John R. Prensner

A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κΒ pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types. Two Articles in this issue add major data sets to the growing picture of the cancer genome. Bignell et al. analysed a large number of homozygous gene deletions in a collection of 746 publicly available cancer cell lines. Combined with information about hemizygous deletions of the same genes, the data suggest that many deletions found in cancer reflect the position of a gene at a fragile site in the genome, rather than as a recessive cancer gene whose loss confers a selective growth advantage. Beroukhim et al. present the largest data set to date on somatic copy-number variations across more than 3,000 specimens of human primary cancers. Many alterations are shared between multiple tumour types. Functional experiments demonstrate an oncogenic role for the apoptosis genes MCL1 and BCL2L1 that are associated with amplifications found in many cancers. One way of discovering genes with key roles in cancer development is to identify genomic regions that are frequently altered in human cancers. Here, high-resolution analyses of somatic copy-number alterations (SCNAs) in numerous cancer specimens provide an overview of regions of focal SCNA that are altered at significant frequency across several cancer types. An oncogenic function is also found for the anti-apoptosis genes MCL1 and BCL2L1, which reside in amplified genome regions in many cancers.

Long noncoding RNAs (lncRNAs) are emerging as important regulators of tissue physiology and disease processes including cancer. To delineate genome-wide lncRNA expression, we curated 7,256 RNA sequencing (RNA-seq) libraries from tumors, normal tissues and cell lines comprising over 43 Tb of sequence from 25 independent studies. We applied ab initio assembly methodology to this data set, yielding a consensus human transcriptome of 91,013 expressed genes. Over 68% (58,648) of genes were classified as lncRNAs, of which 79% were previously unannotated. About 1% (597) of the lncRNAs harbored ultraconserved elements, and 7% (3,900) overlapped disease-associated SNPs. To prioritize lineage-specific, disease-associated lncRNA expression, we employed non-parametric differential expression testing and nominated 7,942 lineage- or cancer-associated lncRNA genes. The lncRNA landscape characterized here may shed light on normal biology and cancer pathogenesis and may be valuable for future biomarker development.

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.

The discovery of numerous noncoding RNA (ncRNA) transcripts in species from yeast to mammals has dramatically altered our understanding of cell biology, especially the biology of diseases such as cancer. In humans, the identification of abundant long ncRNA (lncRNA) >200 bp has catalyzed their characterization as critical components of cancer biology. Recently, roles for lncRNAs as drivers of tumor suppressive and oncogenic functions have appeared in prevalent cancer types, such as breast and prostate cancer. In this review, we highlight the emerging impact of ncRNAs in cancer research, with a particular focus on the mechanisms and functions of lncRNAs.

Comprehensive knowledge of the genomic alterations that underlie cancer is a critical foundation for diagnostics, prognostics, and targeted therapeutics. Systematic efforts to analyze cancer genomes are underway, but the analysis is hampered by the lack of a statistical framework to distinguish meaningful events from random background aberrations. Here we describe a systematic method, called Genomic Identification of Significant Targets in Cancer (GISTIC), designed for analyzing chromosomal aberrations in cancer. We use it to study chromosomal aberrations in 141 gliomas and compare the results with two prior studies. Traditional methods highlight hundreds of altered regions with little concordance between studies. The new approach reveals a highly concordant picture involving ≈35 significant events, including 16–18 broad events near chromosome-arm size and 16–21 focal events. Approximately half of these events correspond to known cancer-related genes, only some of which have been previously tied to glioma. We also show that superimposed broad and focal events may have different biological consequences. Specifically, gliomas with broad amplification of chromosome 7 have properties different from those with overlapping focal EGFR amplification: the broad events act in part through effects on MET and its ligand HGF and correlate with MET dependence in vitro . Our results support the feasibility and utility of systematic characterization of the cancer genome.

Noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease and to reveal uncharacterized aspects of tumor biology. Here we discover 121 unannotated prostate cancer-associated ncRNA transcripts (PCATs) by ab initio assembly of high-throughput sequencing of polyA(+) RNA (RNA-Seq) from a cohort of 102 prostate tissues and cells lines. We characterized one ncRNA, PCAT-1, as a prostate-specific regulator of cell proliferation and show that it is a target of the Polycomb Repressive Complex 2 (PRC2). We further found that patterns of PCAT-1 and PRC2 expression stratified patient tissues into molecular subtypes distinguished by expression signatures of PCAT-1-repressed target genes. Taken together, our findings suggest that PCAT-1 is a transcriptional repressor implicated in a subset of prostate cancer patients. These findings establish the utility of RNA-Seq to identify disease-associated ncRNAs that may improve the stratification of cancer subtypes.

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

Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority. The molecular basis for this clinical heterogeneity remains incompletely understood. Here we characterize a long noncoding RNA termed SChLAP1 (second chromosome locus associated with prostate-1; also called LINC00913) that is overexpressed in a subset of prostate cancers. SChLAP1 levels independently predict poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo gain-of-function and loss-of-function experiments indicate that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, SChLAP1 antagonizes the genome-wide localization and regulatory functions of the SWI/SNF chromatin-modifying complex. These results suggest that SChLAP1 contributes to the development of lethal cancer at least in part by antagonizing the tumor-suppressive functions of the SWI/SNF complex.

Prostate cancer biomarkers that improve upon the PSA test should address unmet clinical needs.

<h3>Importance</h3> Cancer is caused by a diverse array of somatic and germline genomic aberrations. Advances in genomic sequencing technologies have improved the ability to detect these molecular aberrations with greater sensitivity. However, integrating them into clinical management in an individualized manner has proven challenging. <h3>Objective</h3> To evaluate the use of integrative clinical sequencing and genetic counseling in the assessment and treatment of children and young adults with cancer. <h3>Design, Setting, and Participants</h3> Single-site, observational, consecutive case series (May 2012-October 2014) involving 102 children and young adults (mean age, 10.6 years; median age, 11.5 years, range, 0-22 years) with relapsed, refractory, or rare cancer. <h3>Exposures</h3> Participants underwent integrative clinical exome (tumor and germline DNA) and transcriptome (tumor RNA) sequencing and genetic counseling. Results were discussed by a precision medicine tumor board, which made recommendations to families and their physicians. <h3>Main Outcomes and Measures</h3> Proportion of patients with potentially actionable findings, results of clinical actions based on integrative clinical sequencing, and estimated proportion of patients or their families at risk of future cancer. <h3>Results</h3> Of the 104 screened patients, 102 enrolled with 91 (89%) having adequate tumor tissue to complete sequencing. Only the 91 patients were included in all calculations, including 28 (31%) with hematological malignancies and 63 (69%) with solid tumors. Forty-two patients (46%) had actionable findings that changed their cancer management: 15 of 28 (54%) with hematological malignancies and 27 of 63 (43%) with solid tumors. Individualized actions were taken in 23 of the 91 (25%) based on actionable integrative clinical sequencing findings, including change in treatment for 14 patients (15%) and genetic counseling for future risk for 9 patients (10%). Nine of 91 (10%) of the personalized clinical interventions resulted in ongoing partial clinical remission of 8 to 16 months or helped sustain complete clinical remission of 6 to 21 months. All 9 patients and families with actionable incidental genetic findings agreed to genetic counseling and screening. <h3>Conclusions and Relevance</h3> In this single-center case series involving young patients with relapsed or refractory cancer, incorporation of integrative clinical sequencing data into clinical management was feasible, revealed potentially actionable findings in 46% of patients, and was associated with change in treatment and family genetic counseling for a small proportion of patients. The lack of a control group limited assessing whether better clinical outcomes resulted from this approach than outcomes that would have occurred with standard care.

