract Prostate cancer is a common and clinically heterogeneous disease with marked variability in progression. The recent identification of gene fusions of the 5′-untranslated region of TMPRSS2 (21q22.3) with the ETS transcription factor family members, either ERG (21q22.2), ETV1 (7p21.2), or ETV4 (17q21), suggests a mechanism for overexpression of the ETS genes in the majority of prostate cancers. In the current study using fluorescence in situ hybridization (FISH), we identified the TMPRSS2:ERG rearrangements in 49.2% of 118 primary prostate cancers and 41.2% of 18 hormone-naive lymph node metastases. The FISH assay detected intronic deletions between ERG and TMPRSS2 resulting in TMPRSS2:ERG fusion in 60.3% (35 of 58) of the primary TMPRSS2:ERG prostate cancers and 42.9% (3 of 7) of the TMPRSS2:ERG hormone-naive lymph node metastases. A significant association was observed between TMPRSS2:ERG rearranged tumors through deletions and higher tumor stage and the presence of metastatic disease involving pelvic lymph nodes. Using 100K oligonucleotide single nucleotide polymorphism arrays, a homogeneous deletion site between ERG and TMPRSS2 on chromosome 21q22.2-3 was identified with two distinct subclasses distinguished by the start point of the deletion at either 38.765 or 38.911 Mb. This study confirms that TMPRSS2:ERG is fused in approximately half of the prostate cancers through deletion of genomic DNA between ERG and TMPRSS2. The deletion as cause of TMPRSS2:ERG fusion is associated with clinical features for prostate cancer progression compared with tumors that lack the TMPRSS2:ERG rearrangement. (Cancer Res 2006; 66(17): 8337-41)

Several reports have recently documented that CXCR7/RDC1 functions as a chemokine receptor for SDF-1/CXCL12, which regulates a spectrum of normal and pathological processes. In this study, the role of CXCR7/RDC1 in prostate cancer (PCa) was explored. Staining of high density tissue microarrays demonstrates that the levels of CXCR7/RDC1 expression increase as the tumors become more aggressive. In vitro and in vivo studies with PCa cell lines suggest that alterations in CXCR7/RDC1 expression are associated with enhanced adhesive and invasive activities in addition to a survival advantage. In addition, it was observed that CXCR7/RDC1 levels are regulated by CXCR4. Among the potential downstream targets of CXCR7/RDC1 are CD44 and cadherin-11, which are likely to contribute to the invasiveness of PCa cells. CXCR7/RDC1 also regulates the expression of the proangiogenic factors interleukin-8 or vascular endothelial growth factor, which are likely to participate in the regulation of tumor angiogenesis. Finally, we found that signaling by CXCR7/RDC1 activates AKT pathways. Together, these data demonstrate a role for CXCR7/RDC1 in PCa metastasis and progression and suggest potential targets for therapeutic intervention.

Human prostate cancers (PCa) express great variability in their ability to metastasize to bone. The identification of molecules associated with aggressive phenotypes will help to define PCa subsets and will ultimately lead to better treatment strategies. The chemokine stromal-derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 are now known to modulate the migration and survival of an increasing array of normal and malignant cell types including breast, pancreatic cancers, glioblastomas, and others. The present investigation extends our previous investigations by determining the expression of CXCR4 and CXCL12 in humans using high-density tissue microarrays constructed from clinical samples obtained from a cohort of over 600 patients. These data demonstrate that CXCR4 protein expression is significantly elevated in localized and metastastic cancers. At the RNA level, human PCa tumors also express CXCR4 and message, but overall, they were not significantly different suggesting post-transcriptional regulation of the receptor plays a major role in regulating protein expression. Similar observations were made for CXCL12 message, but in this case more CXCL12 message was expressed by metastastic lesions as compared to normal tissues. PCa cell lines also express CXCL12 mRNA, and regulate mRNA expression in response to CXCL12 and secrete biologically active protein. Furthermore, neutralizing antibody to CXCL12 decreased the proliferation of bone homing LNCaP C4-2B and PC3 metastastic tumor cells. These investigations provide important new information pertaining to the molecular basis of how tumors may 'home' to bone, and the mechanisms that may account for their growth in selected end organs.

The evolution of cooperation has a well established theoretical framework based on game theory. This approach has made valuable contributions to a wide variety of disciplines, including political science, economics, and evolutionary biology. Existing cancer theory suggests that individual clones of cancer cells evolve independently from one another, acquiring all of the genetic traits or hallmarks necessary to form a malignant tumor. It is also now recognized that tumors are heterotypic, with cancer cells interacting with normal stromal cells within the tissue microenvironment, including endothelial, stromal, and nerve cells. This tumor cell-stromal cell interaction in itself is a form of commensalism, because it has been demonstrated that these nonmalignant cells support and even enable tumor growth. Here, we add to this theory by regarding tumor cells as game players whose interactions help to determine their Darwinian fitness. We marshal evidence that tumor cells overcome certain host defenses by means of diffusible products. Our original contribution is to raise the possibility that two nearby cells can protect each other from a set of host defenses that neither could survive alone. Cooperation can evolve as by-product mutualism among genetically diverse tumor cells. Our hypothesis supplements, but does not supplant, the traditional view of carcinogenesis in which one clonal population of cells develops all of the necessary genetic traits independently to form a tumor. Cooperation through the sharing of diffusible products raises new questions about tumorigenesis and has implications for understanding observed phenomena, designing new experiments, and developing new therapeutic approaches.

