Anirban Sahu

The Landscape of Long Noncoding RNAs in the Human Transcriptome

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.

The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex

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.

RNA biomarkers associated with metastatic progression in prostate cancer: a multi-institutional high-throughput analysis of SChLAP1.

BACKGROUND 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. METHODS 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. FINDINGS 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). INTERPRETATION 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. FUNDING Prostate Cancer Foundation, National Institutes of Health, Department of Defense, Early Detection Research Network, Doris Duke Charitable Foundation, and Howard Hughes Medical Institute.

The lncRNA PCAT29 Inhibits Oncogenic Phenotypes in Prostate Cancer

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. Visual Overview

A Novel RNA In Situ Hybridization Assay for the Long Noncoding RNA SChLAP1 Predicts Poor Clinical Outcome After Radical Prostatectomy in Clinically Localized Prostate Cancer

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.

Insights into Chinese prostate cancer with RNA-seq

The prevalence of certain driver mutations in prostate cancer varies across ethnic populations. A recent study uses RNA-seq to profile the mutational landscape of prostate cancer in a Chinese population to gain additional insights on ethnic variation in this disease.

Abstract 541: The role of long noncoding RNA SChLAP1 in prostate cancer

Prostate cancer is the second most common epithelial cancer and second leading cause of cancer death in men. Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority of patients. The molecular basis for this clinical heterogeneity remains incompletely understood. Long noncoding RNAs (lncRNAs) have emerged as a prominent layer of transcriptional regulation implicated in various biological and disease processes, including several types of cancer. Therefore, we hypothesized that lncRNAs may play a role in mediating aggressive prostate cancer. Previously, our lab utilized RNA sequencing across a cohort of prostate tissues to discover differentially expressed lncRNAs in cancer versus benign samples. We identifed a novel transcript termed SChLAP1 (Second Chromosome Locus Associated with Prostate-1) overexpressed in a subset of localized and metastatic cancers. Clinical analyses showed that SChLAP1 levels independently predict poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo experiments indicated that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, we found that SChLAP1 binds to and antagonizes the genome-wide localization of the tumor-suppressive SWI/SNF nucleosome-remodeling complex. Given the scaffolding capabilities of lncRNAs, previous studies describing an inhibitory role of SWI/SNF on PRC2 function, and the known oncogenic role of PRC2 in prostate cancer, we hypothesized that SChLAP1 may mediate PRC2 activity in conjunction with SWI/SNF to promote aggressive disease. Here, we show that a SChLAP1-associated gene signature nominates PRC2-related concepts, and in vitro studies indicate an interaction between SChLAP1 and PRC2 that enhances PRC2 genome-wide localization as well as its histone methyltransferase activity. These results suggest a mechanistic model in which SChLAP1 simultaneously engages multiple epigenetic complexes, inhibiting tumor-suppressive functions of SWI/SNF while enhancing oncogenic activity of PRC2, which leads to aggressive prostate cancer. Citation Format: Anirban Sahu, Matthew K. Iyer, John R. Prensner, Arul M. Chinnaiyan. The role of long noncoding RNA SChLAP1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 541. doi:10.1158/1538-7445.AM2014-541

Abstract 5214: Reconstructing targetable pathways in KRAS dependent lung cancer by integrating transcriptome, proteome and phosphoproteome.