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

Pseudogene transcripts can provide a novel tier of gene regulation through generation of endogenous siRNAs or miRNA-binding sites. Characterization of pseudogene expression, however, has remained confined to anecdotal observations due to analytical challenges posed by the extremely close sequence similarity with their counterpart coding genes. Here, we describe a systematic analysis of pseudogene "transcription" from an RNA-Seq resource of 293 samples, representing 13 cancer and normal tissue types, and observe a surprisingly prevalent, genome-wide expression of pseudogenes that could be categorized as ubiquitously expressed or lineage and/or cancer specific. Further, we explore disease subtype specificity and functions of selected expressed pseudogenes. Taken together, we provide evidence that transcribed pseudogenes are a significant contributor to the transcriptional landscape of cells and are positioned to play significant roles in cellular differentiation and cancer progression, especially in light of the recently described ceRNA networks. Our work provides a transcriptome resource that enables high-throughput analyses of pseudogene expression.

Ewing's sarcoma family of tumors (ESFT) refers to aggressive malignancies which frequently harbor characteristic EWS-FLI1 or EWS-ERG genomic fusions. Here, we report that these fusion products interact with the DNA damage response protein and transcriptional coregulator PARP-1. ESFT cells, primary tumor xenografts, and tumor metastases were all highly sensitive to PARP1 inhibition. Addition of a PARP1 inhibitor to the second-line chemotherapeutic agent temozolamide resulted in complete responses of all treated tumors in an EWS-FLI1-driven mouse xenograft model of ESFT. Mechanistic investigations revealed that DNA damage induced by expression of EWS-FLI1 or EWS-ERG fusion genes was potentiated by PARP1 inhibition in ESFT cell lines. Notably, EWS-FLI1 fusion genes acted in a positive feedback loop to maintain the expression of PARP1, which was required for EWS-FLI-mediated transcription, thereby enforcing oncogene-dependent sensitivity to PARP-1 inhibition. Together, our findings offer a strong preclinical rationale to target the EWS-FLI1:PARP1 intersection as a therapeutic strategy to improve the treatment of ESFTs.

Abstract Impairment of double-stranded DNA break (DSB) repair is essential to many cancers. However, although mutations in DSB repair proteins are common in hereditary cancers, mechanisms of impaired DSB repair in sporadic cancers remain incompletely understood. Here, we describe the first role for a long noncoding RNA (lncRNA) in DSB repair in prostate cancer. We identify PCAT-1, a prostate cancer outlier lncRNA, which regulates cell response to genotoxic stress. PCAT-1 expression produces a functional deficiency in homologous recombination through its repression of the BRCA2 tumor suppressor, which, in turn, imparts a high sensitivity to small-molecule inhibitors of PARP1. These effects reflected a posttranscriptional repression of the BRCA2 3′UTR by PCAT-1. Our observations thus offer a novel mechanism of “BRCAness” in sporadic cancers. Cancer Res; 74(6); 1651–60. ©2014 AACR.

Improved clinical predictors for disease progression are needed for localised prostate cancer, since only a subset of patients develop recurrent or refractory disease after first-line treatment. Therefore, we undertook an unbiased analysis to identify RNA biomarkers associated with metastatic progression after prostatectomy.Prostate cancer samples from patients treated with radical prostatectomy at three academic institutions were analysed for gene expression by a high-density Affymetrix GeneChip platform, encompassing more than 1 million genomic loci. In a discovery cohort, all protein-coding genes and known long non-coding RNAs were ranked by fold change in expression between tumours that subsequently metastasised versus those that did not. The top ranked gene was then validated for its prognostic value for metastatic progression in three additional independent cohorts. 95% of the gene expression assays were done in a Clinical Laboratory Improvements Amendments certified laboratory facility. All genes were assessed for their ability to predict metastatic progression by receiver-operating-curve area-under-the-curve analyses. Multivariate analyses were done for the primary endpoint of metastatic progression, with variables including Gleason score, preoperative prostate-specific antigen concentration, seminal vesicle invasion, surgical margin status, extracapsular extension, lymph node invasion, and expression of the highest ranked gene.1008 patients were included in the study: 545 in the discovery cohort and 463 in the validation cohorts. The long non-coding RNA SChLAP1 was identified as the highest-ranked overexpressed gene in cancers with metastatic progression. Validation in three independent cohorts confirmed the prognostic value of SChLAP1 for metastatic progression. On multivariate modelling, SChLAP1 expression (high vs low) independently predicted metastasis within 10 years (odds ratio [OR] 2·45, 95% CI 1·70-3·53; p<0·0001). The only other variable that independently predicted metastasis within 10 years was Gleason score (8-10 vs 5-7; OR 2·14, 95% CI 1·77-2·58; p<0·0001).We identified and validated high SChLAP1 expression as significantly prognostic for metastatic disease progression of prostate cancer. Our findings suggest that further development of SChLAP1 as a potential biomarker, for treatment intensification in aggressive prostate cancer, warrants future study.Prostate Cancer Foundation, National Institutes of Health, Department of Defense, Early Detection Research Network, Doris Duke Charitable Foundation, and Howard Hughes Medical Institute.

Long non-coding RNAs (lncRNAs) represent an emerging layer of cancer biology, contributing to tumor proliferation, invasion, and metastasis. Here, we describe a role for the oncogenic lncRNA PCAT-1 in prostate cancer proliferation through cMyc. We find that PCAT-1–mediated proliferation is dependent on cMyc protein stabilization, and using expression profiling, we observed that cMyc is required for a subset of PCAT-1–induced expression changes. The PCAT-1–cMyc relationship is mediated through the post-transcriptional activity of the MYC 3′ untranslated region, and we characterize a role for PCAT-1 in the disruption of MYC-targeting microRNAs. To further elucidate a role for post-transcriptional regulation, we demonstrate that targeting PCAT-1 with miR-3667-3p, which does not target MYC, is able to reverse the stabilization of cMyc by PCAT-1. This work establishes a basis for the oncogenic role of PCAT-1 in cancer cell proliferation and is the first study to implicate lncRNAs in the regulation of cMyc in prostate cancer.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

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

Abstract Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise &lt;1% of the genome. Here we leverage a compendium of 58,648 long noncoding RNAs (lncRNAs) to subtype 947 breast cancer samples. We show that lncRNA-based profiling categorizes breast tumours by their known molecular subtypes in breast cancer. We identify a cohort of breast cancer-associated and oestrogen-regulated lncRNAs, and investigate the role of the top prioritized oestrogen receptor (ER)-regulated lncRNA, DSCAM-AS1 . We demonstrate that DSCAM-AS1 mediates tumour progression and tamoxifen resistance and identify hnRNPL as an interacting protein involved in the mechanism of DSCAM-AS1 action. By highlighting the role of DSCAM-AS1 in breast cancer biology and treatment resistance, this study provides insight into the potential clinical implications of lncRNAs in breast cancer.

Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration-resistant prostate cancer (CRPC). Although prior work has focused on targeting AR directly, co-activators of AR signaling, which may represent new therapeutic targets, are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin-MLL subunit. Menin expression is higher in CRPC than in both hormone-naive prostate cancer and benign prostate tissue, and high menin expression correlates with poor overall survival of individuals diagnosed with prostate cancer. Treatment with a small-molecule inhibitor of menin-MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a crucial co-activator of AR and a potential therapeutic target in advanced prostate cancer.

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome.

