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

Supplementary Figures 1-2 from Inactivation of Apc in the Mouse Prostate Causes Prostate Carcinoma

Supplementary Methods, Figures, Tables, and References from Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in the Western hemisphere. Tumor-specific chromosomal translocations, characteristic findings in several human malignancies that directly lead to malignant transformation, have not been identified in CLL. Using paired-end transcriptome sequencing, we identified recurrent and reciprocal RNA chimeras involving yippee like 5 ( YPEL5 ) and serine/threonine-protein phosphatase PP1-beta-catalytic subunit ( PPP1CB ) in CLL. Two of seven index cases (28%) harbored the reciprocal RNA chimeras in our initial screening. Using quantitative real-time PCR (q real-time PCR), YPEL5/PPP1CB and PPP1CB/YPEL5 fusion transcripts were detected in 97 of 103 CLL samples (95%) but not in paired normal samples, benign lymphocytes, or various unrelated cancers. Whole-genome sequencing and Southern blotting demonstrated no evidence for a genomic fusion between YPEL5 and PPP1CB . YPEL5/PPP1CB chimera, when introduced into mammalian cells, expressed a truncated PPP1CB protein that demonstrated diminished phosphatase activity. PPP1CB silencing resulted in enhanced proliferation and colony formation of MEC1 and JVM3 cells, implying a role in the pathogenesis of mature B-cell leukemia. These studies uncover a potential role for recurrent RNA chimeras involving phosphatases in the pathogenesis of a common form of leukemia.

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 long non-coding RNA (lncRNA) species >250 bp in length. To illustrate this approach, we employed RNA-Seq on a cohort of 102 prostate tissues and cell lines and performed ab initio transcriptome assembly to discover unannotated ncRNAs with cancer-specific expression patterns. In total, we observed that ∼20% of the prostate cancer transcriptome represents unannotated, polyadenylated RNA species that were enriched for activate chromatin marks (H3K4me3, RNA polymerase II) defined by ChIP-Seq data. From 1,859 intergenic lncRNAs overall, we nominated 121 such Prostate Cancer Associated Transcripts (PCATs) that collectively performed as well as known biomarkers in differentiating benign, cancerous, and metastatic tissues. Among these, we identified an uncharacterized two-exon, polyadenylated lncRNA, PCAT-1, as a prostate cancer outlier and a prostate-specific driver of cancer cell proliferation. Mechanistically, microarray profiling following modulation of PCAT-1 expression in vitro defined a core set of genes repressed by PCAT-1, identifying this lncRNA as a transcriptional repressor of genes implicated in DNA maintenance processes, such as CENPF and BRCA2. Interestingly, when PCAT-1 expression is low, it is negatively regulated by the Polycomb Repressive Complex 2 (PRC2), which directly binds the PCAT-1 promoter in vitro. Supporting this, human tumor samples with high PCAT-1 or high PRC2 expression were exclusive and defined distinct molecular subtypes distinguished by gene expression signatures of PCAT-1-repressed target genes. PRC2-based repression of PCAT-1 was reversible by inhibition of PRC2. BRCA2 inactivation is known to sensitize cancer cells to small molecular inhibitors of the PARP1 DNA repair enzyme by impairing homologous recombation of double-stranded DNA breaks, suggesting that PCAT-1 expression may sensitize prostate cells to PARP1 inhibitors. To test this, we generated two isogenic overexpression models of PCAT-1, and one stable knockdown model, in prostate cell lines. PCAT-1-overexpressing cell lines (Du145 and RWPE) showed a marked increase in sensitivity to two PARP inhibitors, whereas the stable knockdown cells (LNCaP) showed decreased sensitivity to PARP inhibitors. This was accompanied by a decrease in RAD51 foci formation, a hallmark of DNA repair via homologous recombination, in PCAT-1 overexpressing cells, and an increase in RAD51 foci formation in PCAT-1 knockdown cells. MCF7 breast cancer cells overexpressing PCAT-1 did not recapitulate these phenotypes, supporting our observation that PCAT-1 is a prostate-specific gene. These data define PCAT-1 as a novel prostate cancer-associated lncRNA functionally involved in tumor cell proliferation and sensitivity to PARP inhibitor therapy by suppressing homologous recombination. High PCAT-1 expression may therefore represent a potential biomarker for patient response to PARP targeted therapy. The findings presented herein establish the utility of RNA-Seq to comprehensively identify disease-associated lncRNAs and suggest that clinical translation of these findings may improve the stratification of cancer subtypes. Citation Format: John R. Prensner, Hari K. Iyer, Christopher A. Maher, Felix Feng, Arul M. Chinnaiyan, Matthew K. Iyer, Wei Chen, O. Alejandro Balbin, Saravana M. Dhanasekaran, Qi Cao, Xuhong Cao, Xiaojun Jing, Daniel Robinson. Transcriptome sequencing identifies PCAT-1, a novel lncRNA implicated in prostate cancer progression and therapeutic response to PARP inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C36.