Prostate cancer continues to be the most common lethal malignancy diagnosed in American men and the second leading cause of male cancer mortality. The American Cancer Society estimates that during 2005, ∼232,090 new cases of prostate cancer will be diagnosed in the United States and 30,350 men will die of metastatic disease (1). Approximately 1 man in 5 will be diagnosed with prostate cancer during his lifetime, and 1 man in 33 will die of this disease. As the population ages, these numbers are expected to increase. Initially, almost all metastatic prostate cancers require testosterone for growth, and the role of androgen deprivation as a first-line therapy for metastatic prostate cancer has been recognized for more than 60 years (2, 3). Hormone deprivation is accomplished by surgical (orchiectomy) or medical (luteinizing hormone-releasing hormone agonists, antiandrogens) castration. Hormonal therapy leads to remissions lasting 2 to 3 years; however, virtually all patients progress to a clinically androgen-independent state resulting in death in ∼16 to 18 months (4–9).Androgens are primary regulators of normal prostate as well as prostate cancer cell growth and proliferation. During androgen-dependent progression, prostate cancer cells depend on the androgen receptor as the primary mediator of growth and survival (10–12). When testosterone enters the cell, it is converted by the enzyme 5α-reductase to its active metabolite, dihydrotestosterone, a more active hormone with a 5- to 10-fold higher affinity for the androgen receptor. Dihydrotestosterone binds androgen receptors in the cytoplasm, causing phosphorylation, dimerization, and subsequent translocation into the nucleus, thereby binding to the androgen-response elements within the DNA, with consequent activation of genes involved in cell growth and survival (10). During androgen-independent progression, prostate cancer cells develop a variety of cellular pathways to survive and flourish in an androgen-depleted environment. Postulated and documented mechanisms include androgen receptor (AR) gene amplification, AR gene mutations, involvement of coregulators, ligand-independent activation of the androgen receptor, and the involvement of tumor stem cells (Fig. 1; refs. 8, 10–14).Hypersensitive pathway. One way in which prostate cancer cells circumvent the effects of androgen blockade is by developing the ability to use very low levels of androgen for growth (14–16). Hence, they do not become androgen independent in the classic sense, but rather castration independent. There are several proposed mechanisms that could explain response to lower levels of androgen.One such mechanism is increased expression of the androgen receptor, allowing enhanced ligand binding. The existence of this pathway is supported by studies of hormone-refractory tumors that have shown increased expression of androgen receptor compared with androgen-dependent tumors (14, 17–22). Increased production of androgen receptor is likely secondary to gene amplification as a result of mutation or through selective pressure of the androgen-depleted environment, causing the cells with fewer androgen receptors to die off and the clonal expansion of cells with more androgen receptor. Chen et al. (14) have shown, through comprehensive gene profiling of seven human prostate cancer xenograft models, that AR gene expression was up-regulated in the progression from androgen-dependent to castration-independent growth. Chen et al. (14) also showed that androgen-independent cells require 80% lower concentrations of androgen than androgen-dependent cells for growth.Increased sensitivity of the androgen receptor to androgens has been proposed as another mechanism for castration independence (15). Using the prostate cancer cell line LNCaP, which has a mutated androgen receptor, Gregory and coworkers have shown high level expression of the androgen receptor, increased stability of the androgen receptor, and enhanced nuclear localization of the androgen receptor in recurrent prostate tumors associated with an increased sensitivity to the growth-promoting effects of dihydrotestosterone. The concentration of dihydrotestosterone required for growth stimulation of androgen-independent cells was 4 orders of magnitude lower than that required for androgen-dependent LNCaP cells (15).A third hypersensitive mechanism proposed is increased local production of androgens by prostate cancer cells themselves. This most likely occurs by increased rate of conversion of testosterone to dihydrotestosterone by increasing 5α-reductase activity (10). Evidence in support of this mechanism includes (a) ethnic groups with higher levels of 5α-reductase activity have a higher rate of prostate cancer (23); (b) after androgen ablation therapy, serum testosterone levels decrease by 95%, but concentration of dihydrotestosterone in prostatic tissue is reduced by only 60% (24); and (c) genes involved in steroid biosynthesis have been found to be overexpressed in recurrent human prostate tumors (22).Promiscuous receptor. Although the wild-type androgen receptor is activated only by testosterone and dihydrotestosterone, the specificity of the androgen receptor has been shown to be broadened by mutations. The majority of mutations discovered leading to binding promiscuity are clustered in the ligand-binding domains (25, 26). These mutations allow the androgen receptor to be activated by nonandrogenic steroid molecules normally present in the circulation as well as antiandrogens (10–12, 14, 27–31). Such mutations have been shown to be more frequent in androgen-independent prostate cancer cells and form the basis of antiandrogen withdrawal syndromes in which ∼10% to 30% of patients treated with long-term antiandrogens will show disease regression with drug withdrawal (32–35).Outlaw pathway. It has also been determined that activation of androgen receptor can be accomplished by ligand-dependent binding of nonsteroid molecules or activation of downstream signaling of the androgen receptor by ligand-independent mechanisms. Growth and proliferation of tumor cells is therefore no longer under androgen control, although the androgen receptor machinery remains active. This type of activation has been coined outlaw activation (10–13). Deregulated growth factors, including insulin-like growth factor, keratinocyte growth factor, and epidermal growth factor, and cytokines, including IL-6, have been shown to directly phosphorylate and activate the androgen receptor (36, 37). In addition, it has been shown that androgen receptor–dependent genes can be activated by deregulation of signal transduction pathways (38, 39). For example, overexpression of the receptor tyrosine kinase HER-2/neu has been shown to activate androgen receptor–dependent genes in the absence of a ligand (38, 39).The activation of outlaw pathways spotlights the potential importance of tumor-microenvironment interactions in the development of castration-independent growth (40, 41). Paget (42) recognized that a “fertile soil” was necessary for the successful growth of cancer metastases. For example, many factors contribute to both androgen-dependent and independent growth of prostate cancer in the bone (41). The interaction of prostate cancer cells with bone stromal cells, osteoblasts, and osteoclasts, as well as the remodeling of bone extracellular matrix, results in the release and deregulation of multiple growth factors that the prostate cancer cells can use to become androgen independent.Coactivators and corepressors. A large number of coactivators and corepressors involved in the regulation of androgen receptor–driven transcription have been identified (43). They function as signaling intermediaries between the androgen receptor and general transcriptional machinery. Alterations in the balance between coactivators and corepressors have been shown to influence androgen receptor activation, although the precise mechanisms remain unknown (10, 44–47). An increase in coactivator levels has been shown in androgen-independent disease (44, 48–51). Coactivator proteins have been shown to enhance the activity of the androgen receptor to alternative ligands, sensitize the receptor to lower levels of native and nonnative ligands, and to allow ligand-independent activation (44, 52).Bypass pathway. Alternatively, the androgen receptor pathway can be bypassed completely and prostate cancer cells develop the ability to survive independent of ligand-mediated or non-ligand-mediated androgen receptor activation. The best known bypass mechanism involves modulation of apoptosis. In androgen-dependent prostate cancer cells, androgen receptor activation stimulates cell proliferation and depletion of androgen leads to apoptosis of these cells. Androgen-independent prostate cancer cells have been shown to frequently up-regulate antiapoptotic molecules (53–56). Inactivation of the tumor-suppressor gene phosphatase and tensin homologue with subsequent activation of Akt is one way in which androgen-independent prostate cancer cells escape apoptosis in an androgen-depleted environment (12). Another postulated bypass mechanism is the neuroendocrine differentiation of prostate cancer cells (12). Neuroendocrines cells have a low rate of proliferation and are more prevalent in androgen-refractory prostate cancer (12). They also secrete neuropeptides, such as serotonin and bombesin, which can increase the proliferation of neighboring cancer cells, allowing progression in a low-androgen environment (12).Prostate cancer stem cells. In contrast to the stochastic model of tumorigenesis, in which every cell within the tumor is potentially tumor-initiating, the stem cell model of cancer, or the hierarchical model, postulates that only a rare subset of cells are tumorigenic (54). A population of cells comprising 0.1% of prostate tumors, which are CD44+/α2β1/CD133+ and do not express androgen receptors, has been identified and is thought to be prostate cancer stem or progenitor cells (57). Another potential mechanism for survival in the androgen-depleted environment is the presence of prostate cancer stem cells that continually resupply the tumor cell population, despite therapy. Although these cells remain a small percentage of the actual percentage of the tumor, they are not affected by androgen-depletion therapy. These stem cells differentiate into androgen-dependent and independent cells, resulting in the heterogeneous androgen receptor phenotype that is observed in androgen-independent patients (8).Novel strategies to target hormone refractory prostate cancer. Ultimately, understanding the mechanisms is not enough. Understanding the pathways to what we now know as castration-resistant prostate cancer offers specific therapeutic opportunities based on these mechanisms (40, 41, 58). As in many cancers, novel therapies are being approached with two different strategies. The first is the identification of critical pathways and targets. The second is the development of multitargeted approaches that are directed not only at the cancer cell but also at the surrounding microenvironment of the tumor (Fig. 2).Critical pathways. Bypass pathways that promote cell survival are being targeted with a variety of strategies. Targeting the Bcl-2 molecule-microtubule complex with the agent docetaxel has led to the first shown survival benefit for patients with androgen-independent prostate cancer (58). In a trial involving 1,006 men with hormone refractory prostate cancer, Tannock et al. (59) reported in 2004 that 75 mg/m2 of docetaxel given every 3 weeks plus daily prednisone led to a median survival rate of 18.9 months, compared with a survival rate of 16.4 months in patients who received 12 mg/m2 of mitoxantrone given every 3 weeks plus daily prednisone (P = 0.009). As compared with the mitoxantrone group, those given docetaxel every 3 weeks had a hazard ratio for death of 0.76 (95% confidence interval, 0.62-0.94; P = 0.009). The every-3-week schedule of docetaxel showed an advantage in median survival when compared with mitoxantrone (18.9 versus 16.4 months; P = 0.009). Docetaxel every 3 weeks plus prednisone therapy also resulted in improvement in pain, a decrease in prostate-specific antigen, and improved quality of life compared with mitoxantrone plus prednisone.G3139 (oblimersen sodium) is an antisense oligonucleotide directed against Bcl-2 mRNA that seems to have clinical activity in conjunction with docetaxel (60). Several trials are under way with the polyphenol gossypol and its derivatives inhibit Bcl-2, Bcl-xL, and cyclin D1 (61). An antisense molecule to clusterin, a prosurvival gene that is up-regulated in androgen-independent prostate cancer, is currently in clinical trials (62). 1,25-Dihydroxycholecalciferol (calcitriol) potentiates the activity of chemotherapeutic agents by causing G0-G1 arrest and inducing apoptosis. The combination of calcitriol and docetaxel seems to have increased antitumor activity as compared with docetaxel alone and is the subject of ongoing investigation (63).Although new chemotherapeutic drugs such as satraplatin and the epothilones are in clinical trials in advanced prostate cancer, the most exciting progress is being made in targeting outlaw signal transduction pathways that are deregulated in prostate cancer (58, 64–66). Outlaw pathways often rely on the activation of the tyrosine kinase growth factor receptors. Antibodies and small molecules that target growth factor receptors on prostate cancer cells include those directed against epidermal growth factor receptor (gefitinib, cetuximab, trastuzumab, lapatinib, and erlotinib) and platelet-derived growth factor receptors (imantinib and leflunomide; refs. 41, 67). Other tyrosine receptor pathway inhibitors include BAY43-9006, reolysin, LErafAON, and GEM 231. Many of these pathways can also be targeted in the supporting cells of the tumor. The now classic example of this is the use of agents that target the vascular endothelial growth factor pathway to interrupt the blood supply to the tumor cells (68). A phase III trial of bevacizumab (Avastin) and docetaxel is being done by the Eastern Cooperative Oncology Group. Another such agent is atrasentan, which targets the endothelin-1 axis in osteoblasts (69). This agent, in combination with docetaxel, is the subject of a phase III trial by the SouthWest Oncology Group. As the role of the androgen receptor in androgen-resistant disease has been recognized, it has become a focus of therapeutic development.In focusing on targeting the hypersensitive pathway, one approach has been to try to lower androgen receptor protein levels. Ansamycin antibiotics, such as 17-allylamino-17-demethoxygeldanamycin, have been shown in preclinical models to induce degradation of steroid receptors, including the androgen receptor, by inhibiting heat shock protein 90, a chaperone required for the folding of these proteins, and are currently in phase I trials (70–73). In preclinical studies, Eder et al. (74) were able to inhibit androgen receptor expression in LNCaP tumor cells using antisense androgen receptor oligodeoxynucleotides resulting in decreased growth, decreased production of prostate-specific antigen, and increased apoptosis. Further advances are also being made to reduce testosterone beyond the currently known second-line and third-line hormonal agents; it was recognized that much less ligand is needed for androgen receptor activation in hormone-refractory prostate cancer cells (75).A large number of coactivators and corepressors involved in the regulation of androgen receptor–driven transcription have been identified (43). Because it is known that ligand-dependent and ligand-independent androgen receptor transcriptional activity is dependent on coactivator recruitment, modification of these coregulators or their binding targets may be important therapeutically. Peptide antagonists that target specific nuclear receptor coactivator binding surfaces have been developed in the preclinical setting and have been shown to inhibit androgen receptor transcriptional activity by disrupting androgen receptor-coactivator interactions (76).The signal transduction pathways associated with tumor hypoxia has been the subject of intense scrutiny because they involve the outlaw, bypass, and cofactor pathways (77). CCI-779 and RAD001 target the mammalian target of rapamycin and upstream regulator of hypoxia-inducible factor 1α (78–80). Hypoxia-inducible factor 1α is also regulated through its binding to heat shock protein 90, which prevents its degradation by the proteosome complex (80). Heat shock protein 90 also stabilizes multiple other enzymes that are part of tyrosine kinase signaling cascades, as well as other proteins, such as steroid receptors including the androgen receptor (81). The geldanamycin derivative 17-(allylamino)-17-demethoxygeldanamycin is an antibiotic that inhibits heat shock protein 90 function, leading to proteosome degradation of these multiple signaling targets (77–81).Multitargeted attack of the cancer cell and the tumor microenvironment. While other molecular targets, including the mitotic kinesins, histone deacetylases, the small-molecule inhibitor suberoylanilide hydroxamic acid, and cyclic GMP phosphodiesterases, are being pursued, the combination of multiple agents that simultaneously attack the tumor and its microenvironment are being developed as well as already being used in the clinic (41, 82, 83). Prostate cancer cells in the bone interact with the extracellular matrix, stromal cells, osteoblasts, osteoclasts, and endothelial cells to coordinate a sophisticated series of interactions to promote tumor cell survival and proliferation (41, 84). The Food and Drug Administration–approved bisphosphonate zoledronic acid binds to the hydroxyapatite of damaged bone and inhibits osteoclast function, leading to a decrease in skeletal related events, such as fractures, in patients with hormone refractory prostate cancer (85, 86). Similarly, the radioisotopes samarium and strontium have been Food and Drug Administration approved for patients with bone metastases because they, too, bind to hydroxyapatite and deliver radiation to the entire tumor microenvironment, damaging not only the cancer cells but all of the supporting cells of the microenvironment (87, 88). The benefits of these agents in decreasing the morbidity of metastatic prostate cancer have been clearly delineated. Agents that deliver radiation to all prostate cancer cells, whether they reside in bone or soft tissue sites, are in clinical trials. Prostate-specific membrane antigen is a transmembrane glycoprotein that is highly expressed by prostate cancers and trials using antibodies to prostate-specific membrane antigen conjugated to multiple different radiopharmaceuticals and chemotherapy agents are under way (89).All of these approaches can be complemented by strategies that exploit the immune system. Several vaccine strategies that either increase the antigenicity of prostate cancer cells or sensitize dendritic cells to the presence of prostate cancer cells are in clinical trials. These include APC8015 (Provenge), an investigational therapeutic vaccine that uses autologous antigen-presenting cells loaded with a recombinant fusion protein of prostatic acid phosphatase linked to a molecule that specifically targets a receptor expressed on the surface of human antigen-presenting cells, GVAX, an autologous tumor vaccine in which cells from the patient's tumor are transduced in vitro with an engineered adenovirus containing the GM-CSF gene and poxvirus-TRICOM combinations (90–92).The treatment of hormone-refractory prostate cancer is evolving rapidly. The definition of the multiple mechanisms by which prostate cancer cells can become resistant to castration has led to the identification of several new leads and pathways that are being quickly exploited. Combining this knowledge with a multifaceted attack on the tumor microenvironment that includes therapy involving endothelial cells, osteoblasts, osteoclasts, tumor stroma, and immune modulation should lead to a rapid increase in survival in patients with hormone-refractory prostate cancer in the near future.

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.

To delineate the role of SDF-1 and CXCR4 in metastatic prostate cancer (CaP), positive correlations were established between SDF-1 levels and tumor metastasis. Neutralization of CXCR4 limited the number and the growth of intraosseous metastasis in vivo. Together, these in vivo metastasis data provide critical support that SDF-1/CXCR4 plays a role in skeletal metastasis.Previously we determined that the stromal-derived factor-1 (SDF-1)/CXCR4 chemokine axis is activated in prostate cancer (CaP) metastasis to bone. To delineate the role of SDF-1/CXCR4 in CaP, we evaluated SDF-1 levels in a variety of tissues and whether neutralization of SDF-1 prevented metastasis and/or intraosseous growth of CaPs.SDF-1 levels were established in various mouse tissues by ELISA, immunohistochemistry, and in situ hybridization. To assess the role of SDF-1/CXCR4 in metastasis, bone metastases were established by administering CaP cells into the left cardiac ventricle of nude animals in the presence or absence of neutralizing CXCR4 antibody. The effect of SDF-1 on intraosseous growth of CaP cells was determined using intratibial injections and anti-CXCR4 antibodies and peptides.There was a positive correlation between the levels of SDF-1 and tissues in which metastatic CaP lesions were observed. SDF-1 levels were highest in the pelvis, tibia, femur, liver, and adrenal/kidneys compared with the lungs, tongue, and eye, suggesting a selective effect. SDF-1 staining was generally low or undetectable in the center of the marrow and in the diaphysis. SDF-1 mRNA was localized to the metaphysis of the long bones nearest to the growth plate where intense expression was observed near the endosteal surfaces covered by osteoblastic and lining cells. Antibody to CXCR4 significantly reduced the total metastatic load compared with IgG control-treated animals. Direct intratibial injection of tumor cells followed by neutralizing CXCR4 antibody or a specific peptide that blocks CXCR4 also decreased the size of the tumors compared with controls.These data provide critical support for a role of SDF-1/CXCR4 in skeletal metastasis. Importantly, these data show that SDF-1/CXCR4 participate in localizing tumors to the bone marrow for prostate cancer.

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.

Few intervention studies have been conducted to help couples manage the effects of prostate cancer and maintain their quality of life. The objective of this study was to determine whether a family-based intervention could improve appraisal variables (appraisal of illness or caregiving, uncertainty, hopelessness), coping resources (coping strategies, self-efficacy, communication), symptom distress, and quality of life in men with prostate cancer and their spouses.For this clinical trial, 263 patient-spouse dyads were stratified by research site, phase of illness, and treatment; then, they were randomized to the control group (standard care) or the experimental group (standard care plus a 5-session family intervention). The intervention targeted couples' communication, hope, coping, uncertainty, and symptom management. The final sample consisted of 235 couples: 123 couples in the control group and 112 couples in the experimental group. Data collection occurred at baseline before randomization and at 4 months, 8 months, and 12 months.At 4-month follow-up, intervention patients reported less uncertainty and better communication with spouses than control patients, but they reported no other effects. Intervention spouses reported higher quality of life, more self-efficacy, better communication, and less negative appraisal of caregiving, uncertainty, hopelessness, and symptom distress at 4 months compared with controls, and some effects were sustained to 8 months and 12 months.Men with prostate cancer and their spouses reported positive outcomes from a family intervention that offered them information and support. Programs of care need to be extended to spouses who likely will experience multiple benefits from intervention.

The identification of novel tumor-interactive chemokines and the associated insights into the molecular and cellular basis of tumor-microenvironment interactions have continued to stimulate the development of targeted cancer therapeutics. Recently, we have identified monocyte chemoattractant protein 1 (MCP-1; CCL2) as a prominent regulator of prostate cancer growth and metastasis. Using neutralizing antibodies to human CCL2 (CNTO888) and the mouse homologue CCL2/JE (C1142), we show that treatment with anti-CCL2/JE antibody (2 mg/kg, twice weekly i.p.) attenuated PC-3Luc-mediated overall tumor burden in our in vivo model of prostate cancer metastasis by 96% at 5 weeks postintracardiac injection. Anti-CCL2 inhibition was not as effective as docetaxel (40 mg/kg, every week for 3 weeks) as a single agent, but inhibition of CCL2 in combination with docetaxel significantly reduced overall tumor burden compared with docetaxel alone, and induced tumor regression relative to initial tumor burden. These data suggest an interaction between tumor-derived chemokines and host-derived chemokines acting in cooperation to promote tumor cell survival, proliferation, and metastasis.