Beginning with precursor lesions, aberrant DNA methylation marks the entire spectrum of prostate cancer progression. We mapped the global DNA methylation patterns in select prostate tissues and cell lines using MethylPlex-next-generation sequencing (M-NGS). Hidden Markov model-based next-generation sequence analysis identified ∼68,000 methylated regions per sample. While global CpG island (CGI) methylation was not differential between benign adjacent and cancer samples, overall promoter CGI methylation significantly increased from ~12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues, respectively (P-value < 2 × 10(-16)). We found distinct patterns of promoter methylation around transcription start sites, where methylation occurred not only on the CGIs, but also on flanking regions and CGI sparse promoters. Among the 6691 methylated promoters in prostate tissues, 2481 differentially methylated regions (DMRs) are cancer-specific, including numerous novel DMRs. A novel cancer-specific DMR in the WFDC2 promoter showed frequent methylation in cancer (17/22 tissues, 6/6 cell lines), but not in the benign tissues (0/10) and normal PrEC cells. Integration of LNCaP DNA methylation and H3K4me3 data suggested an epigenetic mechanism for alternate transcription start site utilization, and these modifications segregated into distinct regions when present on the same promoter. Finally, we observed differences in repeat element methylation, particularly LINE-1, between ERG gene fusion-positive and -negative cancers, and we confirmed this observation using pyrosequencing on a tissue panel. This comprehensive methylome map will further our understanding of epigenetic regulation in prostate cancer progression.

High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts' (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found consistent antisense expression in at least 38% of annotated transcripts, which in general is positively correlated with sense gene expression. Investigation of sense/antisense pair expressions across tissue types revealed lineage-specific, ubiquitous and cancer-specific antisense loci transcription. Comparisons between tumor and normal samples identified both concordant (same direction) and discordant (opposite direction) sense/antisense expression patterns. Finally, we provide OncoNAT, a catalog of cancer-related genes with significant antisense transcription, which will enable future investigations of sense/antisense regulation in cancer. Using OncoNAT we identified several functional NATs, including NKX2-1-AS1 that regulates the NKX2-1 oncogene and cell proliferation in lung cancer cells. Overall, this study provides a comprehensive account of NATs and supports a role for NATs' regulation of tumor suppressors and oncogenes in cancer biology.

Abstract Long noncoding RNAs (lncRNA) have recently been associated with the development and progression of a variety of human cancers. However, to date, the interplay between known oncogenic or tumor-suppressive events and lncRNAs has not been well described. Here, the novel lncRNA, prostate cancer–associated transcript 29 (PCAT29), is characterized along with its relationship to the androgen receptor. PCAT29 is suppressed by DHT and upregulated upon castration therapy in a prostate cancer xenograft model. PCAT29 knockdown significantly increased proliferation and migration of prostate cancer cells, whereas PCAT29 overexpression conferred the opposite effect and suppressed growth and metastases of prostate tumors in chick chorioallantoic membrane assays. Finally, in prostate cancer patient specimens, low PCAT29 expression correlated with poor prognostic outcomes. Taken together, these data expose PCAT29 as an androgen-regulated tumor suppressor in prostate cancer. Implications: This study identifies PCAT29 as the first androgen receptor–repressed lncRNA that functions as a tumor suppressor and that its loss may identify a subset of patients at higher risk for disease recurrence. Visual Overview: http://mcr.aacrjournals.org/content/early/2014/07/31/1541-7786.MCR-14-0257/F1.large.jpg. Mol Cancer Res; 12(8); 1081–7. ©2014 AACR.

IntroductionThe aim of this study was to examine the effects of KRAS mutant subtypes on the outcome of patients with resected lung adenocarcinoma (AC).MethodsUsing clinical and sequencing data, we identified 179 patients with resected lung AC for whom KRAS mutational status was determined. A multivariate Cox model was used to identify factors associated with disease-free survival (DFS) and overall survival (OS). Publicly available mutation and gene-expression data from lung cancer cell lines and lung AC were used to assess whether distinct KRAS mutant variants have a different profile.ResultsPatients with KRAS mutation had a significantly shorter DFS compared with those with KRAS wild-type (p = 0.009). Patients with KRAS-G12C mutant tumors had significantly shorter DFS compared with other KRAS mutants and KRAS wild-type tumors (p < 0.001). In the multivariate Cox model, KRAS-G12C remained as an independent prognostic marker for DFS (Hazard ratio = 2.46, 95% confidence interval 1.51–4.00, p < 0.001) and for OS (Hazard ratio = 2.35, 95% confidence interval 1.35–4.10, p = 0.003). No genes were statistically significant when comparing the mutational or transcriptional profile of lung cancer cell lines and lung AC harboring KRAS-G12C with other KRAS mutant subtypes. Gene set enrichment analysis revealed that KRAS-G12C mutants overexpressed epithelial to mesenchymal transition genes and expressed lower levels of genes predicting KRAS dependency.ConclusionsKRAS-G12C mutation is associated with worse DFS and OS in resected lung AC. Gene-expression profiles in lung cancer cell lines and surgically resected lung AC revealed that KRAS-G12C mutants had an epithelial to mesenchymal transition and a KRAS-independent phenotype.

Long noncoding RNAs (IncRNAs) are increasingly implicated in cancer biology, contributing to essential cancer cell functions such as proliferation, invasion, and metastasis. In prostate cancer, several lncRNAs have been nominated as critical actors in disease pathogenesis. Among these, expression of PCGEM1 and PRNCR1 has been identified as a possible component in disease progression through the coordination of androgen receptor (AR) signaling (Yang et al., Nature 2013, see ref. [1]). However, concerns regarding the robustness of these findings have been suggested. Here, we sought to evaluate whether PCGEM1 and PRNCR1 are associated with prostate cancer. Through a comprehensive analysis of RNA-sequencing data (RNA-seq), we find evidence that PCGEM1 but not PRNCR1 is associated with prostate cancer. We employ a large cohort of >230 high-risk prostate cancer patients with long-term outcomes data to show that, in contrast to prior reports, neither gene is associated with poor patient outcomes. We further observe no evidence that PCGEM1 nor PRNCR1 interact with AR, and neither gene is a component of AR signaling. Thus, we conclusively demonstrate that PCGEM1 and PRNCR1 are not prognostic lncRNAs in prostate cancer and we refute suggestions that these lncRNAs interact in AR signaling.

The maturation of genomic technologies has enabled new discoveries in disease pathogenesis as well as new approaches to patient care. In pediatric oncology, patients may now receive individualized genomic analysis to identify molecular aberrations of relevance for diagnosis and/or treatment. In this context, several recent clinical studies have begun to explore the feasibility and utility of genomics-driven precision medicine. Here, we review the major developments in this field, discuss current limitations, and explore aspects of the clinical implementation of precision medicine, which lack consensus. Lastly, we discuss ongoing scientific efforts in this arena, which may yield future clinical applications.

Ribosome profiling (Ribo-Seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of noncanonical sites of ribosome translation outside the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7000 noncanonical ORFs are translated, which, at first glance, has the potential to expand the number of human protein CDSs by 30%, from ∼19,500 annotated CDSs to over 26,000 annotated CDSs. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of noncanonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome but searching for guidance on how to proceed. Here, we discuss the current state of noncanonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein coding."

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs. Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs.

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

Long noncoding RNAs (lncRNAs) are an emerging class of oncogenic molecules implicated in a diverse range of human malignancies. We recently identified SChLAP1 as a novel lncRNA that demonstrates outlier expression in a subset of prostate cancers, promotes tumor cell invasion and metastasis, and associates with lethal disease. Based on these findings, we sought to develop an RNA in situ hybridization (ISH) assay for SChLAP1 to 1) investigate the spectrum of SChLAP1 expression from benign prostatic tissue to metastatic castration-resistant prostate cancer and 2) to determine whether SChLAP1 expression by ISH is associated with outcome after radical prostatectomy in patients with clinically localized disease. The results from our current study demonstrate that SChLAP1 expression increases with prostate cancer progression, and high SChLAP1 expression by ISH is associated with poor outcome after radical prostatectomy in patients with clinically localized prostate cancer by both univariate (hazard ratio = 2.343, P = .005) and multivariate (hazard ratio = 1.99, P = .032) Cox regression analyses. This study highlights a potential clinical utility for SChLAP1 ISH as a novel tissue-based biomarker assay for outcome prognostication after radical prostatectomy.