It is difficult to imagine a field that is changing as rapidly as pathology. A convergence of factors including not only scientific and technological advances but also changes in business models is transforming the field, particularly in the area of cancer diagnostics. The authors examine 8 themes, or "forces of change," in pathology and speculate on how these will affect pathology sign-out and the future role of pathologists in patient care.

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.

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

Abstract Long intervening non-coding RNAs (lincRNAs) have been implicated in diverse biological processes including p53 signaling and chromatin remodeling, but have not been thoroughly profiled in human cancers. Here, we have developed an approach for ab initio reconstruction of poly-A+ transcriptome sequencing (RNA-seq) data for the unbiased discovery of novel transcripts. To accomplish this, we developed AssemblyLine, a method that clusters and filters large collections of transcripts to produce a consensus transcriptome. To demonstrate AssemblyLine, we sequenced a cohort comprised of 81 prostatic tissues (20 benign, 47 localized tumors, and 14 metastases) and 21 prostatic cell lines using the Illumina Genome Analyzer II and generated 1.723 billion sequence fragments. We successfully aligned 1.42 billion reads with Tophat – a program capable of ab initio splice junction discovery – and then used Cufflinks to model sample-specific transcriptomes totaling 8.25 million transcripts. AssemblyLine condensed the 8.25 million original transcripts into 35,415 distinct transcriptional loci, of which 1,859 (5.2%) represented candidate lincRNAs that lacked genomic overlap with known gene annotations. These putative RNAs lacked robust open reading frames suggesting that the vast majority were non-coding. Further, they exhibited evolutionary conservation and were enriched with histone modifications supporting independent transcriptional start sites and active transcription. Together, these results add confidence to AssemblyLine's nomination process and suggest that these novel lincRNAs may be transcriptionally active in prostate cancer. We then selected 106 transcripts that were differentially expressed in localized prostate cancer when compared to benign adjacent tissue (False Discovery Rate < 0.05), and 15 transcripts with profound cancer outlier expression profiles for further study. These 121 Prostate Cancer Associated Transcripts (PCATs) accurately classified benign, localized, and metastatic prostate cancer tissues by unsupervised hierarchical clustering. Consistent with AssemblyLine's nominations, PCR-based experiments on selected transcripts in an independent tissue cohort showed high validation rates for the transcript structure and expression level predictions. Furthermore, in vitro studies of PCAT-1, a novel lincRNA observed in our dataset as highly upregulated in prostate cancer, revealed direct regulation by the histone methyltransferase EZH2. siRNA knockdown of PCAT-1 in LNCaP, a prostate cancer cell line, caused a 25-50% decrease in cell proliferation. Thus, this study establishes a paradigm for ab initio transcriptome annotation and discovery of novel lincRNAs in cancer tissues. Further, these results provide intriguing evidence that lincRNAs are aberrantly expressed and may play a role in prostate cancer progression. 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 929. doi:10.1158/1538-7445.AM2011-929