Novel recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS family members ERG, ETV1, or ETV4 have been recently identified as a common molecular event in prostate cancer development. We comprehensively analyzed the frequency and risk of disease progression for the TMPRSS2 and ETS family genes rearrangements in a cohort of 96 American men surgically treated for clinically localized prostate cancer. Using three break apart (TMPRSS2, ERG, ETV4) and one fusion (TMPRSS:ETV1) fluorescence in situ hybridization (FISH) assays, we identified rearrangements in TMPRSS2, ERG, ETV1, and ETV4 in 65, 55, 2, and 2% of cases, respectively. Overall, 54 and 2% of cases demonstrated TMPRSS2:ERG and TMPRSS2:ETV1 fusions, respectively. As intronic loss of genomic DNA between TMPRSS2 and ERG has been identified as a mechanism of TMPRSS2:ERG fusion, our assays allowed us to detect deletion of the 3′ end of TMPRSS2 and the 5′ end of ERG in 41 and 39% of cases rearranged for respective genes. Prostate cancers demonstrating TMPRSS2 gene rearrangement were associated with high pathologic stage (P=0.04). Our results confirm that recurrent chromosomal aberrations in TMPRSS2 and/or ETS family members are found in about 70% of prostate cancers. Importantly, we define a novel approach to study these gene fusions and identified cases where TMPRSS2 was rearranged without rearrangement of ERG, ETV1 or ETV4 and cases with ETS family gene rearrangement without TMPRSS2 rearrangement, suggesting that novel 5′ and 3′ partners may be involved in gene fusions in prostate cancer.

The niche microenvironment in which cancer cells reside plays a prominent role in the growth of cancer. It is therefore imperative to mimic the in vivo tumor niche in vitro to better understand cancer and enhance development of therapeutics. Here, we engineer a 3D metastatic prostate cancer model that includes the types of surrounding cells in the bone microenvironment that the metastatic prostate cancer cells reside in. Specifically, we used a two-layer microfluidic system to culture 3D multi-cell type spheroids of fluorescently labeled metastatic prostate cancer cells (PC-3 cell line), osteoblasts and endothelial cells. This method ensures uniform incorporation of all co-culture cell types into each spheroid and keeps the spheroids stationary for easy tracking of individual spheroids and the PC-3's residing inside them over the course of at least a week. This culture system greatly decreased the proliferation rate of PC-3 cells without reducing viability and may more faithfully recapitulate the in vivo growth behavior of malignant cancer cells within the bone metastatic prostate cancer microenvironment.

Our recent studies have shown that annexin II, expressed on the cell surface of osteoblasts, plays an important role in the adhesion of hematopoietic stem cells (HSCs) to the endosteal niche. Similarly, prostate cancer (PCa) cells express the annexin II receptor and seem to use the stem cell niche for homing to the bone marrow. The role of the niche is thought to be the induction and sustenance of HSC dormancy. If metastatic PCa cells occupy a similar or the same ecological niche as HSCs, then it is likely that the initial role of the HSC niche will be to induce dormancy in metastatic cells. In this study, we demonstrate that the binding of PCa to annexin II induces the expression of the growth arrest-specific 6 (GAS6) receptors AXL, Sky, and Mer, which, in the hematopoietic system, induce dormancy. In addition, GAS6 produced by osteoblasts prevents PCa proliferation and protects PCa from chemotherapy-induced apoptosis. Our results suggest that the activation of GAS6 receptors on PCa in the bone marrow environment may play a critical role as a molecular switch, establishing metastatic tumor cell dormancy.

Abstract Recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS transcription factor family members ERG, ETV1, and ETV4 have been identified as a critical event in prostate cancer development. In this study, we characterized the prevalence and diversity of these rearrangements in hormone-refractory metastatic prostate cancer. We used a fluorescence in situ hybridization (FISH) split probe strategy to comprehensively evaluate TMPRSS2-ETS aberrations across 97 nonosseous metastatic sites of prostate cancer from 30 rapid autopsies of men who died of androgen-independent disease. Tissue microarrays were constructed representing multiple metastatic sites from each patient, and split signal FISH probes for TMPRSS2, ERG, ETV1, and ETV4 were used to assess for TMPRSS2-ETS rearrangements. In patients exhibiting these aberrations, multiple sites from an individual case harbored the same gene fusion molecular subtype suggesting clonal expansion of disease. The most common prostate cancer gene fusion, TMPRSS2-ERG, can be generated by the mechanism of interstitial deletion (Edel) about 39% to 60% of the time in clinically localized disease. Interestingly, we observed that all of the androgen-independent metastatic prostate cancer sites harboring TMPRSS2-ERG were associated with Edel. These findings suggest that TMPRSS2-ERG with Edel is an aggressive and, in this study, uniformly lethal molecular subtype of prostate cancer associated with androgen-independent disease. [Cancer Res 2008;68(10):3584–90]

CCL2 is a chemokine known to recruit monocytes and macrophages to sites of inflammation. A growing body of research suggests CCL2 is progressively overexpressed in tumor beds and may play a role in the clinical progression of solid tumors. Cancer cells derived from several solid tumor types demonstrate functional receptors for CCL2, suggesting this chemokine may achieve tumorigenicity through direct effects on malignant cells; however, a variety of normal host cells that co-exist with cancer in the tumor microenvironment also respond to CCL2. These cells include macrophages, osteoclasts, endothelial cells, T-lymphocytes, and myeloid-derived immune suppressor cells (MDSCs). CCL2 mediated interactions between normal and malignant cells in the tumor microenvironment and plays a multi-faceted role in tumor progression.

Cell cooperation promotes many of the hallmarks of cancer via the secretion of diffusible factors that can affect cancer cells or stromal cells in the tumour microenvironment. This cooperation cannot be explained simply as the collective action of cells for the benefit of the tumour because non-cooperative subclones can constantly invade and free-ride on the diffusible factors produced by the cooperative cells. A full understanding of cooperation among the cells of a tumour requires methods and concepts from evolutionary game theory, which has been used successfully in other areas of biology to understand similar problems but has been underutilized in cancer research. Game theory can provide insights into the stability of cooperation among cells in a tumour and into the design of potentially evolution-proof therapies that disrupt this cooperation.

Prostate cancer (PCa) metastasizes selectively to bone through unknown mechanisms. In the current study, we identified exosome-mediated transfer of pyruvate kinase M2 (PKM2) from PCa cells into bone marrow stromal cells (BMSCs) as a novel mechanism through which primary tumor-derived exosomes promote premetastatic niche formation. We found that PKM2 up-regulates BMSC CXCL12 production in a HIF-1α-dependent fashion, which subsequently enhances PCa seeding and growth in the bone marrow. Furthermore, serum-derived exosomes from patients with either primary PCa or PCa metastasis, as opposed to healthy men, reveal that increased exosome PKM2 expression is associated with metastasis, suggesting clinical relevance of exosome PKM2 in PCa. Targeting the exosome-induced CXCL12 axis diminished exosome-mediated bone metastasis. In summary, primary PCa cells educate the bone marrow to create a premetastatic niche through primary PCa exosome-mediated transfer of PKM2 into BMSCs and subsequent up-regulation of CXCL12. This novel mechanism indicates the potential for exosome PKM2 as a biomarker and suggests therapeutic targets for PCa bone metastasis.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.The initial STOMP and ORIOLE trial reports suggested that metastasis-directed therapy (MDT) in oligometastatic castration-sensitive prostate cancer (omCSPC) was associated with improved treatment outcomes. Here, we present long-term outcomes of MDT in omCSPC by pooling STOMP and ORIOLE and assess the ability of a high-risk mutational signature to risk stratify outcomes after MDT. The primary end point was progression-free survival (PFS) calculated using the Kaplan-Meier method. High-risk mutations were defined as pathogenic somatic mutations within ATM, BRCA1/2, Rb1, or TP53. The median follow-up for the whole group was 52.5 months. Median PFS was prolonged with MDT compared with observation (pooled hazard ratio [HR], 0.44; 95% CI, 0.29 to 0.66; P value < .001), with the largest benefit of MDT in patients with a high-risk mutation (HR high-risk, 0.05; HR no high-risk, 0.42; P value for interaction: .12). Within the MDT cohort, the PFS was 13.4 months in those without a high-risk mutation, compared with 7.5 months in those with a high-risk mutation (HR, 0.53; 95% CI, 0.25 to 1.11; P = .09). Long-term outcomes from the only two randomized trials in omCSPC suggest a sustained clinical benefit to MDT over observation. A high-risk mutational signature may help risk stratify treatment outcomes after MDT.

High-density tissue microarrays (TMA) are useful for profiling protein expression in a large number of samples but their use for clinical biomarker studies may be limited in heterogeneous tumors like prostate cancer. In this study, the optimization and validation of a tumor sampling strategy for a prostate cancer outcomes TMA is performed. Prostate cancer proliferation determined by Ki-67 immunohistochemistry was tested. Ten replicate measurements of proliferation using digital image analysis (CAS200, Bacus Labs, Lombard, IL, USA) were made on 10 regions of prostate cancer from a standard glass slide. Five matching tissue microarray sample cores (0.6 mm diameter) were sampled from each of the 10 regions in the parallel study. A bootstrap resampling analysis was used to statistically simulate all possible permutations of TMA sample number per region or sample. Statistical analysis compared TMA samples with Ki-67 expression in standard pathology immunohistochemistry slides. The optimal sampling for TMA cores was reached at 3 as fewer TMA samples significantly increased Ki-67 variability and a larger number did not significantly improve accuracy. To validate these results, a prostate cancer outcomes tissue microarray containing 10 replicate tumor samples from 88 cases was constructed. Similar to the initial study, 1 to 10 randomly selected cores were used to evaluate the Ki-67 expression for each case, computing the 90th percentile of the expression from all samples used in each model. Using this value, a Cox proportional hazards analysis was performed to determine predictors of time until prostate-specific antigen (PSA) recurrence after radical prostatectomy for clinically localized prostate cancer. Examination of multiple models demonstrated that 4 cores was optimal. Using a model with 4 cores, a Cox regression model demonstrated that Ki-67 expression, preoperative PSA, and surgical margin status predicted time to PSA recurrence with hazard ratios of 1.49 (95% confidence interval [CI] 1.01-2.20, p = 0.047), 2.36 (95% CI 1.15-4.85, p = 0.020), and 9.04 (95% CI 2.42-33.81, p = 0.001), respectively. Models with 3 cores to determine Ki-67 expression were also found to predict outcome. In summary, 3 cores were required to optimally represent Ki-67 expression with respect to the standard tumor slide. Three to 4 cores gave the optimal predictive value in a prostate cancer outcomes array. Sampling strategies with fewer than 3 cores may not accurately represent tumor protein expression. Conversely, more than 4 cores will not add significant information. This prostate cancer outcomes array should be useful in evaluating other putative prostate cancer biomarkers.

Metastases from primary tumors are responsible for most cancer deaths. It has been shown that circulating tumor cells (CTCs) can be detected in the peripheral blood of patients with a variety of metastatic cancers and that the presence of these cells is associated with poor clinical outcomes. Characterization of CTCs in metastatic cancer patients could provide additional information to augment management of the disease. Here, we describe a novel approach for the identification of molecular markers to detect and characterize CTCs in peripheral blood. Using an integrated platform to immunomagnetically isolate and immunofluorescently detect CTCs, we obtained blood containing > or = 100 CTCs from one metastatic colorectal, one metastatic prostate, and one metastatic breast cancer patient. Using the RNA extracted from the CTC-enriched portion of the sample and comparing it with the RNA extracted from the corresponding CTC-depleted portion, for the first time, global gene expression profiles from CTCs were generated and a list of cancer-specific, CTC-specific genes was obtained. Subsequently, samples immunomagnetically enriched for CTCs from 74 metastatic cancer patients and 50 normal donors were used to confirm by quantitative real-time reverse transcription-PCR CTC-specific expression of selected genes and to show that gene expression profiles for CTCs may be used to distinguish normal donors from advanced cancer patients as well as to differentiate among the three different metastatic cancers. Genes such as AGR2, S100A14, S100A16, FABP1, and others were found useful for detection of CTCs in peripheral blood of advanced cancer patients.