Global 'multi-omics' profiling of cancer cells harbours the potential for characterizing the signalling networks associated with specific oncogenes. Here we profile the transcriptome, proteome and phosphoproteome in a panel of non-small cell lung cancer (NSCLC) cell lines in order to reconstruct targetable networks associated with KRAS dependency. We develop a two-step bioinformatics strategy addressing the challenge of integrating these disparate data sets. We first define an 'abundance-score' combining transcript, protein and phospho-protein abundances to nominate differentially abundant proteins and then use the Prize Collecting Steiner Tree algorithm to identify functional sub-networks. We identify three modules centred on KRAS and MET, LCK and PAK1 and β-Catenin. We validate activation of these proteins in KRAS-dependent (KRAS-Dep) cells and perform functional studies defining LCK as a critical gene for cell proliferation in KRAS-Dep but not KRAS-independent NSCLCs. These results suggest that LCK is a potential druggable target protein in KRAS-Dep lung cancers.

The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in 15 neurodegenerative diseases, termed tauopathies. One way to treat tauopathies may be to accelerate tau clearance, but the molecular mechanisms governing tau stability are not yet clear. We recently identified chemical probes that markedly accelerate the clearance of tau in cellular and animal models. In the current study, we used one of these probes in combination with immunoprecipitation and mass spectrometry to identify 48 proteins whose association with tau changes during the first 10 min after treatment. These proteins included known modifiers of tau proteotoxicity, such as ILF-2 (NFAT), ILF-3, and ataxin-2. A striking observation from the data set was that tau binding to heat shock protein 70 (Hsp70) decreased, whereas binding to Hsp90 significantly increased. Both chaperones have been linked to tau homeostasis, but their mechanisms have not been established. Using peptide arrays and binding assays, we found that Hsp70 and Hsp90 appeared to compete for binding to shared sites on tau. Further, the Hsp90-bound complex proved to be important in initiating tau clearance in cells. These results suggest that the relative levels of Hsp70 and Hsp90 may help determine whether tau is retained or degraded. Consistent with this model, analysis of reported microarray expression data from Alzheimer's disease patients and age-matched controls showed that the levels of Hsp90 are reduced in the diseased hippocampus. These studies suggest that Hsp70 and Hsp90 work together to coordinate tau homeostasis.

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

New discoveries regarding recurrent chromosomal aberrations in epithelial tumors have challenged the view that gene fusions play a minor role in these cancers. It is now known that recurrent fusions characterize significant subsets of prostate, breast, lung and renal-cell carcinomas, among others. This work has generated new insights into the molecular subtypes of tumors and highlighted important advances in bioinformatics, sequencing, and microarray technology as tools for gene fusion discovery. Given the ubiquity of tyrosine kinases and transcription factors in gene fusions, further interest in the potential 'druggability' of gene fusions with targeted therapeutics has also flourished. Nevertheless, the majority of chromosomal abnormalities in epithelial cancers remain uncharacterized, underscoring the limitations of our knowledge of carcinogenesis and the requirement for further research.

ABSTRACT Ribosome profiling (Ribo-seq) has catalyzed a paradigm shift in our understanding of the translational ‘vocabulary’ of the human genome, discovering thousands of translated open reading frames (ORFs) within long non-coding RNAs and presumed untranslated regions of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs due to uncertainties about their experimental reproducibility and physiological roles. Yet, it is indisputable that certain Ribo-seq ORFs make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unannotated by reference biological databases, this lack of characterisation will thwart research efforts examining their roles. Here, we outline the initial stages of a community-led effort supported by GENCODE / Ensembl, HGNC and UniProt to produce a consolidated catalog of human Ribo-seq ORFs.

Abstract In the decades following the discovery that genes encode proteins, scientists have tried to exhaustively and comprehensively characterize the human genome. Recent advances in computational methods along with transcriptomic and proteomic techniques have now shown that historically non‐coding genomic regions may contain non‐canonical open reading frames (ncORFs), which may encode functional miniproteins or otherwise exert regulatory activity through coding‐independent functions. Increasingly, it is clear that these ncORFs may play critical roles in major human diseases such as cancer. In this review, we summarize the history and current progress of ncORF research and explore the known functions of ncORFs and the miniproteins they may encode. We particularly highlight the emerging body of evidence supporting a role for ncORFs and miniproteins contributions in cancer. Finally, we provide a blueprint for high‐priority areas of future research for ncORFs in cancer, focusing on ncORF detection, functional characterization, and therapeutic intervention.

In the past decade, biomarker discovery has become ubiquitous in cancer research. However, despite this interest in biomarker research, few newly-characterized biomarkers have emerged as clinically-used entities. Here, we review the current state of biomarker research in cancer and identify challenges that stall many biomarker discovery efforts. We outline a model for systematic biomarker discovery, exemplified by recent efforts in prostate cancer, in which bioinformatics plays a central role in identifying promising new candidate biomarkers. Finally, we review the role of the National Cancer Institute’s Early Detection Research Network (EDRN) in biomarker studies and the importance of EDRN-led efforts to establish a research standard for more effective biomarker discovery efforts.

Abstract Systemic therapy for pediatric desmoid tumors has been challenged by a lack of high‐quality clinical evidence and potential adverse effects. The gamma‐secretase inhibitor nirogacestat has shown promising efficacy in adults. We report four cases of pediatric and young adult desmoid tumor patients (three with familial adenomatous polyposis [FAP] syndrome) who received nirogacestat on compassionate use. After a median of 13.5 months (range 6‐18), three had durable benefit: a complete response (Case 1); a partial response (Case 2); stable disease (Case 3). The fourth had disease progression after a partial response. No patient experienced grade 3 or 4 adverse events.

Abstract Congenital disorders of glycosylation (CDGs) are clinically heterogeneous disorders defined by a decreased ability to modify biomolecules with oligosaccharides. Critical disruptions in protein recognition, interaction, binding, and anchoring lead to broad physiological effects. Patients present with endocrinopathy, immunodeficiency, hepatopathy, coagulopathy, and neurodevelopmental impairment. Patients may experience mortality/morbidity associated with shock physiology that is frequently culture negative and poorly responsive to standard care. Oedema, pleural and pericardial effusions, ascites, proteinuria, and protein‐losing enteropathy are observed with an exaggerated inflammatory response. The negative serum protein steady state results from several mechanisms including reduced hepatic synthesis and secretion, increased consumption, and extravasation. Disruption of the glycocalyx, a layer of glycosylated proteins that lines the endothelium preventing thrombosis and extravasation, is a suspected cause of endothelial dysfunction in CDG patients. We performed a retrospective review of CDG patients admitted to our institution with acute illness over the past 2 years. Longitudinal clinical and laboratory data collected during the sick and well states were assessed for biomarkers of inflammation and efficacy of interventions. Six patients representing 4 CDG subtypes and 14 hospitalisations were identified. Acute D‐dimer elevation, proteinuria, decreased serum total protein levels, coagulation proteins, and albumin were observed with acute illness. Infusion of fresh frozen plasma, and in some cases protein C concentrate, was associated with clinical and biomarker improvement. This was notable with intra‐patient comparison of treated vs untreated courses. Use of endothelial barrier support therapy may reduce endothelial permeability by restoring both regulatory serum protein homeostasis and supporting the glycocalyx and is likely a critical component of care for this population.