Abstract In cancer, the genome undergoes global DNA methylation changes, which contributes to disease progression. We mapped the global DNA methylation patterns in prostate tissues and cells (n=19) from fifty nanograms of genomic DNA. The methylated regions enriched by Methylplex technology, were subjected -Next Generation Sequencing (M-NGS). A total of 28 lanes of next generation sequencing yielded approximately 530 million sequence reads. The reads uniquely mapped to human genome were analyzed by H-Peak program to identify 1.8 million methylated regions from the 28 runs. While twenty percent of all CpG islands (CGIs) (68,508) were methylated in tissues, the promoter CGI methylation gradually increased from ∼12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues. However surprisingly, the total methylation events in intergenic/intronic regions between benign adjacent and cancer tissues were largely comparable. We found distinct patterns in promoter methylation around transcription start sites where methylation occurred directly on the CGIs, flanking regions and on CGI sparse promoters. Among the 6,691 methylated promoters in prostate tissues, 2481 differentially methylated regions (DMRs) were cancer specific and several previously studied targets were among them. A novel cancer specific DMR in WFDC2 promoter showed 77% methylation in cancer (17/22), 100% methylation in transformed prostate cell lines (6/6), none in the benign tissues (0/10) and normal PrEC cells. Integration of LNCaP DNA methylation and H3K4me3 data suggested a role for DNA methylation in alternate transcription start site utilization. Specifically, we detail the regulation of RASSF1, a gene previously reported to be silenced through DNA methylation in PCa and NDRG2. We show that variant-1 of RASSF1, but not variant-2 or -3, is specifically silenced in LNCaP through promoter methylation, and normal cells do not exhibit this phenomenon. Treatment with 5-Aza-2-deoxycytidine preferentially induced re-expression of variant-1 of RASSF1 in LNCaP, confirming transcriptional regulation of this isoform through DNA methylation. The methylated promoters lacking CGIs showed sparse H3K4me3 modification, interestingly methylated promoters containing CGIs showed a mutually exclusive H3K4me3/DNA methylation marks. Finally, we observed a difference in the methylation of LINE-1 elements between transcription factor ERG positive and negative cancers. The comprehensive methylome map presented here will further our understanding of epigenetic regulation of the prostate cancer genome. 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 4840. doi:10.1158/1538-7445.AM2011-4840

The human skeleton is affected by mutations in low-density lipoprotein receptor-related protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with osteocyte-specific expression of inducible Lrp5 mutations that cause high and low bone mass phenotypes in humans. We found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also induced an Lrp5 mutation in cells that form the appendicular skeleton but not in cells that form the axial skeleton; we observed that bone properties were altered in the limb but not in the spine. These data indicate that Lrp5 signaling functions locally, and they suggest that increasing LRP5 signaling in mature bone cells may be a strategy for treating human disorders associated with low bone mass, such as osteoporosis.

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 >=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

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.

Canonical Wnt signaling has emerged as an important pathway that underlies the initia nottion of prostate cancer. Both human cancers and mouse models have confirmed that mutations or altered expression of components of this pathway are associated with prostate tumors. Additionally, several reports suggest that this pathway plays a key role in the establishment of skeletal metastasis. This review discusses our current knowledge of the Wnt signaling pathway in the development of prostate cancer. First, we will overview the Wnt signaling pathway to provide background for the rest of the discussion. We will then review the literature on the role of this pathway and the down notstream effector, beta-catenin, in the development and progression of prostate cancer and skeletal metastasis. We will also discuss reports that suggest that beta-catenin can directly interact with the androgen receptor to modulate its activity. These recent developments may provide insight into how tumor growth can be achieved under androgen deprivation. Finally, we speculate on how the pathway may be targeted for therapeutic treatment and what agents may be available to achieve this goal.

Abstract Alterations of the Wnt/β-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/β-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre–mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of β-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of β-catenin associated with prostate carcinoma suggests a role for β-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis. [Cancer Res 2007;67(6):2490–6]