Prostate cancer is the most common cancer diagnosed in U.S. men and remains incurable once it has metastasized. Many stages of the metastatic cascade involve cellular interactions mediated by cell surface components, such as carbohydrate-binding proteins, including galactoside-binding lectins (galectins). Modified citrus pectin (pH-modified), a soluble component of plant fiber derived from citrus fruit, has been shown to interfere with cell-cell interactions mediated by cell surface carbohydrate-binding galectin-3 molecules.The aim of this study was to determine whether modified citrus pectin, a complex polysaccharide rich in galactosyl residues, could inhibit spontaneous metastasis of prostate adenocarcinoma cells in the rat.The ability of modified citrus pectin to inhibit the adhesion of Dunning rat prostate cancer MAT-LyLu cells to rat endothelial cells was measured by 51Cr-labeling. Modified citrus pectin inhibition of MAT-LyLu cell anchorage-independent growth was measured by colony formation in agarose. The presence of galectin-3 in rat MAT-LyLu cells and human prostate carcinoma was demonstrated by immunoblotting and immunohistochemistry. One million MAT-LyLu cells were injected subcutaneously into the hind limb of male Copenhagen rats on day 0. Rats were given 0.0%, 0.01%, 0.1%, or 1.0% (wt/vol) modified citrus pectin continuously in their drinking water (from day 4 until necropsy on day 30). The number of MAT-LyLu tumor colonies in the lungs were counted.Compared with 15 or 16 control rats that had lung metastases on day 30, seven of 14 rats in the 0.1% and nine of 16 rats in the 1.0% modified citrus-pectin group had statistically significant (two-sided; P < .03 and P < .001, respectively) reductions in lung metastases. The lungs of the 1.0% modified citrus pectin-treated rats had significantly (two-sided; P < .05) fewer metastatic colonies than control groups (9 colonies +/- 4 [mean +/- SE] in the control group compared with 1 colony +/- 1 in the treated group). Modified citrus pectin had no effect on the growth of the primary tumors. In vitro, modified citrus pectin inhibited MAT-LyLu cell adhesion to rat endothelial cells in a time- and dose-dependent manner as well as their colony formation in semisolid medium.We present a novel therapy in which oral intake of modified citrus pectin acts as a potent inhibitor of spontaneous prostate carcinoma metastasis in the Copenhagen rat.Further investigations are warranted to determine the following: 1) the role of galectin-3 in normal and cancerous prostate tissues and 2) the ability of modified citrus pectin to inhibit human prostate metastasis in nude mice.

Tumor cells in the bone interact with the microenvironment to promote tumor cell survival and proliferation, resulting in a lethal phenotype for patients with advanced prostate cancer. Monocyte chemoattractant protein 1 (CCL2) is a member of the CC chemokine family and is known to promote monocyte chemotaxis to sites of inflammation. Here we have shown that human bone marrow endothelial (HBME) cells secrete significantly higher levels of CCL2 compared to human aortic endothelial cells and human dermal microvascular endothelial cells. Furthermore, we demonstrate that CCL2 is a potent chemoattractant of prostate cancer epithelial cells, that stimulation of PC-3 and VCaP cells resulted in a dose-dependent activation of PI3 kinase/Akt signaling pathway. Activation of the PI3 kinase/Akt pathway was found to be vital to the proliferative effects of CCL2 stimulation of both PC-3 and VCaP cells. Additionally, CCL2 stimulated the phosphorylation of p70-S6 kinase (a downstream target of Akt) and induced actin rearrangement, resulting in a dynamic morphologic change indicative of microspike formation. These data suggest that bone marrow endothelial cells are a major source of CCL2, that an elevated secretion of CCL2 recruits prostate cancer epithelial cells to the bone microenvironment and regulates their proliferation rate.

The ability of CCL2 to influence prostate cancer tumorigenesis and metastasis may occur through two distinct mechanisms: 1) a direct effect on tumor cell growth and function, and 2) an indirect effect on the tumor microenvironment by the regulation of macrophage mobilization and infiltration into the tumor bed. We have previously demonstrated that CCL2 exerts a direct effect on prostate cancer epithelial cells by the regulation of their growth, invasion, and migration, resulting in enhanced tumorigenesis and metastasis. Here we describe an indirect effect of CCL2 on prostate cancer growth and metastasis by regulating monocyte/macrophage infiltration into the tumor microenvironment and by stimulating a phenotypic change within these immune cells to promote tumor growth (tumor-associated macrophages). VCaP prostate cancer cells were subcutaneously injected in male SCID mice and monitored for tumor volume, CD68+ macrophage infiltration, and microvascular density. Systemic administration of antiCCL2 neutralizing antibodies (CNT0888 and C1142) significantly retarded tumor growth and attenuated CD68` macrophage infiltration, which was accompanied by a significant decrease in microvascular density. These data suggest that CCL2 contributes to prostate cancer growth through the regulation of macrophage infiltration and enhanced angiogenesis within the tumor.

One of the most life-threatening complications of prostate cancer is skeletal metastasis. In order to develop treatment for metastasis, it is important to understand its molecular mechanisms. Our work in this field has drawn parallels between hematopoietic stem cell and prostate cancer homing to the marrow. Our recent work demonstrated that annexin II expressed by osteoblasts and endothelial cells plays a critical role in niche selection. In this study, we demonstrate that annexin II and its receptor play a crucial role in establishing metastasis of prostate cancer. Prostate cancer cell lines migrate toward annexin II and the adhesion of prostate cancer to osteoblasts and endothelial cells was inhibited by annexin II. By blocking annexin II or its receptor in animal models, short-term and long-term localization of prostate cancers are limited. Annexin II may also facilitate the growth of prostate cancer in vitro and in vivo by the MAPK pathway. These data strongly suggest that annexin II and its receptor axis plays a central role in prostate cancer metastasis, and that prostate cancer utilize the hematopoietic stem cell homing mechanisms to gain access to the niche.

Purpose Despite the high prevalence of prostate cancer, little information is available on the quality of life of men and their spouses during the phases of illness. This study assessed patients' and spouses' quality of life, appraisal of illness, resources, symptoms, and risk for distress across three phases of prostate cancer: newly diagnosed, biochemical recurrence, and advanced. Patients and Methods The sample consisted of 263 patient/spouse dyads. A stress-appraisal conceptual model guided the selection of variables which were then assessed with established instruments. Study variables were examined for phase effects (differences in dyads across three phases), role effects (patients v spouses), and phase-by-role interactions (differences within dyads across phases) using analysis of variance (ANOVA). Results More phase effects than role effects were found, indicating that the psychosocial experiences of patients and their spouses were similar, but differed from dyads in other phases. Dyads in the advanced phase were at highest risk for distress. These patients had the lowest physical quality of life, and their spouses had the lowest emotional quality of life of all participants. Dyads in the biochemical recurrence and advanced phases had more negative appraisals of illness and caregiving, greater uncertainty, and more hopelessness compared with dyads in the newly diagnosed phase. Spouses, in contrast to patients, had less confidence in their ability to manage the illness and perceived less support across all phases of illness. Conclusion Phase-specific programs of care are needed to assist both men with prostate cancer and their spouses to manage the effects of illness.

Prostate cancer recurrence, evidenced by rising prostate-specific antigen (PSA) levels after radical prostatectomy, is an increasingly prevalent clinical problem in need of new treatment options. Preclinical studies have suggested that for tumors in general, settings of minimal cancer volume may be uniquely suitable for recombinant vaccine therapy targeting tumor-associated antigens. A clinical study was undertaken to evaluate the safety and biologic effects of vaccinia-PSA (PROSTVAC) administered to subjects with postprostatectomy recurrence of prostate cancer and to assess the feasibility of interrupted androgen deprivation as a tool for modulating expression of the vaccine target antigen, as well as detecting vaccine bioactivity in vivo.A limited Phase I clinical trial was conducted to evaluate the safety and biologic effects of vaccinia-PSA administered in 6 patients with androgen-modulated recurrence of prostate cancer after radical prostatectomy. End points included toxicity, serum PSA rise related to serum testosterone restoration, and immunologic effects measured by Western blot analysis for anti-PSA antibody induction.Toxicity was minimal, and dose-limiting toxicity was not observed. Noteworthy variability in time required for testosterone restoration (after interruption of androgen deprivation therapy) was observed. One subject showed continued undetectable serum PSA (less than 0.2 ng/mL) for over 8 months after testosterone restoration, an interval longer than those reported in previous androgen deprivation interruption studies. Primary anti-PSA IgG antibody activity was induced after vaccinia-PSA immunization in 1 subject, although such antibodies were detectable in several subjects at baseline.Interrupted androgen deprivation may be a useful tool for modulating prostate cancer bioactivity in clinical trials developing novel biologic therapies. Immune responses against PSA may be present among some patients with prostate cancer at baseline and may be induced in others through vaccinia-PSA immunization.

CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein-1) has been demonstrated to recruit monocytes to tumor sites. Monocytes are capable of being differentiated into tumor-associated macrophages (TAMs) and osteoclasts (OCs). TAMs have been shown to promote tumor growth in several cancer types. Osteoclasts have also been known to play an important role in cancer bone metastasis. To investigate the effects of CCL2 on tumorigenesis and its potential effects on bone metastasis of human prostate cancer, CCL2 was overexpressed into a luciferase-tagged human prostate cancer cell line PC-3. In vitro, the conditioned medium of CCL2 overexpressing PC-3luc cells (PC-3(lucCCL2)) was a potent chemoattractant for mouse monocytes in comparison to a conditioned medium from PC-3(lucMock). In addition, CCL2 overexpression increased the growth of transplanted xenografts and increased the accumulation of macrophages in vivo. In a tumor dissemination model, PC-3(lucCCL2) enhanced the growth of bone metastasis, which was associated with more functional OCs. Neutralizing antibodies targeting both human and mouse CCL2 inhibited the growth of PC-3(luc), which was accompanied by a decrease in macrophage recruitment to the tumor. These findings suggest that CCL2 increases tumor growth and bone metastasis through recruitment of macrophages and OCs to the tumor site.

Prostate cancer continues to be the most common nonskin cancer diagnosed and the second leading cause of cancer death in men in the United States. Prostate cancer that has metastasized to bone remains incurable. The interactions between prostate cancer cells and the various cells of the host microenvironment result in enhanced growth of tumor cells and activation of host cells that together culminate in osteoblastic bone metastases. These dynamic tumor-host interactions are mediated by cancer and host-produced cytokines and chemokines. Among them, chemokine (C-C motif) ligand 2 (CCL2) has been identified as a prominent modulator of metastatic growth in the bone microenvironment. CCL2 is produced by bone marrow osteoblasts, endothelial cells, stromal cells, and prostate cancer cells. It has been demonstrated to modulate tumor-associated macrophage migration and promote osteoclast maturation. In addition, CCL2 functions through binding to its receptor CCR2 to induce prostate cell proliferation, migration, and invasion in both autocrine and paracrine manners. CCL2 protects prostate cancer cells from autophagic death by activating survivin through a PI3K/AKT (phosphatidylinositol 3-kinase/protein kinase B)-dependent mechanism. Inhibition of CCL2 substantially decreases macrophage infiltration, decreases osteoclast function, and inhibits prostate cancer growth in bone in preclinical animal models. The multiple roles of CCL2 in the tumor microenvironment make it an attractive therapeutic target in metastatic prostate cancer as well as in other cancers.

Abstract Mutations in the androgen receptor (AR) that enable activation by antiandrogens occur in hormone-refractory prostate cancer, suggesting that mutant ARs are selected by treatment. To validate this hypothesis, we compared AR variants in metastases obtained by rapid autopsy of patients treated with flutamide or bicalutamide, or by excision of lymph node metastases from hormone-naïve patients. AR mutations occurred at low levels in all specimens, reflecting genetic heterogeneity of prostate cancer. Base changes recurring in multiple samples or multiple times per sample were considered putative selected mutations. Of 26 recurring missense mutations, most in the NH2-terminal domain (NTD) occurred in multiple tumors, whereas those in the ligand binding domain (LBD) were case specific. Hormone-naïve tumors had few recurring mutations and none in the LBD. Several AR variants were assessed for mechanisms that might underlie treatment resistance. Selection was evident for the promiscuous receptor AR-V716M, which dominated three metastases from one flutamide-treated patient. For the inactive cytoplasmically restricted splice variant AR23, coexpression with AR enhanced ligand response, supporting a decoy function. A novel NTD mutation, W435L, in a motif involved in intramolecular interaction influenced promoter-selective, cell-dependent transactivation. AR-E255K, mutated in a domain that interacts with an E3 ubiquitin ligase, led to increased protein stability and nuclear localization in the absence of ligand. Thus, treatment with antiandrogens selects for gain-of-function AR mutations with altered stability, promoter preference, or ligand specificity. These processes reveal multiple targets for effective therapies regardless of AR mutation. [Cancer Res 2009;69(10):4434–42]

Purpose To define methodology to show clinical benefit for patients in the state of a rising prostate-specific antigen (PSA). Results Hypothesis. A clinical states framework was used to address the hypothesis that definitive phase III trials could not be conducted in this patient population. Patient Population. The Group focused on men with systemic (nonlocalized) recurrence and a defined risk of developing clinically detectable metastases. Models to define systemic versus local recurrence, and risk of metastatic progression were discussed. Intervention. Therapies that have shown favorable effects in more advanced clinical states; meaningful biologic surrogates of activity linked with efficacy in other tumor types; and/or effects on a target or pathway known to contribute to prostate cancer progression in this state can be considered for evaluation. Outcomes. An intervention-specific posttherapy PSA-based outcome definition that would justify further testing should be described at the outset. Reporting. Trial reports should include a table showing the number of patients who achieve a specific PSA-based outcome, the number who remain enrolled onto the trial, and the number who came off study at different time points. The term PSA response should be abandoned. Trial Design. The phases of drug development for this state are optimizing dose and schedule, demonstration of a treatment effect, and clinical benefit. To move a drug forward should require a high bar that includes no rise in PSA in a defined proportion of patients for a specified period of time at a minimum. Agents that do not produce this effect can only be tested in combination. The preferred end point of clinical benefit is prostate cancer–specific survival; the time to development of metastatic disease is an alternative. Conclusion Methodology to show that an intervention alters the natural history of prostate cancer is described. At each stage of development, only agents with sufficient activity should be moved forward.