Ribosome profiling (Ribo-seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of non-canonical sites of ribosome translation outside of the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7,000 non-canonical open reading frames (ORFs) are translated, which, at first glance, has the potential to expand the number of human protein-coding sequences by 30%, from ∼19,500 annotated CDSs to over 26,000. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of non-canonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome, but searching for guidance on how to proceed. Here, we discuss the current state of non-canonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein-coding".The human genome encodes thousands of non-canonical open reading frames (ORFs) in addition to protein-coding genes. As a nascent field, many questions remain regarding non-canonical ORFs. How many exist? Do they encode proteins? What level of evidence is needed for their verification? Central to these debates has been the advent of ribosome profiling (Ribo-seq) as a method to discern genome-wide ribosome occupancy, and immunopeptidomics as a method to detect peptides that are processed and presented by MHC molecules and not observed in traditional proteomics experiments. This article provides a synthesis of the current state of non-canonical ORF research and proposes standards for their future investigation and reporting.Combined use of Ribo-seq and proteomics-based methods enables optimal confidence in detecting non-canonical ORFs and their protein products.Ribo-seq can provide more sensitive detection of non-canonical ORFs, but data quality and analytical pipelines will impact results.Non-canonical ORF catalogs are diverse and span both high-stringency and low-stringency ORF nominations.A framework for standardized non-canonical ORF evidence will advance the research field.

The biological process of RNA translation is fundamental to cellular life and has wide-ranging implications for human disease. Yet, accurately delineating the variation in RNA translation represents a significant challenge. Here, we develop RiboTIE, a transformer model-based approach to map global RNA translation. We find that RiboTIE offers unparalleled precision and sensitivity for ribosome profiling data. Application of RiboTIE to normal brain and medulloblastoma cancer samples enables high-resolution insights into disease regulation of RNA translation.

Purpose Brain tumors have become the leading cause of cancer-related mortality in young patients. Novel effective therapies on the basis of the unique biology of each tumor are urgently needed. The goal of this study was to evaluate the feasibility, utility, and clinical impact of integrative clinical sequencing and genetic counseling in children and young adults with high-risk brain tumors. Patients and Methods Fifty-two children and young adults with brain tumors designated by the treating neuro-oncologist to be high risk (&gt; 25% chance for treatment failure; mean age, 10.2 years; range, 0 to 39 years) were enrolled in a prospective, observational, consecutive case series, in which participants underwent integrative clinical exome (tumor and germline DNA) and transcriptome (tumor RNA) sequencing and genetic counseling. Results were discussed in a multi-institutional brain tumor precision medicine teleconference. Results Sequencing revealed a potentially actionable germline or tumor alteration in 25 (63%) of 40 tumors with adequate tissue, of which 21 (53%) resulted in an impact on treatment or change of diagnosis. Platelet-derived growth factor receptor or fibroblast growth factor receptor pathway alterations were seen in nine of 20 (45%) glial tumors. Eight (20%) sequenced tumors harbored an oncogenic fusion isolated on RNA sequencing. Seventeen of 20 patients (85%) with glial tumors were found to have a potentially actionable result, which resulted in change of therapy in 14 (70%) patients. Patients with recurrent brain tumors receiving targeted therapy had a median progression-free survival (from time on therapy) of 4 months. Conclusion Selection of personalized agents for children and young adults with high-risk brain tumors on the basis of integrative clinical sequencing is feasible and resulted in a change in therapy in more than two thirds of children and young adults with high-risk glial tumors.

(Cell 171, 1559–1572.e1–e20; December 14, 2017) In the original Figure 4E, nucleotide numbers shown in the schematic representation of THOR were incorrect. The positions labeled as 143 and 196 should be 196 and 388, respectively. The corrected Figure 4E is shown below. Also, in the original figure legend of Figure S2, the order of the figure description was incorrect. The alphabetical labels (E), (F), (G), (H), (I), (J), and (K) should be (K), (J), (E), (F), (G), (H), and (I), respectively. The corrected legend of Figure S2 is shown below. The authors regret these errors and apologize for any confusion they may have caused. Figure S2. Characterization of THOR Transcript, Coding Potential, and Tissue Expression, Related to Figure 1 (A) Northern blot of endogenous THOR in H1299 cells, and of H1437 cells expressing LacZ control, THOR, and THOR with the addition of siRNA targeting THOR. Blot of gapdh provided as a control. (B) Bar plot depicting the qPCR expression of the long versus short THOR isoform. (C) qPCR expression of the long THOR isoform following addition of siRNA. (D) Northern blot of THOR in zebrafish kidney and testis. Blot of GAPDH provided as a control. (E) Coding probability scores for the transcripts were assessed by Coding Potential Assessment Tool (CPAT). NRAS and TP53 used as positive control, and SCHLAP1 as a negative control. (F) Coding probability scores for the PhyloCSF and CPC tools for THOR and MYC. Values less than 0 suggest a lack of coding potential. (G) Genome browser depiction of the THOR locus with aggregate ribosomal profiling track (red), aggregate poly-A RNA-seq track (green) and GENCODE v22 genome annotation obtained from the GWIPS-viz ribo-seq genome browser. (H) H&E image of the testis and surrounding tissue architecture. (I) H&E (left) and THOR ISH (right) for the human testis, rete, and adipose. (J) 3’ RACE for the THOR transcripts expressed by the lentiviral system. PCR agarose gel (left) shows two bands utilized in Sanger sequencing (right). (K) 5’ RACE for the THOR transcripts expressed by the lentiviral system. PCR agarose gel (left) confirms single band used in Sanger sequencing (right). Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNAHosono et al.CellDecember 14, 2017In BriefAn ultraconserved lncRNA promotes oncogenesis. Full-Text PDF Open Archive

Abstract Background: Long noncoding RNAs (lncRNAs) have recently been associated with the development and progression of a variety of human cancers. To date, the interplay between known oncogenic drivers, such as estrogen receptor (ER), and lncRNAs has not been well described. In this study, we identify M41 as the top outlier lncRNA in ER-positive vs ER-negative breast cancer and investigate its role in preclinical cancer phenotypes and clinical outcomes. Methods and Materials: RNA sequencing was performed on 89 breast cancer samples and cell lines, including 42 ER+ cases, and a modified cancer outlier analysis was used to identify lncRNAs enriched in ER-positive disease. To assess ER regulation of the top enriched lncRNA (M41), ChIP-Seq and ChIP-PCR was used to detect binding of ER to M41 promoter and qPCR was used to determine changes in M41 expression following 10 nM estradiol treatment in MCF7 and T47D cells. Following knockdown via siRNA, the impact of M41 expression was assessed on cell invasion, migration, proliferation, and anchorage-independent growth. The impact of M41 knockdown on tamoxifen sensitivity was assessed by cell proliferation studies in MCF7 cells with acquired tamoxifen resistance. Lastly, clinical associations between M41 expression and grade/node status, as well as event-free survival (EFS), was determined using ANOVA and Kaplan-Meier analyses of TCGA samples. Results: M41, an uncharacterized lncRNA located on chr21q22.2, was identified as the top outlier lncRNA in ER-positive vs ER-negative breast cancer. M41 demonstrated outlier expression (RPKM values&amp;gt;50) in 15% of ER-positive cancers, and was not significantly expressed in normal breast tissue. ChIP studies show that ER robustly binds to the M41 promoter. Estradiol stimulation significantly increased M41 expression in a time-dependent manner. Knockdown of M41 significantly inhibited all assessed oncogenic phenotypes in the ER-positive MCF7 and T47D cells, with a 60-80% decrease in both invasion and anchorage-independent growth, but had no effect in the ER-negative MDA-MB-231 cell line (which has minimal M41 expression). M41 expression was greater than 10-fold higher in tamoxifen-resistant MCF7 cells compared to parental controls (p&amp;lt;0.001), and knockdown of M41 restored tamoxifen sensitivity on cell proliferation studies; studies on the mechanism of M41-mediated tamoxifen resistance are ongoing. M41 overexpression was significantly correlated with node positivity, increasing grade, and luminal B subtype in ER-positive breast cancer samples (p&amp;lt;0.001). In TCGA samples, M41 overexpression was significantly associated with decreased EFS (p=0.003). Conclusion: We have identified M41 as an ER-associated oncogenic lncRNA that contributes to preclinical cancer phenotype, promotes tamoxifen resistance in cell line models, and associates with poor outcomes in clinical samples. We suggest that M41 represents a novel biomarker candidate for the prognosis of ER-positive breast cancers and provides new insight into the biological complexity of breast tumor biology. Citation Format: Felix Y Feng, Teng Ma, Corey Speers, Matthew K Iyer, Shuang Zhao, John R Prensner, James M Rae, Lori J Pierce, Arul M Chinnaiyan. The long noncoding RNA M41 promotes aggressiveness and tamoxifen resistance in ER-positive breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-1.

Menorrhagia is a common gynecologic complaint among adolescents, which rarely is secondary to malignancy. Burkitt lymphoma can mimic gynecologic malignancy, however it is rarely seen in adolescents. Burkitt lymphoma of the gynecologic tract requires early diagnosis and intervention for optimal outcomes.We report a case of a 15-year-old adolescent who had multiple admissions for menorrhagia that was thought to be secondary to anovulatory bleeding until pelvic ultrasound revealed a large 8-cm vaginal/cervical mass. Histologic examination of the biopsy specimen revealed Burkitt lymphoma, which was treated with chemotherapy leading to resolution of her menorrhagia.Burkitt lymphoma presenting as a vaginal/cervical mass is exceedingly rare, especially in the adolescent patient. Burkitt lymphoma is generally highly responsive to chemotherapy, and symptoms rapidly improve after initiation of treatment.

A key question in genome research is whether biologically active proteins are restricted to the ∼20,000 canonical, well-annotated genes, or rather extend to the many non-canonical open reading frames (ORFs) predicted by genomic analyses. To address this, we experimentally interrogated 553 ORFs nominated in ribosome profiling datasets. Of these 553 ORFs, 57 (10%) induced a viability defect when the endogenous ORF was knocked out using CRISPR/Cas9 in 8 human cancer cell lines, 257 (46%) showed evidence of protein translation when ectopically expressed in HEK293T cells, and 401 (73%) induced gene expression changes measured by transcriptional profiling following ectopic expression across 4 cell types. CRISPR tiling and start codon mutagenesis indicated that the biological effects of these non-canonical ORFs required their translation as opposed to RNA-mediated effects. We selected one of these ORFs, G029442 --renamed GREP1 (Glycine-Rich Extracellular Protein-1) -- for further characterization. We found that GREP1 encodes a secreted protein highly expressed in breast cancer, and its knock-out in 263 cancer cell lines showed preferential essentiality in breast cancer derived lines. Analysis of the secretome of GREP1-expressing cells showed increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth inhibitory effect of GREP1 knock-out. Taken together, these experiments suggest that the non-canonical ORFeome is surprisingly rich in biologically active proteins and potential cancer therapeutic targets deserving of further study.

Abstract High-throughput sequencing of polyA+ RNA (RNA-Seq) in human cancer shows remarkable potential to identify both novel disease-specific markers for clinical uses and uncharacterized aspects of tumor biology, particularly non-coding RNA (ncRNA) species. To illustrate this approach, we employed RNA-Seq on a cohort of 102 prostate tissues and cells lines. We found that aberrant expression profiles of novel tissue-specific ncRNAs distinguished benign, cancerous, and metastatic tumors, and we defined a core set of 121 novel ncRNAs whose dysregulation characterizes prostate cancer. Among these, a novel prostate-cancer specific ncRNA (termed PCAT-1) defined a subset of aggressive cancers with low expression of the epigenetic regulator EZH2, a component of the Polycomb Repressive Complex 2 (PRC2) commonly upregulated in metastatic cancers. In vitro chromatin immunoprecipitation, RNA immunoprecipitation, and drug treatment assays for core PRC2 genes indicated that the PRC2 complex directly binds and represses PCAT-1, and that PCAT-1 transcript reciprocally binds PRC2. By contrast, in vitro models with high levels of endogenous PCAT-1 transcript did not recapitulate PRC2-mediated repression, and in these cells siRNA-mediated knockdown of PCAT-1 showed a 25 – 50% decrease in cell proliferation. Using gene expression arrays, we determined that PCAT-1 contributes to the transcriptional regulation of genes in several key biological processes, including cell cycle. These data suggest that PCAT-1 exhibits two biological states: a PRC2-repressed state and an active state that promotes proliferation. Next, we showed that novel ncRNAs may serve a clinical purpose for the non-invasive detection and stratification of prostate cancer patients. We performed qPCR on patient urine samples (n=108) and found that a custom ncRNA expression signature, which includes PCAT-1, both diagnosed prostate cancer effectively and yielded prognostic information. Indeed, a high ncRNA expression signature value correlated with high-grade histology (Gleason score &amp;gt;=7 vs. Gleason score =6; p = 0.01). Taken together, the findings presented herein establish the utility of RNA-Seq to comprehensively identify unannotated ncRNAs, such as PCAT-1, implicated in cancer. Our data suggest that PCAT-1 promotes cell proliferation, that in its inactive state PCAT-1 is mechanistically repressed by PRC2, and that PCAT-1 may serve as a candidate biomarker for non-invasive clinical tests. We further speculate that applying these methodologies to other diseases may reveal key aspects of disease biology and clinically important biomarkers, particularly for diseases that currently lack good non-invasive tests in fluids such as blood serum or urine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4707. doi:10.1158/1538-7445.AM2011-4707

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&lt;div&gt;Abstract&lt;p&gt;Ewing's sarcoma family of tumors (ESFT) refers to aggressive malignancies which frequently harbor characteristic EWS-FLI1 or EWS-ERG genomic fusions. Here, we report that these fusion products interact with the DNA damage response protein and transcriptional coregulator PARP-1. ESFT cells, primary tumor xenografts, and tumor metastases were all highly sensitive to PARP1 inhibition. Addition of a PARP1 inhibitor to the second-line chemotherapeutic agent temozolamide resulted in complete responses of all treated tumors in an EWS-FLI1–driven mouse xenograft model of ESFT. Mechanistic investigations revealed that DNA damage induced by expression of EWS-FLI1 or EWS-ERG fusion genes was potentiated by PARP1 inhibition in ESFT cell lines. Notably, EWS-FLI1 fusion genes acted in a positive feedback loop to maintain the expression of PARP1, which was required for EWS-FLI–mediated transcription, thereby enforcing oncogene-dependent sensitivity to PARP-1 inhibition. Together, our findings offer a strong preclinical rationale to target the EWS-FLI1:PARP1 intersection as a therapeutic strategy to improve the treatment of ESFTs. &lt;i&gt;Cancer Res; 72(7); 1608–13. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