In post-fetal life, hematopoiesis occurs in unique microenvironments or ‘niches’ in the marrow. Niches facilitate the maintenance of hematopoietic stem cells (HSCs) as unipotent, while supporting lineage commitment of the expanding blood populations. As the physical locale that regulates HSC function, the niche function is vitally important to the survival of the organism. This places considerable selective pressure on HSCs, as only those that are able to engage the niche in the appropriate context are likely to be maintained as stem cells. Since niches are central regulators of stem cell function, it is not surprising that molecular parasites like neoplasms are likely to seek out opportunities to harvest resources from the niche environment. As such, the niche may unwittingly participate in tumorigenesis as a leukemic or neoplastic niche. The niche may also promote metastasis or chemo-resistance of hematogenous neoplasms or solid tumors. This review focuses on what is known about the physical structures of the niche, how the niche participates in hematopoiesis and neoplastic growth and what molecules are involved. Further understanding of the interactions between stem cells and the niche may be useful for developing therapeutic strategies.

Bone metastasis is a common untreatable complication associated with prostate cancer. Metastatic cells seed in skeletal sites under active turnover containing dense marrow cellularity. We hypothesized that differences in these skeletal-specific processes are among the critical factors that facilitate the preferential localization of metastatic prostate cancer in bone. To test this, athymic mice were administered PTH to induce bone turnover and increase marrow cellularity daily 1 wk before and after intracardiac inoculation of luciferase-tagged PC-3 cells. Tumor localization was monitored by bioluminescence imaging weekly for 5 wk. At the time of tumor inoculation, PTH-treated mice demonstrated significant increases in serum levels of bone turnover markers such as osteocalcin and tartrate-resistant acid phosphatase 5b and in the number of tartrate-resistant acid phosphatase-positive osteoclasts per millimeter of bone when compared with the other groups. Likewise, PTH treatment stimulated a qualitative increase in marrow cellular proliferation as determined by 5-bromo-2'-deoxyuridine immunostaining. Skeletal metastases formed in the hind limb and craniofacial regions of young mice with no difference between groups. In adult mice, however, bioluminescent signals in the hind limb and craniofacial regions were 3-fold higher in PTH-treated mice vs. controls. Fluorochrome labeling revealed increased bone formation activity in trabecular bone adjacent to tumors. When zoledronic acid, a nitrogen-containing bisphosphonate that inhibits osteoclast-mediated bone resorption, was administered concurrently with PTH, a significant reduction in the incidence of bone tumors was observed. Overall, these studies provide new evidence that skeletal sites rich in marrow cellularity under active turnover offer a more congenial microenvironment to facilitate cancer localization in the skeleton.

The majority of men with progressive prostate cancer develop metastases with the skeleton being the most prevalent metastatic site. Unlike many other tumors that metastasize to bone and form osteolytic lesions, prostate carcinomas form osteoblastic lesions. However, histological evaluation of these lesions reveals the presence of underlying osteoclastic activity. These lesions are painful, resulting in diminished quality of life of the patient. There is emerging evidence that prostate carcinomas establish and thrive in the skeleton due to cross-talk between the bone microenvironment and tumor cells. Bone provides chemotactic factors, adhesion factors, and growth factors that allow the prostate carcinoma cells to target and proliferate in the skeleton. The prostate carcinoma cells reciprocate through production of osteoblastic and osteolytic factors that modulate bone remodeling. The prostate carcinoma-induced osteolysis promotes release of the many growth factors within the bone extracellular matrix thus further enhancing the progression of the metastases. This review focuses on the interaction between the bone and the prostate carcinoma cells that allow for development and progression of prostate carcinoma skeletal metastases.

Prostate cancer is the most commonly diagnosed non-cutaneous neoplasm among American males and is the second leading cause of cancer-related death. Prostate specific antigen screening has resulted in earlier disease detection, yet approximately 30% of men will die of metastatic disease. Slow disease progression, an aging population and associated morbidity and mortality underscore the need for improved disease classification and therapies. To address these issues, we analyzed a cohort of patients using array comparative genomic hybridization (aCGH). The cohort comprises 64 patients, half of whom recurred postoperatively. Analysis of the aCGH profiles revealed numerous recurrent genomic copy number aberrations. Specific loss at 8p23.2 was associated with advanced stage disease, and gain at 11q13.1 was found to be predictive of postoperative recurrence independent of stage and grade. Moreover, comparison with an independent set of metastases revealed approximately 40 candidate markers associated with metastatic potential. Copy number aberrations at these loci may define metastatic genotypes.

To test the use of 1 mg/day of oral diethylstilbesterol (DES) as a treatment for patients with advanced prostate cancer who had failed primary hormonal therapy. Approximately 40,000 men this year will experience first-line hormonal therapy failure for their metastatic prostate cancer. At this time there is no standard therapy for men whose first-line hormonal manipulation has failed. This clinical problem has been exacerbated by the use of prostate-specific antigen (PSA) as a proved biomarker to follow disease progression. Patients who are experiencing hormonal therapy failure now present with a rising PSA, and virtually all are asymptomatic. The dilemma of how to treat these patients represents a new clinical problem for the medical oncologist and urologist that needs to be answered.We conducted a Phase II trial of oral DES in 21 patients. Patients were followed for response by PSA criteria and toxicity. A decrease in two serial measurements of PSA of greater than 50% from baseline was judged to be a partial response.Nine of 21 patients achieved a PSA response (43% response rate with 95% confidence intervals of 22% to 64%) leading to early cessation of this Phase II trial. Eight of 13 patients (62%) who had only one prior hormone manipulation that failed demonstrated a PSA response, whereas only 1 of 8 patients (13%) who had received two or more hormone treatments responded (P = 0.07). The median follow-up is 82 weeks (range 8 to 122) among 16 surviving patients. The survival rate at 2 years is 63% (95% confidence interval 41% to 99%).DES appears to be an active agent for second-line hormone therapy for metastatic prostate cancer. Because it has been taken off the market for economic reasons, DES should be considered for development under the orphan drug strategy.

Abstract Antiapoptotic members of the Bcl-2 family proteins are overexpressed in prostate cancer and are promising molecular targets for modulating chemoresistance of prostate cancer. (-)-Gossypol, a natural BH3 mimetic, is a small-molecule inhibitor of Bcl-2/Bcl-xL/Mcl-1 currently in phase II clinical trials as an adjuvant therapy for human prostate cancer. Our objective is to examine the chemosensitization potential of (-)-gossypol in prostate cancer and its molecular mechanisms of action. (-)-Gossypol inhibited cell growth and induced apoptosis through mitochondria pathway in human prostate cancer PC-3 cells and synergistically enhanced the antitumor activity of docetaxel both in vitro and in vivo in PC-3 xenograft model in nude mouse. (-)-Gossypol blocked the interactions of Bcl-xL with Bax or Bad in cancer cells by fluorescence resonance energy transfer assay and overcame the Bcl-xL protection of FL5.12 model cells on interleukin-3 withdrawal. Western blot and real-time PCR studies showed that a dose-dependent increase of the proapoptotic BH3-only proteins Noxa and Puma contributed to the cell death induced by (-)-gossypol and to the synergistic effects of (-)-gossypol and docetaxel. The small interfering RNA knockdown studies showed that Noxa and Puma are required in the (-)-gossypol-induced cell death. Taken together, these data suggest that (-)-gossypol exerts its antitumor activity through inhibition of the antiapoptotic protein Bcl-xL accompanied by an increase of proapoptotic Noxa and Puma. (-)-Gossypol significantly enhances the antitumor activity of chemotherapy in vitro and in vivo, representing a promising new regime for the treatment of human hormone-refractory prostate cancer with Bcl-2/Bcl-xL/Mcl-1 overexpression. [Mol Cancer Ther 2008;7(7):2192–202]

In this report, we challenge a common perception that tumor embolism is a size-limited event of mechanical arrest, occurring in the first capillary bed encountered by blood-borne metastatic cells. We tested the hypothesis that mechanical entrapment alone, in the absence of tumor cell adhesion to blood vessel walls, is not sufficient for metastatic cell arrest in target organ microvasculature. The in vivo metastatic deposit formation assay was used to assess the number and location of fluorescently labeled tumor cells lodged in selected organs and tissues following intravenous inoculation. We report that a significant fraction of breast and prostate cancer cells escapes arrest in a lung capillary bed and lodges successfully in other organs and tissues. Monoclonal antibodies and carbohydrate-based compounds (anti-Thomsen-Friedenreich antigen antibody, anti-galectin-3 antibody, modified citrus pectin, and lactulosyl-l-leucine), targeting specifically beta-galactoside-mediated tumor-endothelial cell adhesive interactions, inhibited by >90% the in vivo formation of breast and prostate carcinoma metastatic deposits in mouse lung and bones. Our results indicate that metastatic cell arrest in target organ microvessels is not a consequence of mechanical trapping, but is supported predominantly by intercellular adhesive interactions mediated by cancer-associated Thomsen-Friedenreich glycoantigen and beta-galactoside-binding lectin galectin-3. Efficient blocking of beta-galactoside-mediated adhesion precludes malignant cell lodging in target organs.

Abstract BACKGROUND Stromal cell‐derived factor‐1 (SDF‐1 or CXCL12) and CXCR4 are key elements in the metastasis of prostate cancer cells to bone—but the mechanisms as to how it localizes to the marrow remains unclear. METHODS Prostate cancer cell lines were stimulated with SDF‐1 and evaluated for alterations in the expression of adhesion molecules using microarrays, FACs, and Western blotting to identify α v β 3 receptors. Cell–cell adhesion and invasion assays were used to verify that activation of the receptor is responsive to SDF‐1. RESULTS We demonstrate that SDF‐1 transiently regulates the number and affinity of α v β 3 receptors by prostate cancer cells to enhance their metastatic behavior by increasing adhesiveness and invasiveness. SDF‐1 transiently increased the expression of β 3 receptor subunit and increased its phosphorylation in metastatic but not nonmetastatic cells. CONCLUSIONS The transition from a locally invasive phenotype to a metastatic phenotype may be primed by the elevated expression of α v β 3 receptors. Activation and increased expression of α v β 3 within SDF‐1‐rich organs may participate in metastatic localization. Prostate 67:61–73, 2007. © 2006 Wiley‐Liss, Inc.

Prostate cancer continues to be the most common lethal malignancy diagnosed in American men and the second leading cause of male cancer mortality. Over 60 years ago, Huggins and Hodges discovered androgen deprivation as a first-line therapy for metastatic prostate cancer, which leads to remissions typically lasting 2 to 3 years, but in most men prostate cancer ultimately progresses to an androgen-independent state resulting in death due to widespread metastases. Multiple mechanisms of androgen independence have now been documented, including amplification of the androgen receptor as well as signal transduction pathways that bypass the androgen receptor completely. In 2004, two landmark studies demonstrated a survival advantage in androgen-independent prostate cancer patients utilizing docetaxel chemotherapy, setting a new standard of care for this disease. In addition, treatments with the bisphosphonate zoledronic acid and systemic radioisotopes have also been shown to have palliative benefits in this population. Building on these advances, several new traditional chemotherapeutic agents as well as new targeted therapies are under development.

Advanced prostate cancer can progress to systemic metastatic tumors, which are generally androgen insensitive and ultimately lethal. Here, we report a comprehensive genomic survey for somatic events in systemic metastatic prostate tumors using both high-resolution copy number analysis and targeted mutational survey of 3508 exons from 577 cancer-related genes using next generation sequencing. Focal homozygous deletions were detected at 8p22, 10q23.31, 13q13.1, 13q14.11, and 13q14.12. Key genes mapping within these deleted regions include PTEN, BRCA2, C13ORF15, and SIAH3. Focal high-level amplifications were detected at 5p13.2-p12, 14q21.1, 7q22.1, and Xq12. Key amplified genes mapping within these regions include SKP2, FOXA1, and AR. Furthermore, targeted mutational analysis of normal-tumor pairs has identified somatic mutations in genes known to be associated with prostate cancer including AR and TP53, but has also revealed novel somatic point mutations in genes including MTOR, BRCA2, ARHGEF12, and CHD5. Finally, in one patient where multiple independent metastatic tumors were available, we show common and divergent somatic alterations that occur at both the copy number and point mutation level, supporting a model for a common clonal progenitor with metastatic tumor-specific divergence. Our study represents a deep genomic analysis of advanced metastatic prostate tumors and has revealed candidate somatic alterations, possibly contributing to lethal prostate cancer.