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&lt;div&gt;Abstract&lt;p&gt;Recurrent gene fusions involving oncogenic ETS transcription factors (including &lt;i&gt;ERG, ETV1&lt;/i&gt;, and &lt;i&gt;ETV4&lt;/i&gt;) have been identified in a large fraction of prostate cancers. The most common fusions contain the 5′ untranslated region of &lt;i&gt;TMPRSS2&lt;/i&gt; fused to &lt;i&gt;ERG&lt;/i&gt;. Recently, we identified additional 5′ partners in &lt;i&gt;ETV1&lt;/i&gt; fusions, including &lt;i&gt;TMPRSS2, SLC45A3, HERV-K_22q11.23, C15ORF21&lt;/i&gt;, and &lt;i&gt;HNRPA2B1&lt;/i&gt;. Here, we identify &lt;i&gt;ETV5&lt;/i&gt; as the fourth ETS family member involved in recurrent gene rearrangements in prostate cancer. Characterization of two cases with &lt;i&gt;ETV5&lt;/i&gt; outlier expression by RNA ligase–mediated rapid amplification of cDNA ends identified one case with a &lt;i&gt;TMPRSS2:ETV5&lt;/i&gt; fusion and one case with a &lt;i&gt;SLC45A3:ETV5&lt;/i&gt; fusion. We confirmed the presence of these fusions by quantitative PCR and fluorescence &lt;i&gt;in situ&lt;/i&gt; hybridization. &lt;i&gt;In vitro&lt;/i&gt; recapitulation of &lt;i&gt;ETV5&lt;/i&gt; overexpression induced invasion in RWPE cells, a benign immortalized prostatic epithelial cell line. Expression profiling and an integrative molecular concepts analysis of RWPE-&lt;i&gt;ETV5&lt;/i&gt; cells also revealed the induction of an invasive transcriptional program, consistent with &lt;i&gt;ERG&lt;/i&gt; and &lt;i&gt;ETV1&lt;/i&gt; overexpression in RWPE cells, emphasizing the functional redundancy of ETS rearrangements. Together, our results suggest that the family of 5′ partners previously identified in &lt;i&gt;ETV1&lt;/i&gt; gene fusions can fuse with other ETS family members, suggesting numerous rare gene fusion permutations in prostate cancer. [Cancer Res 2008;68(1):73–80]&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Recurrent gene fusions involving oncogenic ETS transcription factors (including &lt;i&gt;ERG, ETV1&lt;/i&gt;, and &lt;i&gt;ETV4&lt;/i&gt;) have been identified in a large fraction of prostate cancers. The most common fusions contain the 5′ untranslated region of &lt;i&gt;TMPRSS2&lt;/i&gt; fused to &lt;i&gt;ERG&lt;/i&gt;. Recently, we identified additional 5′ partners in &lt;i&gt;ETV1&lt;/i&gt; fusions, including &lt;i&gt;TMPRSS2, SLC45A3, HERV-K_22q11.23, C15ORF21&lt;/i&gt;, and &lt;i&gt;HNRPA2B1&lt;/i&gt;. Here, we identify &lt;i&gt;ETV5&lt;/i&gt; as the fourth ETS family member involved in recurrent gene rearrangements in prostate cancer. Characterization of two cases with &lt;i&gt;ETV5&lt;/i&gt; outlier expression by RNA ligase–mediated rapid amplification of cDNA ends identified one case with a &lt;i&gt;TMPRSS2:ETV5&lt;/i&gt; fusion and one case with a &lt;i&gt;SLC45A3:ETV5&lt;/i&gt; fusion. We confirmed the presence of these fusions by quantitative PCR and fluorescence &lt;i&gt;in situ&lt;/i&gt; hybridization. &lt;i&gt;In vitro&lt;/i&gt; recapitulation of &lt;i&gt;ETV5&lt;/i&gt; overexpression induced invasion in RWPE cells, a benign immortalized prostatic epithelial cell line. Expression profiling and an integrative molecular concepts analysis of RWPE-&lt;i&gt;ETV5&lt;/i&gt; cells also revealed the induction of an invasive transcriptional program, consistent with &lt;i&gt;ERG&lt;/i&gt; and &lt;i&gt;ETV1&lt;/i&gt; overexpression in RWPE cells, emphasizing the functional redundancy of ETS rearrangements. Together, our results suggest that the family of 5′ partners previously identified in &lt;i&gt;ETV1&lt;/i&gt; gene fusions can fuse with other ETS family members, suggesting numerous rare gene fusion permutations in prostate cancer. [Cancer Res 2008;68(1):73–80]&lt;/p&gt;&lt;/div&gt;

Supplementary Figures 1-2, Tables 1-2 from Characterization of TMPRSS2:ETV5 and SLC45A3:ETV5 Gene Fusions in Prostate Cancer

&lt;div&gt;Abstract&lt;p&gt;Ewing's sarcoma family of tumors (ESFT) refers to aggressive malignancies which frequently harbor characteristic EWS-FLI1 or EWS-ERG genomic fusions. Here, we report that these fusion products interact with the DNA damage response protein and transcriptional coregulator PARP-1. ESFT cells, primary tumor xenografts, and tumor metastases were all highly sensitive to PARP1 inhibition. Addition of a PARP1 inhibitor to the second-line chemotherapeutic agent temozolamide resulted in complete responses of all treated tumors in an EWS-FLI1–driven mouse xenograft model of ESFT. Mechanistic investigations revealed that DNA damage induced by expression of EWS-FLI1 or EWS-ERG fusion genes was potentiated by PARP1 inhibition in ESFT cell lines. Notably, EWS-FLI1 fusion genes acted in a positive feedback loop to maintain the expression of PARP1, which was required for EWS-FLI–mediated transcription, thereby enforcing oncogene-dependent sensitivity to PARP-1 inhibition. Together, our findings offer a strong preclinical rationale to target the EWS-FLI1:PARP1 intersection as a therapeutic strategy to improve the treatment of ESFTs. &lt;i&gt;Cancer Res; 72(7); 1608–13. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

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Supplementary Figures 1-2, Tables 1-2 from Characterization of TMPRSS2:ETV5 and SLC45A3:ETV5 Gene Fusions in Prostate Cancer

&lt;div&gt;Abstract&lt;p&gt;Impairment of double-stranded DNA break (DSB) repair is essential to many cancers. However, although mutations in DSB repair proteins are common in hereditary cancers, mechanisms of impaired DSB repair in sporadic cancers remain incompletely understood. Here, we describe the first role for a long noncoding RNA (lncRNA) in DSB repair in prostate cancer. We identify &lt;i&gt;PCAT-1&lt;/i&gt;, a prostate cancer outlier lncRNA, which regulates cell response to genotoxic stress. &lt;i&gt;PCAT-1&lt;/i&gt; expression produces a functional deficiency in homologous recombination through its repression of the &lt;i&gt;BRCA2&lt;/i&gt; tumor suppressor, which, in turn, imparts a high sensitivity to small-molecule inhibitors of &lt;i&gt;PARP1&lt;/i&gt;. These effects reflected a posttranscriptional repression of the &lt;i&gt;BRCA2&lt;/i&gt; 3′UTR by &lt;i&gt;PCAT-1&lt;/i&gt;. Our observations thus offer a novel mechanism of “BRCAness” in sporadic cancers. &lt;i&gt;Cancer Res; 74(6); 1651–60. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;PDF file - 2687KB, Supplementary Figure S1: Effect of PCAT-1 on RAD51 foci formation. Supplementary Figure S2: PCAT-1 impairs homologous recombination. Supplementary Figure S3: PCAT-1 expression alters gamma-H2AX formation. Supplementary Figure S4: PCAT-1 expression effects clonogenic survival when treated with PARP inhibitors Olaparib or ABT-888. Supplementary Figure S5: PCAT-1 expression effects clonogenic survival when treated with ionizing radiation. Supplementary Figure S6: Treatment of PCAT-1 isogenic cells with ABT-888. Supplementary Figure S7: PCAT-1 overexpression in RWPE engenders sensitivity to genotoxic stress. Supplementary Figure 8: BRCA2 knockdown rescues RAD51 foci formation in LNCAP shPCAT1 cells after PARP inhibition. Supplementary Figure 9: PCAT-1 expression does not impact cell cycle distribution. Supplementary Figure S10: Effect of PCAT-1 in vivo on Olaparib treatment. Supplementary Figure S11: PCAT-1 does not operate via an epigenetic or STAU1-based mechanism. Supplementary Figure 12: PCAT-1 is cytoplasmic and effects target mRNA stability. Supplementary Figure 13: PCAT-1 overexpression in RWPE cells. Supplementary Figure 14: A model of PCAT-1 function in homologous recombination in prostate cancer. Supplementary Table 1: Primer Pairs used in this study.&lt;/p&gt;