Resistance to cell death is a hallmark of cancer. Autophagy is a survival mechanism activated in response to nutrient deprivation; however, excessive autophagy will ultimately induce cell death in a nonapoptotic manner. The present study demonstrates that CCL2 protects prostate cancer PC3 cells from autophagic death, allowing prolonged survival in serum-free conditions. Upon serum starvation, CCL2 induced survivin up-regulation in PC3, DU 145, and C4-2B prostate cancer cells. Both cell survival and survivin expression were stunted in CCL2-stimulated PC3 cells when treated either with the phosphatidylinositol 3-kinase inhibitor LY294002 (2 microm) or the Akt-specific inhibitor-X (Akti-X; 2.5 microm). Furthermore, CCL2 significantly reduced light chain 3-II (LC3-II) in serum-starved PC3; in contrast, treatment with LY294002 or Akti-X reversed the effect of CCL2 on LC3-II levels, suggesting that CCL2 signaling limits autophagy in these cells. Upon serum deprivation, the analysis of LC3 localization by immunofluorescence revealed a remarkable reduction in LC3 punctate after CCL2 stimulation. CCL2 treatment also resulted in a higher sustained mTORC1 activity as measured by an increase in phospho-p70S6 kinase (Thr389). Rapamycin, an inducer of autophagy, both down-regulated survivin and decreased PC3 cell viability in serum-deprived conditions. Treatment with CCL2, however, allowed cells to partially resist rapamycin-induced death, which correlated with survivin protein levels. In two stable transfectants expressing survivin-specific short hairpin RNA, generated from PC3, survivin protein levels controlled both cell viability and LC3 localization in response to CCL2 treatment. Altogether, these findings indicate that CCL2 protects prostate cancer PC3 cells from autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway and reveal survivin as a critical molecule in this survival mechanism.

The organization of DNA within the nucleus has been demonstrated to be both cell and tissue specific and is arranged in a non-random fashion in both sperm and somatic cells. Nuclear structure has a pivotal role in this three-dimensional organization of DNA and RNA and contributes as well to forming fixed organizing sites for nuclear functions, such as DNA replication, transcription, and RNA processing. In sperm, DNA is also organized in a specific fashion by the nuclear matrix and DNA-protamine interactions. Within somatic cells, the nuclear matrix provides a three-dimensional framework for the tissue specific regulation of genes by directed interaction with transcriptional activators. This differential organization of the DNA by the nuclear matrix, in a tissue specific manner, contributes to tissue specific gene expression. The nuclear matrix is the first link from the DNA to the entire tissue matrix system and provides a direct structural linkage to the cytomatrix and extracellular matrix. In summary, the tissue matrix serves as a dynamic structural framework for the cell which interacts to organize and process spatial and temporal information to coordinate cellular functions and gene expression. The tissue matrix provides a structural system for integrating form and function.

The last decade has seen an explosion in knowledge of the molecular basis and treatment of cancer. The molecular events that define the lethal phenotype of various cancers--the genetic and cellular alterations that lead to a cancer with a poor or incurable prognosis--are being defined. While these studies describe the cellular events of the lethal phenotype of cancer in detail, how these events result in the common clinical syndromes that kill the majority of cancer patients is not well understood. It is clear that the central step that makes most cancers incurable is metastasis. Understanding the traits that a cancer acquires to successfully grow and metastasize to distant sites gives insight into how tumors produce multiple factors that result in multiple different clinical syndromes that are lethal for the patient.

Abstract The generation of an ‘angiogenic switch’ is essential for tumor growth, yet its regulation is poorly understood. In this investigation, we explored the linkage between metastasis and angiogenesis through CXCL12/CXCR4 signaling. We found that CXCR4 regulates the expression and secretion of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1). Overexpression of PGK1 reduced the secretion of vascular endothelial growth factor and interleukin-8 and increased the generation of angiostatin. At metastatic sites, however, high levels of CXCL12 signaling through CXCR4 reduced PGK1 expression, releasing the angiogenic response for metastastic growth. These data suggest that PGK1 is a critical downstream target of the chemokine axis and an important regulator of an ‘angiogenic switch’ that is essential for tumor and metastatic growth. [Cancer Res 2007;67(1):149–59]

Abstract Monocyte chemoattractant protein 1 (CCL2) is a recently identified prominent regulator of prostate cancer growth and metastasis. The purpose of this study was to investigate the mechanistic role of CCL2 in prostate cancer growth in bone. The present study found that CCL2 was up-regulated in osteoblasts (3-fold by PC-3 and 2-fold by VCaP conditioned medium) and endothelial cells (2-fold by PC-3 and VCaP conditioned medium). Parathyroid hormone-related protein (PTHrP) treatment of osteoblastic cells up-regulated CCL2 and was blocked by a PTHrP antagonist, suggesting that prostate cancer–derived PTHrP plays an important role in elevation of osteoblast-derived CCL2. CCL2 indirectly increased blood vessel formation in endothelial cells through vascular endothelial growth factor-A, which was up-regulated 2-fold with administration of CCL2 in prostate cancer cells. In vivo, anti-CCL2 treatment suppressed tumor growth in bone. The decreased tumor burden was associated with decreased bone resorption (serum TRAP5b levels were decreased by 50–60% in anti–CCL2-treated animals from VCaP or PC-3 cell osseous lesions) and microvessel density was decreased by 70% in anti–CCL2-treated animals with bone lesions from VCaP cells. These data suggest that a destructive cascade is driven by tumor cell–derived, PTHrP-mediated induction of CCL2, which facilitates tumor growth via enhanced osteoclastic and endothelial cell activity in bone marrow. Taken together, CCL2 mediates the interaction between tumor-derived factors and host-derived chemokines acting in cooperation to promote skeletal metastasis. [Cancer Res 2009;69(4):1685–92]

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.

It is well established that bleeding activates the hematopoietic system to regenerate the loss of mature blood elements. We have shown that hematopoietic stem cells (HSCs) isolated from animals challenged with an acute bleed regulate osteoblast differentiation from marrow stromal cells. This suggests that HSCs participate in bone formation where the molecular basis for this activity is the production of BMP2 and BMP6 by HSCs. Yet, what stimulates HSCs to produce BMPs is unclear.In this study, we demonstrate that erythropoietin (Epo) activates Jak-Stat signaling pathways in HSCs which leads to the production of BMPs. Critically, Epo also directly activates mesenchymal cells to form osteoblasts in vitro, which in vivo leads to bone formation. Importantly, Epo first activates osteoclastogenesis which is later followed by osteoblastogenesis that is induced by either Epo directly or the expression of BMPs by HSCs to form bone.These data for the first time demonstrate that Epo regulates the formation of bone by both direct and indirect pathways, and further demonstrates the exquisite coupling between hematopoiesis and osteopoiesis in the marrow.

For metastasis to occur, tumor cells must first detach from their tissue of origin. This requires altering both the tissue of origin and the cancer cell. Once detached, cancer cells in circulation must also acquire survival mechanisms. Although many may successfully disseminate, variation exists in the efficiency with which circulating tumor cells home to and invade the bone marrow as metastastic seeds. Disseminated tumor cells that do successfully invade the marrow are secured by cell-cell and cell-extracellular matrix adhesion. However, establishing a foothold in the marrow is not sufficient for disseminated tumor cells to create metastases. A significant latent phase must be overcome by either rescuing cellular proliferation or attenuating micrometastatic mass dormancy programs. Finally, growing metastases fuel osteolysis, osteoblastogenesis and T-cell differentiation, creating a variety of tumor phenotypes. Each step in the metastatic cascade is rich in biological targets and mechanistic pathways.

Since the effectiveness of androgen deprivation for treatment of advanced prostate cancer was first demonstrated, prevention strategies and medical therapies for prostate cancer have been based on understanding the biologic underpinnings of the disease.Prostate cancer treatment is one of the best examples of a systematic therapeutic approach to target not only the cancer cells themselves, but the microenvironment in which they are proliferating.As the population ages and prostate cancer prevalence increases, challenges remain in the diagnosis of clinically relevant prostate cancer as well as the management of the metastatic and androgen-independent metastatic disease states.

Abstract BACKGROUND Prostate cancer (PCa) frequently metastasizes to the bone and induces osteoblastic lesions. We previously demonstrated through over‐expression of the Wnt inhibitor dickkopf‐1 (DKK‐1) that Wnts contribute to the osteoblastic component of PCa osseous lesions in vivo. METHODS To test the clinical significance of DKK‐1 expression during PCa progression, tissue microarrays were stained for DKK‐1 protein by immunohistochemistry. RESULTS DKK‐1 expression index (EI) was found to increase in PIN and primary lesions compared to non‐neoplastic tissue (106 ± 10 vs. 19 ± 6, respectively, where the EI is the product of the percent expression and staining intensity). DKK‐1 expression was also found to be higher in all PCa metastatic lesions (56 ± 21 EI) compared to non‐neoplastic tissues but was significantly decreased versus primary PCa lesions ( P &lt; 0.008). The decline in DKK‐1 correlated with a shift of β‐catenin staining from the nucleus to the cytoplasm suggesting possible mechanism for the observed decrease in DKK‐1 levels during PCa progression. Within metastatic lesions, DKK‐1 expression was least abundant in PCa bone metastases relative to all soft tissue PCa metastatic lesions except lymph node metastases. High DKK‐1 expression within PCa metastases was further associated with shorter over‐all patient survival. CONCLUSIONS Taken together, these data demonstrate that elevated DKK‐1 expression is an early event in PCa and that as PCa progresses DKK‐1 expression declines, particularly in advanced bone metastases. The decline of DKK‐1 in bone metastases can unmask Wnts' osteoblastic activity. These data support a model in which DKK‐1 is a molecular switch that transitions the phenotype of PCa osseous lesions from osteolytic to osteoblastic. Prostate 68: 1396–1404, 2008. © 2008 Wiley‐Liss, Inc.

Prostate cancer continues to be a major cause of morbidity and mortality in men around the world. The field of prostate cancer research continues to be hindered by the lack of relevant preclinical models to study tumorigenesis and to further development of effective prevention and therapeutic strategies. The Prostate Cancer Foundation held a Prostate Cancer Models Working Group (PCMWG) Summit on August 6th and 7th, 2007 to address these issues. The PCMWG reviewed the state of prostate cancer preclinical models and identified the current limitations of cell line, xenograft and genetically engineered mouse models that have hampered the transition of scientific findings from these models to human clinical trials. In addition the PCMWG identified administrative issues that inhibit the exchange of models and impede greater interactions between academic centers and these centers with industry. The PCMWG identified potential solutions for discovery bottlenecks that include: (1) insufficient number of models with insufficient molecular and biologic diversity to reflect human cancer, (2) a lack of understanding of the molecular events that define tumorigenesis, (3) a lack of tools for studying tumor-host interactions, (4) difficulty in accessing model systems across institutions, and (5) addressing why preclinical studies appear not to be predictive of human clinical trials. It should be possible to apply the knowledge gained molecular and epigenetic studies to develop new cell lines and models that mimic progressive and fatal prostate cancer and ultimately improve interventions.

Abstract We previously reported the development of a simple, user‐friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti‐cancer drug sensitivity testing. The 384 hanging drop array plate allows for high‐throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell‐based high‐throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence‐ and colorimetric‐based assays through Z ‐factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. Biotechnol. Bioeng. 2012; 109:1293–1304. © 2011 Wiley Periodicals, Inc.

Abstract Progression of primary prostate cancer to castration-resistant prostate cancer (CRPC) is associated with numerous genetic and epigenetic alterations that are thought to promote survival at metastatic sites. In this study, we investigated gene copy number and CpG methylation status in CRPC to gain insight into specific pathophysiologic pathways that are active in this advanced form of prostate cancer. Our analysis defined and validated 495 genes exhibiting significant differences in CRPC in gene copy number, including gains in androgen receptor (AR) and losses of PTEN and retinoblastoma 1 (RB1). Significant copy number differences existed between tumors with or without AR gene amplification, including a common loss of AR repressors in AR-unamplified tumors. Simultaneous gene methylation and allelic deletion occurred frequently in RB1 and HSD17B2, the latter of which is involved in testosterone metabolism. Lastly, genomic DNA from most CRPC was hypermethylated compared with benign prostate tissue. Our findings establish a comprehensive methylation signature that couples epigenomic and structural analyses, thereby offering insights into the genomic alterations in CRPC that are associated with a circumvention of hormonal therapy. Genes identified in this integrated genomic study point to new drug targets in CRPC, an incurable disease state which remains the chief therapeutic challenge. Cancer Res; 72(3); 616–25. ©2011 AACR.

Using stereolithography, 20 different structural variations comprised of millimeter diameter holes surrounded by trenches, plateaus, or micro-ring structures were prepared and tested for their ability to stably hold arrays of microliter sized droplets within the structures over an extended period of time. The micro-ring structures were the most effective in stabilizing droplets against mechanical and chemical perturbations. After confirming the importance of micro-ring structures using rapid prototyping, we developed an injection molding tool for mass production of polystyrene 3D cell culture plates with an array of 384 such micro-ring surrounded through-hole structures. These newly designed and injection molded polystyrene 384 hanging drop array plates with micro-rings were stable and robust against mechanical perturbations as well as surface fouling-facilitated droplet spreading making them capable of long term cell spheroid culture of up to 22 days within the droplet array. This is a significant improvement over previously reported 384 hanging drop array plates which are susceptible to small mechanical shocks and could not reliably maintain hanging drops for longer than a few days. With enhanced droplet stability, the hanging drop array plates with micro-ring structures provide better platforms and open up new opportunities for high-throughput preparation of microscale 3D cell constructs for drug screening and cell analysis.

It is well known that the gastrointestinal (GI) microbiota can influence the metabolism, pharmacokinetics, and toxicity of cancer therapies. Conversely, the effect of cancer treatments on the composition of the GI microbiota is poorly understood. We hypothesized that oral androgen receptor axis-targeted therapies (ATT), including bicalutamide, enzalutamide, and abiraterone acetate, may be associated with compositional differences in the GI microbiota.We profiled the fecal microbiota in a cross-sectional study of 30 patients that included healthy male volunteers and men with different clinical states of prostate cancer (i.e., localized, biochemically recurrent, and metastatic disease) using 16S rDNA amplicon sequencing. Functional inference of identified taxa was performed using PICRUSt.We report a significant difference in alpha diversity in GI microbiota among men with versus without a prostate cancer diagnosis. Further analysis identified significant compositional differences in the GI microbiota of men taking ATT, including a greater abundance of species previously linked to response to anti-PD-1 immunotherapy such as Akkermansia muciniphila and Ruminococcaceae spp. In functional analyses, we found an enriched representation of bacterial gene pathways involved in steroid biosynthesis and steroid hormone biosynthesis in the fecal microbiota of men taking oral ATT.There are measurable differences in the GI microbiota of men receiving oral ATT. We speculate that oral hormonal therapies for prostate cancer may alter the GI microbiota, influence clinical responses to ATT, and/or potentially modulate the antitumor effects of future therapies including immunotherapy. Given our findings, larger, longitudinal studies are warranted.

The receptor tyrosine kinase Axl is overexpressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our laboratory indicate that Axl and its ligand growth arrest-specific 6 (GAS6) may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer cell lines PC3 and DU145 and has negligible levels of expression in a nonmetastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial-mesenchymal transition in prostate cancer cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were downregulated. Moreover, CoCl(2), a hypoxia mimicking agent, prevented GAS6-mediated downregulation of Axl in these cell lines. Immunochemical staining of human prostate cancer tissue microarrays showed that Axl, GAS6, and hypoxia-inducible factor-1α (Hif-1α; indicator of hypoxia) were all coexpressed in prostate cancer and in bone metastases compared with normal tissues. Together, our studies indicate that Axl plays a crucial role in prostate cancer metastasis and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression is maintained leading to enhanced signaling.

We present a unifying theory to explain cancer recurrence, therapeutic resistance, and lethality. The basis of this theory is the formation of simultaneously polyploid and aneuploid cancer cells, polyaneuploid cancer cells (PACCs), that avoid the toxic effects of systemic therapy by entering a state of cell cycle arrest. The theory is independent of which of the classically associated oncogenic mutations have already occurred. PACCs have been generally disregarded as senescent or dying cells. Our theory states that therapeutic resistance is driven by PACC formation that is enabled by accessing a polyploid program that allows an aneuploid cancer cell to double its genomic content, followed by entry into a nondividing cell state to protect DNA integrity and ensure cell survival. Upon removal of stress, e.g., chemotherapy, PACCs undergo depolyploidization and generate resistant progeny that make up the bulk of cancer cells within a tumor.

Emerging data suggest that metastasis is a spectrum of disease burden rather than a binary state, and local therapies, such as radiation, might improve outcomes in oligometastasis. However, current definitions of oligometastasis are solely numerical.To characterize the somatic mutational landscape across the disease spectrum of metastatic castration-sensitive prostate cancer (mCSPC) to elucidate a biological definition of oligometastatic CSPC.This was a retrospective study of men with mCSPC who underwent clinical-grade sequencing of their tumors (269 primary tumor, 25 metastatic sites). Patients were classified as having biochemically recurrent (ie, micrometastatic), metachronous oligometastatic (≤5 lesions), metachronous polymetastatic (>5 lesions), or de novo metastatic (metastasis at diagnosis) disease.We measured the frequency of driver mutations across metastatic classifications and the genomic associations with radiographic progression-free survival (rPFS) and time to castrate-resistant prostate cancer (CRPC).The frequency of driver mutations in TP53 (p = 0.01), WNT (p = 0.08), and cell cycle (p = 0.04) genes increased across the mCSPC spectrum. TP53 mutation was associated with shorter rPFS (26.7 vs 48.6 mo; p = 0.002), and time to CRPC (95.6 vs 155.8 mo; p = 0.02) in men with oligometastasis, and identified men with polymetastasis with better rPFS (TP53 wild-type, 42.7 mo; TP53 mutated, 18.5 mo; p = 0.01). Mutations in TP53 (incidence rate ratio [IRR] 1.45; p = 0.004) and DNA double-strand break repair (IRR 1.61; p < 0.001) were associated with a higher number of metastases. Mutations in TP53 were also independently associated with shorter rPFS (hazard ratio [HR] 1.59; p = 0.03) and the development of CRPC (HR 1.71; p = 0.01) on multivariable analysis. This study was limited by its retrospective nature, sample size, and the use of commercially available sequencing platforms, resulting in a limited predefined set of genes examined.Somatic mutational profiles reveal a spectrum of metastatic biology that helps in redefining oligometastasis beyond a simple binary state of lesion enumeration.Oligometastatic prostate cancer is typically defined as less than three to five metastatic lesions and evidence suggests that using radiation or surgery to treat these sites improves clinical outcomes. As of now, treatment decisions for oligometastasis are solely defined according to the number of lesions. However, this study suggests that tumor mutational profiles can provide a biological definition of oligometastasis and complement currently used numerical definitions.

Cell division and cell death are fundamental processes governing growth and development across the tree of life. This relationship represents an evolutionary link between cell cycle and cell death programs that is present in all cells. Cancer is characterized by aberrant regulation of both, leading to unchecked proliferation and replicative immortality. Conventional anti-cancer therapeutic strategies take advantage of the proliferative dependency of cancer yet, in doing so, are triggering apoptosis, a death pathway to which cancer is inherently resistant. A thorough understanding of how therapeutics kill cancer cells is needed to develop novel, more durable treatment strategies. While cancer evolves cell-intrinsic resistance to physiological cell death pathways, there are opportunities for cell cycle agnostic forms of cell death, for example, necroptosis or ferroptosis. Furthermore, cell cycle independent death programs are immunogenic, potentially licensing host immunity for additional antitumor activity. Identifying cell cycle independent vulnerabilities of cancer is critical for developing alternative strategies that can overcome therapeutic resistance.

Most definitions of cancer broadly conform to the current NCI definition: "Cancer is a disease in which some of the body's cells grow uncontrollably and spread to other parts of the body." These definitions tend to describe what cancer "looks like" or "does" but do not describe what cancer "is" or "has become." While reflecting past insights, current definitions have not kept pace with the understanding that the cancer cell is itself transformed and evolving. We propose a revised definition of cancer: Cancer is a disease of uncontrolled proliferation by transformed cells subject to evolution by natural selection. We believe this definition captures the essence of the majority of previous and current definitions. To the simplest definition of cancer as a disease of uncontrolled proliferation of cells, our definition adds in the adjective "transformed" to capture the many tumorigenic processes that cancer cells adopt to metastasize. To the concept of uncontrolled proliferation of transformed cells, our proposed definition then adds "subject to evolution by natural selection." The subject to evolution by natural selection modernizes the definition to include the genetic and epigenetic changes that accumulate within a population of cancer cells that lead to the lethal phenotype. Cancer is a disease of uncontrolled proliferation by transformed cells subject to evolution by natural selection.

E-cadherin is a calcium 2+-dependent cell-adhesion molecule that determines epithelial development in the embryo and maintains adult differentiated epithelium and homeostasis. Aberrant or decreased expression has been reported to be associated with prostate carcinoma progression. The degree of E-cadherin expression in prostate cancer remains controversial. Some studies have reported decreased expression of E-cadherin as tumors advance and metastasize. Other studies have not demonstrated this relationship. To address these variations, we undertook a study to systematically evaluate E-cadherin expression in a broad range of prostate tissue. Benign prostate, clinically localized prostate cancer, and hormone-refractory metastatic prostate cancer were analyzed under uniform conditions using high-density tissue microarrays (TMA). Formalin-fixed, paraffin-embedded prostate carcinoma from men with clinically localized prostate carcinoma and autopsy material from men who died of widely metastatic, hormone-refractory prostate carcinoma were arrayed into 6 high-density TMA blocks. Benign and atrophic prostate tissue and high-grade prostatic intraepithelial neoplasia (PIN) were also included from the clinically localized cases. Immunohistochemistry was performed using the immunoglobulin G1 mouse monoclonal antibody (HECD-1; Zymed, San Francisco, CA). Membranous staining was recorded as low (aberrant) or high (normal). E-cadherin expression was considered aberrant if less than 70% of the cells had strong membranous staining. A total of 1,220 prostate TMA samples were analyzed. High (normal) E-cadherin expression was seen in 87% of 757 benign, 80% of 41 high-grade PIN, 82% of 325 prostate carcinoma and 90% of 97 hormone-refractory prostate carcinoma TMA samples. Mean E-cadherin expression was determined for each of the 128 clinically localized prostate cancer cases. Aberrant E-cadherin expression showed a statistical trend toward an association with positive surgical margins (P =.012), higher Gleason score (P =.18), and prostate-specific antigen (PSA) failure (Kaplan-Meier analysis, log-rank P =.09). There was a statistically significant association between aberrant E-cadherin expression and larger tumor size (P =.01). No significant associations were seen with extraprostatic extension and seminal vesicle invasion. The current study shows a broad-spectrum approach to evaluating E-cadherin protein expression in prostate carcinoma. Clinically localized prostate tumors, treated with surgery alone, show a high level of E-cadherin expression. Aberrant expression was identified in tumors with positive surgical margins, higher Gleason score, and a higher rate of PSA failure. However, these trends were not statistically significant. A statically significant association between aberrant E-cadherin expression and larger tumor size was identified. In the metastatic hormone-refractory prostate tumors, E-cadherin expression was vastly expressed, and only rare cases had aberrant expression. Therefore, the findings of this study are most consistent with a transient down-regulation of E-cadherin in localized prostate cancer. Metastatic prostate cancer shows strong E-cadherin expression as determined by anti-E-cadherin antibody HECD-1.

Using a modified version of the mRNA differential display technique, five human bladder cancer cell lines from low grade to metastatic were analyzed to identify differences in gene expression. A 316-bp cDNA (C11-300) was isolated that was not expressed in the metastatic cell line TccSuP. Sequence analysis revealed that this gene was identical to KIAA 0429, has a 5.3-kb transcript that mapped to 8q24.1. The protein is predicted to be 356 amino acids in size and has an actin-binding WH2 domain. Northern blot revealed expression in multiple normal tissues, but none in a metastatic breast cancer cell line (SKBR3) or in metastatic prostatic cancer cell lines (LNCaP, PC3). We have named this gene Missing in Metastasis (MIM) and our data suggest that it may be involved in cytoskeletal organization.

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.

Genetic alterations serve as beacons for the involvement of specific pathways in tumorigenesis. It was previously shown that 5% of prostate tumors harbor CTNNB1 mutations, suggesting that this tumor type may involve a deregulated APC/CTNNB1 pathway. To explore this possibility further, we searched for mutations in genes implicated in this pathway in 22 samples that included cell lines, xenografts, and primary tumors. We identified seven alterations: two in CTNNB1, three in APC, and two in hTRCP1 (also known as BTRC) which controls the degradation of CTNNB1. Alterations in the CTNNB1 regulatory domain, APC, and hTRCP1 were mutually exclusive, consistent with their equivalent effects on CTNNB1 stability. These results suggest that CTNNB1 signaling plays a critical role in the development of a significant fraction of prostate cancers. Moreover, they provide the first evidence that hTRCP1 plays a role in human neoplasia.

Abstract Background The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status. Methods In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5‐ml blood samples collected from patients with prostate cancer in evacuated blood‐draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6‐diamidino‐2‐phenylindole. Results In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6‐diamidino‐2‐phenylindole and frequently expressed the apoptosis‐induced, caspase‐cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples. Conclusions The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients. © 2004 Wiley‐Liss, Inc.

Traditionally, prostate cancer treatment, as well as all cancer treatment, has been designed to target the tumor cell directly via various hormonal and chemotherapeutic agents. Recently, the realization that cancer cells exist in complex microenvironments that are essential for the tumorigenic and metastatic potential of the cancer cells is starting the redefine the paradigm for cancer therapy. The propensity of prostate cancer cells to metastasize to bone is leading to the design of novel therapies targeting both the cancer cell as well as the bone microenvironment. Tumor cells in the bone interact with the extracellular matrix, stromal cells, osteoblasts, osteoclasts, and endothelial cells to promote tumor-cell survival and proliferation leading to a lethal phenotype that includes increased morbidity and mortality for patients with advanced prostate cancer. Several strategies are being developed that target these complex tumor cell-microenvironment interactions and target the signal transduction pathways of other cells important to the development of metastases, including the osteoclasts, osteoblasts, and endothelial cells of the bone microenvironment. Current and new therapies in metastatic prostate cancer will comprise a multitargeted approach aimed at both the tumor cell and the tumor microenvironment. Here, we review the current therapeutic strategies for targeting the prostate cancer-bone microenvironment and several single- and multiagent targeted approaches to the treatment of advanced prostate cancer that are under development.