&lt;p&gt;PDF file - 2687KB, Supplementary Figure S1: Effect of PCAT-1 on RAD51 foci formation. Supplementary Figure S2: PCAT-1 impairs homologous recombination. Supplementary Figure S3: PCAT-1 expression alters gamma-H2AX formation. Supplementary Figure S4: PCAT-1 expression effects clonogenic survival when treated with PARP inhibitors Olaparib or ABT-888. Supplementary Figure S5: PCAT-1 expression effects clonogenic survival when treated with ionizing radiation. Supplementary Figure S6: Treatment of PCAT-1 isogenic cells with ABT-888. Supplementary Figure S7: PCAT-1 overexpression in RWPE engenders sensitivity to genotoxic stress. Supplementary Figure 8: BRCA2 knockdown rescues RAD51 foci formation in LNCAP shPCAT1 cells after PARP inhibition. Supplementary Figure 9: PCAT-1 expression does not impact cell cycle distribution. Supplementary Figure S10: Effect of PCAT-1 in vivo on Olaparib treatment. Supplementary Figure S11: PCAT-1 does not operate via an epigenetic or STAU1-based mechanism. Supplementary Figure 12: PCAT-1 is cytoplasmic and effects target mRNA stability. Supplementary Figure 13: PCAT-1 overexpression in RWPE cells. Supplementary Figure 14: A model of PCAT-1 function in homologous recombination in prostate cancer. Supplementary Table 1: Primer Pairs used in this study.&lt;/p&gt;

&lt;p&gt;Supplementary Table S1. shRNA and primer sequence table.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 1. Gene sequence information of PCAT29.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 3. Expression of PCAT29 in VCaP and LNCaP cells.&lt;/p&gt;

&lt;p&gt;Supplementary Table S2. Positive and negative correlations.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 4. Overlap of positive and negative correlations.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 2. Expression of PCAT29 in LNCaP cells.&lt;/p&gt;

Supplementary Methods and Data (posted 7/5/2011) from Characterization of &lt;i&gt;KRAS&lt;/i&gt; Rearrangements in Metastatic Prostate Cancer

Supplementary Methods, Figures, Tables, and References from Characterization of &lt;i&gt;KRAS&lt;/i&gt; Rearrangements in Metastatic Prostate Cancer

&lt;p&gt;Supplementary Table S2. Positive and negative correlations.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 3. Expression of PCAT29 in VCaP and LNCaP cells.&lt;/p&gt;

&lt;p&gt;Supplementary Table S1. shRNA and primer sequence table.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 2. Expression of PCAT29 in LNCaP cells.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 4. Overlap of positive and negative correlations.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 1. Gene sequence information of PCAT29.&lt;/p&gt;

Supplementary Methods, Figures, Tables, and References from Characterization of &lt;i&gt;KRAS&lt;/i&gt; Rearrangements in Metastatic Prostate Cancer

Supplementary Methods and Data (posted 7/5/2011) from Characterization of &lt;i&gt;KRAS&lt;/i&gt; Rearrangements in Metastatic Prostate Cancer

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets.

Abstract High-risk medulloblastoma is one of the most recalcitrant pediatric cancers, and children with MYC-amplified disease frequently succumb to relapsed disease. Extensive analyses of the coding genome in this disease have characterized additional somatic events in some subsets of patients, though most tumors lack targetable mutations and do not yield insights regarding their aggressive behavior. At the same time, medulloblastoma is known to exhibit extensive rewiring of translational control in MYC-driven tumors, consistent with recent genetic evidence that the impact of this transcription factor on control of mRNA translation may be the most critical aspect of its function during tumorigenesis. Therefore, to propose previously unknown mechanisms for this disease, we have investigated the functional impact of translation of non-canonical open reading frames (ORFs) across medulloblastoma model systems. We demonstrate that these ORFs are commonly translated in medulloblastoma model systems and patient tumors, correlating with disease subtype. Using genome-wide CRISPR/Cas9 screens, we found that ORFs are frequently essential for cell survival in medulloblastoma and describe widespread reliance on upstream open reading frames (uORFs) in particular. From these, we identify a uORF in the ASNSD1 gene that is selectively upregulated and required for maintenance of cell survival by coordinating the function of the prefoldin-like complex, a poorly understood complex implicated in post-translational control. Together, our findings provide a blueprint for oncogenic uORFs as critical disease mediators both in medulloblastoma and, by extension, human cancers more broadly.

&lt;div&gt;Abstract&lt;p&gt;Long noncoding RNAs (lncRNA) have recently been associated with the development and progression of a variety of human cancers. However, to date, the interplay between known oncogenic or tumor-suppressive events and lncRNAs has not been well described. Here, the novel lncRNA, prostate cancer–associated transcript 29 (&lt;i&gt;PCAT29&lt;/i&gt;), is characterized along with its relationship to the androgen receptor. &lt;i&gt;PCAT29&lt;/i&gt; is suppressed by DHT and upregulated upon castration therapy in a prostate cancer xenograft model. &lt;i&gt;PCAT29&lt;/i&gt; knockdown significantly increased proliferation and migration of prostate cancer cells, whereas &lt;i&gt;PCAT29&lt;/i&gt; overexpression conferred the opposite effect and suppressed growth and metastases of prostate tumors in chick chorioallantoic membrane assays. Finally, in prostate cancer patient specimens, low &lt;i&gt;PCAT29&lt;/i&gt; expression correlated with poor prognostic outcomes. Taken together, these data expose &lt;i&gt;PCAT29&lt;/i&gt; as an androgen-regulated tumor suppressor in prostate cancer.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Implications:&lt;/b&gt; This study identifies &lt;i&gt;PCAT29&lt;/i&gt; as the first androgen receptor–repressed lncRNA that functions as a tumor suppressor and that its loss may identify a subset of patients at higher risk for disease recurrence.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Visual Overview:&lt;/b&gt; &lt;a href="http://mcr.aacrjournals.org/content/early/2014/07/31/1541-7786.MCR-14-0257/F1.large.jpg" target="_blank"&gt;http://mcr.aacrjournals.org/content/early/2014/07/31/1541-7786.MCR-14-0257/F1.large.jpg&lt;/a&gt;. &lt;i&gt;Mol Cancer Res; 12(8); 1081–7. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Long noncoding RNAs (lncRNA) have recently been associated with the development and progression of a variety of human cancers. However, to date, the interplay between known oncogenic or tumor-suppressive events and lncRNAs has not been well described. Here, the novel lncRNA, prostate cancer–associated transcript 29 (&lt;i&gt;PCAT29&lt;/i&gt;), is characterized along with its relationship to the androgen receptor. &lt;i&gt;PCAT29&lt;/i&gt; is suppressed by DHT and upregulated upon castration therapy in a prostate cancer xenograft model. &lt;i&gt;PCAT29&lt;/i&gt; knockdown significantly increased proliferation and migration of prostate cancer cells, whereas &lt;i&gt;PCAT29&lt;/i&gt; overexpression conferred the opposite effect and suppressed growth and metastases of prostate tumors in chick chorioallantoic membrane assays. Finally, in prostate cancer patient specimens, low &lt;i&gt;PCAT29&lt;/i&gt; expression correlated with poor prognostic outcomes. Taken together, these data expose &lt;i&gt;PCAT29&lt;/i&gt; as an androgen-regulated tumor suppressor in prostate cancer.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Implications:&lt;/b&gt; This study identifies &lt;i&gt;PCAT29&lt;/i&gt; as the first androgen receptor–repressed lncRNA that functions as a tumor suppressor and that its loss may identify a subset of patients at higher risk for disease recurrence.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Visual Overview:&lt;/b&gt; &lt;a href="http://mcr.aacrjournals.org/content/early/2014/07/31/1541-7786.MCR-14-0257/F1.large.jpg" target="_blank"&gt;http://mcr.aacrjournals.org/content/early/2014/07/31/1541-7786.MCR-14-0257/F1.large.jpg&lt;/a&gt;. &lt;i&gt;Mol Cancer Res; 12(8); 1081–7. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

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

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