Abstract Integrative analysis of genomic aberrations in the context of trancriptomic alterations will lead to a more comprehensive perspective on prostate cancer progression. Genome-wide copy number changes were monitored using array comparative genomic hybridization of laser-capture microdissected prostate cancer samples spanning stages of prostate cancer progression, including precursor lesions, clinically localized disease, and metastatic disease. A total of 62 specific cell populations from 38 patients were profiled. Minimal common regions (MCR) of alterations were defined for each sample type, and metastatic samples displayed the most number of alterations. Clinically localized prostate cancer samples with high Gleason grade resembled metastatic samples with respect to the size of altered regions and number of affected genes. A total of 9 out of 13 MCRs in the putative precursor lesion, high-grade prostatic intraepithelial neoplasia (PIN), showed an overlap with prostate cancer cases (amplifications in 3q29, 5q31.3-q32, 6q27, and 8q24.3 and deletions in 6q22.31, 16p12.2, 17q21.2, and 17q21.31), whereas postatrophic hyperplasia (PAH) did not exhibit this overlap. Interestingly, prostate cancers that do not overexpress ETS family members (i.e., gene fusion–negative prostate cancers) harbor differential aberrations in 1q23, 6q16, 6q21, 10q23, and 10q24. Integrative analysis with matched mRNA profiles identified genetic alterations in several proposed candidate genes implicated in prostate cancer progression. [Cancer Res 2007;67(17):8229–39]

How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe X ) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe X and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that α1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLe X and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that α1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism.

Abstract Prostate tumor cells, which characteristically metastasize to bone, initiate binding interactions with bone marrow endothelium under blood flow conditions through binding interactions with E-selectin. We hypothesized that E-selectin ligands on prostate tumor cells are directly associated with bone-metastatic potential. In this report, we elucidate the identity of E-selectin ligands on human metastatic prostate tumor cells and examine their association with prostate tumor progression and metastasis in vivo. To our surprise, we found that the E-selectin-binding form of P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on the human bone-metastatic prostate tumor MDA PCa 2b cell line. Interestingly, we also found that human prostate tumor cells derived from bone, lymph node, and brain metastases expressed another leukocyte E-selectin ligand, E-selectin ligand-1 (ESL-1). Immunohistochemical analysis of PSGL-1 and ESL-1 in normal prostate tissue and in localized and metastatic prostate tumors revealed that ESL-1 was principally localized to intracellular cell membrane and expressed on all normal and malignant prostate tissue, whereas PSGL-1 was notably detected on the surfaces of bone-metastatic prostate tumor cells. These findings implicate a functional role of PSGL-1 in the bone tropism of prostate tumor cells and establish a new perspective into the molecular mechanism of human prostate tumor metastasis.

Galectin-3, a beta-galactoside-binding protein, has been implicated in a variety of biological functions including cell proliferation, apoptosis, angiogenesis, tumor progression, and metastasis. The present study was undertaken to understand the role of galectin-3 in the progression of prostate cancer. Immunohistochemical analysis of galectin-3 expression revealed that galectin-3 was cleaved during the progression of prostate cancer. Galectin-3 knockdown by small interfering RNA (siRNA) was associated with reduced cell migration, invasion, cell proliferation, anchorage-independent colony formation, and tumor growth in the prostates of nude mice. Galectin-3 knockdown in human prostate cancer PC3 cells led to cell-cycle arrest at G(1) phase, up-regulation of nuclear p21, and hypophosphorylation of the retinoblastoma tumor suppressor protein (pRb), with no effect on cyclin D1, cyclin E, cyclin-dependent kinases (CDK2 and CDK4), and p27 protein expression levels. The data obtained here implicate galectin-3 in prostate cancer progression and suggest that galectin-3 may serve as both a diagnostic marker and therapeutic target for future disease treatments.

Prostate cancer (PCa) is the most commonly diagnosed cancer in American men with a subset inevitably presenting with metastatic disease to the bone. A well-recognized limitation in evaluating new treatments for metastatic PCa is the inability to use imaging to objectively assess response therapy. In this study, we evaluated the feasibility of clinically translating the functional diffusion map (fDM) imaging biomarker for quantifying the spatiotemporal effects of bone tumor response in a patient treated for metastatic PCa with bone metastases. A patient beginning therapy was scanned using MRI before treatment and again at 2 and 8 weeks post-treatment initiation to quantify changes in tumor diffusion values. Three metastatic lesions were identified for fDM analysis, all of which all demonstrated an early increase in diffusion values at 2 weeks, which increased further at 8 weeks post-treatment initiation. This finding correlated with a decrease in the patient's prostate-specific antigen (PSA) levels suggestive of patient response. CT, bone scans, and anatomic MRI images obtained posttreatment were found to be uninformative for the assessment of treatment effectiveness. This study presents the feasibility of fDM-measurements in osseous lesions over time and shows that changes in fDM values were consistent with therapeutic response. Thus, the fDM imaging biomarker may provide a quantifiable therapeutic endpoint to assess response in patients with metastatic bone cancer.

Tumorigenesis in humans is thought to be a multistep process where certain mutations confer a selective advantage, allowing lineages derived from the mutated cell to outcompete other cells. Although molecular cell biology has substantially advanced cancer research, our understanding of the evolutionary dynamics that govern tumorigenesis is limited. This paper analyzes the computational implications of cancer progression presented by Hanahan and Weinberg in The Hallmarks of Cancer. We model the complexities of tumor progression as a small set of underlying rules that govern the transformation of normal cells to tumor cells. The rules are implemented in a stochastic multistep model. The model predicts that (i) early-onset cancers proceed through a different sequence of mutation acquisition than late-onset cancers; (ii) tumor heterogeneity varies with acquisition of genetic instability, mutation pathway, and selective pressures during tumorigenesis; (iii) there exists an optimal initial telomere length which lowers cancer incidence and raises time of cancer onset; and (iv) the ability to initiate angiogenesis is an important stage-setting mutation, which is often exploited by other cells. The model offers insight into how the sequence of acquired mutations affects the timing and cellular makeup of the resulting tumor and how the cellular-level population dynamics drive neoplastic evolution.

Conventional cell trapping methods using microwells with small dimensions (10–20 μm) are useful for examining the instantaneous cell response to reagents; however, such wells have insufficient space for longer duration screening tests that require observation of cell attachment and division. Here we describe a flow method that enables single cell trapping in microwells with dimensions of 50 μm, a size sufficient to allow attachment and division of captured cells. Among various geometries tested, triangular microwells were found to be most efficient for single cell trapping while providing ample space for cells to grow and spread. An important trapping mechanism is the formation of fluid streamlines inside, rather than over, the microwells. A strong flow recirculation occurs in the triangular microwell so that it efficiently catches cells. Once a cell is captured, the cell presence in the microwell changes the flow pattern, thereby preventing trapping of other cells. About 62% of microwells were filled with single cells after a 20 min loading procedure. Human prostate cancer cells (PC3) were used for validation of our system.

Angiosarcoma (ASA) in humans, hemangiosarcoma (HSA) in dogs are deadly neoplastic diseases characterized by an aggressive growth of malignant cells with endothelial phenotype, widespread metastasis, poor response to chemotherapy. Galectin-3 (Gal-3), a p-galactoside-binding lectin implicated in tumor progression, metastasis, endothelial cell biology, angiogenesis, regulation of apoptosis, neoplastic cell response to cytotoxic drugs, has not been studied before in tumors arising from malignant endothelia. Here, we tested the hypothesis that Gal-3 could be widely expressed in human ASA, canine HSA, could play an important role in malignant endothelial cell biology. Immunohistochemical analysis demonstrated that 100% of the human ASA (10 of 10), canine HSA (17 of 17) samples analyzed expressed Gal-3. Two carbohydrate-based Gal-3 inhibitors, modified citrus pectin (MCP), lactulosyl-l-leucine (LL), caused a dose-dependent reduction of SVR murine ASA cell clonogenic survival through the inhibition of Gal-3 antiapoptotic function. Furthermore, both MCP, LL sensitized SVR cells to the cytotoxic drug doxorubicin to a degree sufficient to reduce the in vitro IC50 of doxorubicin by 10.7-fold, 3.64old, respectively. These results highlight the important role of Gal-3 in the biology of ASA, identify Gal-3 as a potential therapeutic target in tumors arising from malignant endothelial cells.

Expression of parathyroid hormone-related protein (PTHrP) correlates with prostate cancer skeletal progression; however, the impact of prostate cancer-derived PTHrP on the microenvironment and osteoblastic lesions in skeletal metastasis has not been completely elucidated. In this study, PTHrP overexpressing prostate cancer clones were stably established by transfection of full length rat PTHrP cDNA. Expression and secretion of PTHrP were verified by western blotting and IRMA assay. PTHrP overexpressing prostate cancer cells had higher growth rates in vitro, and generated larger tumors when inoculated subcutaneously into athymic mice. The impact of tumor-derived PTHrP on bone was investigated using a vossicle co-implant model. Histology revealed increased bone mass adjacent to PTHrP overexpressing tumor foci, with increased osteoblastogenesis, osteoclastogenesis and angiogenesis. In vitro analysis demonstrated pro-osteoclastic and pro-osteoblastic effects of PTHrP. PTHrP enhanced proliferation of bone marrow stromal cells and early osteoblast differentiation. PTHrP exerted a pro-angiogenic effect indirectly, as it increased angiogenesis but only in the presence of bone marrow stromal cells. These data suggest PTHrP plays a role in tumorigenesis in prostate cancer, and that PTHrP is a key mediator for communication and interactions between prostate cancer and the bone microenvironment. Prostate cancer-derived PTHrP is actively involved in osteoblastic skeletal progression.

Tumor and stromal interactions in the tumor microenvironment are critical for oncogenesis and cancer progression. Our understanding of the molecular events by which reactive stromal fibroblasts-myofibroblast or cancer-associated fibroblasts (CAF)-affect the growth and invasion of prostate cancer remains unclear. Laser capture microdissection and cDNA microarray analysis of CAFs in prostate tumors revealed strong upregulation of phosphoglycerate kinase-1 (PGK1), an ATP-generating glycolytic enzyme that forms part of the glycolytic pathway and is directly involved in CXCL12-CXCR4 signaling. Normal fibroblasts overexpressing PGK1 resembled myofibroblasts in their expression of smooth muscle alpha-actin, vimentin, and high levels of CXCL12. These cells also displayed a higher proliferative index and the capability to contribute to prostate tumor cell invasion in vitro, possibly through expression of MMP-2 and MMP-3 and activation of the AKT and ERK pathways. Coimplantation of PGK1-overexpressing fibroblasts with prostate tumor cells promoted tumor cell growth in vivo. Collectively, these observations suggest that PGK1 helps support the interactions between cancer and its microenvironment.

Despite significant improvements in therapy, the prognosis for cancer with bone metastasis is generally poor. Therefore, there is a great need for new therapeutic approaches for metastatic disease. It has been appreciated that tumor cells metastasize to bone using mechanisms similar to those of hematopoietic stem cells (HSC) homing to bone marrow (e.g., CXCL12/CXCR4). It was recently found that prostate cancer cells target the bone marrow microenvironment for HSCs, or the HSC niche, during metastasis. Of importance, these disseminated prostate cancer cells can be mobilized out of the niche with the use of HSC mobilizing agents. These findings suggest that the bone marrow HSC niche is a potential therapeutic target for metastatic disease. Therefore, a hypothesis worth considering is that agents that can disrupt the interactions between tumor cells and the HSC niche may be efficacious when used in conjunction with standard chemotherapeutic agents. Although further understanding of the tumor-niche interactions is needed, the concept of targeting the niche in conjunction with chemotherapy could open up new possibilities to eradicate incurable metastatic diseases.

Chemokines are a family of small and secreted proteins that play pleiotropic roles in inflammation-related pathological diseases, including cancer. Among the identified 50 human chemokines, chemokine (C-C motif) ligand 2 (CCL2) is of particular importance in cancer development since it serves as one of the key mediators of interactions between tumor and host cells. CCL2 is produced by cancer cells and multiple different host cells within the tumor microenvironment. CCL2 mediates tumorigenesis in many different cancer types. For example, CCL2 has been reported to promote prostate cancer cell proliferation, migration, invasion, and survival, via binding to its functional receptor CCR2. Furthermore, CCL2 induces the recruitment of macrophages and induces angiogenesis and matrix remodeling. Targeting CCL2 has been demonstrated as an effective therapeutic approach in preclinical prostate cancer models, and currently, neutralizing monoclonal antibody against CCL2 has entered into clinical trials in prostate cancer. In this chapter, targeting CCL2 in prostate cancer will be used as an example to show translation of laboratory findings from cancer molecular biology to the clinic.