Activating mutations in the Ras oncogenes characterize 20-40% of all non-small cell lung cancer (NSCLC), the leading cause of cancer mortality in the United States, which establishes Ras genes as the most commonly mutated oncogenes in this malignancy. Mutations in the KRAS oncogene characterize more than 20% of all NSCLC malignancies and recent studies suggest that those tumors can be divided into KRAS dependent (KRAS-Dep) and KRAS independent (KRAS-Ind), according to their dependency on KRAS mutant for cell survival and proliferation. Although, the KRAS signaling pathway has been considerably studied using high throughput transcriptomic or proteomic technologies in isolation, the specific network of effector genes and proteins that drives carcinogenesis in NSCLC is still far from being understood. Integrative profiling with different omics technologies harbors the potential for characterizing the specific network of effector proteins associated with specific oncogenes. We developed a bioinformatics approach to reconstruct active and targetable networks associated with KRAS dependency in non-small cell lung cancer (NSCLC). By integrating matched global transcriptomics, proteomics and phosphoproteomics datasets, we stratified NSCLCs into those dependent on, or independent of, KRAS signaling by defining the S score that combines transcript, protein and phosphoprotein abundance in order to identify differential expressed proteins. We demonstrate that the S score outperforms naive integration methods and improves the functional specificity of the proteins identified as differential expressed. Next, we reconstruct targetable network modules associated with KRAS dependency by employing the Prize Collecting Steiner Tree (PCST) algorithm to formulate the module reconstruction. This reconstruction identified three conspicuous modules centered on KRAS and MET, LCK and PAK1, and B-Catenin. We validated activation of those proteins in KRAS dependent cell lines and performed functional studies defining LCK as a critical gene to the cell proliferation in KRAS-dependent, but not KRAS-independent, NSCLCs. We further define a functional module of LCK signaling in NSCLC, in which LCK activates downstream proteins such as PAK1 in order to regulate cell death. Finally, we find evidence of LCK signaling in human NSCLCs tissues. These results are the first evidence that suggest LCK as potential targetable protein in the context of KRAS dependency. In summary, our integrative analysis establishes a novel mechanistic basis for KRAS dependency in NSCLC and nominates KRAS-LCK-PAK1 network module as a potentially druggable pathway in KRAS-dependent lung cancers. Citation Format: Alejandro O. Balbin, John Prensner, Anirban Sahu, Anastasia Yocum, Sunita Shankar, Mohan Dhanasekaran, Xuhong Cao, Alexey Nesvizhskii, Arul Chinnaiyan. Reconstructing targetable pathways in KRAS dependent lung cancer by integrating transcriptome, proteome and phosphoproteome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5214. doi:10.1158/1538-7445.AM2013-5214

Abstract LB-35: Reconstructing targetable pathways in lung cancer by integrating transcriptome, proteome and phosphoproteome

Lung cancer is the leading cause of cancer mortality in the United States, and non-small cell lung cancer (NSCLC) is the predominant subtype of this malignancy. Mutations in the KRAS oncogene characterize more than 20% of all NSCLC malignancies, and recent studies suggest that those tumors could be divided into two molecular classifications: KRAS dependent (KRAS-Dep) and KRAS independent (KRAS-Ind), according to their dependency on KRAS mutant for cell survival and proliferation. Although, the KRAS signaling pathway has been extensively characterized using high throughput transcriptomic or proteomic technologies in isolation, the specific network of effector genes and proteins that drives carcinogenesis in NSCLC is still far from being understood. In this study we have generated and integrated a matched global phosphoproteome, proteome and transcriptome data set for a panel KRAS- Dep and KRAS-Ind NSCLC cell lines. Gene expression, protein abundance and protein phosphorylation were quantified for each of the 12 cell lines using RNA-sequencing (RNA-seq) and label-free quantitative tandem mass spectrometry (LC-MS/MS) respectively. We use a Prize Collecting Steiner Tree (PCST) algorithm in order to reconstruct active and targetable pathways associated with KRAS dependency. This algorithm allows us to synthesize transcriptome, proteome and phosphoproteome signatures with a human protein-to-protein interaction network (PPI) derived from public repositories. By using this approach we have nominated, and validated, several druggable kinases that are activated in KRAS-Dep but not in KRAS-Ind NSCLC cell lines. Specifically, this study indicates that KRAS Dep cell lines have an active and druggable pathway constituted by the lymphocyte specific tyrosine kinase (LCK), KRAS, and the p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1). The activation of PAK1 could confer both sustained growth and the ability to modulate cell9s motility, and therefore this pathway could be essential for KRAS dependent cells. In congruence with this, previous reports have suggested moderate to high expression of PAK1 protein in ∼ 20% of NSCLC adenocarcinoma tumors (Ong, C., et al PNAS April 11: 1-6). Additionally, a second but independent pathway constituted by active and stable Beta-catenin (CTNNB1), E-Cadherin (CDH1) and Alpha-Catenin (CTNNA1) also characterizes the KRAS-Dep phenotype. Those proteins belong to the catenin/cadherin/PSEN adhesion complex and play a crucial role in cell differentiation, which could explain previous observations relating KRAS dependency with a more differentiated phenotype (Singh, A., et al Cell 15: 489-500, 2009). In summary in this study, we are establishing a druggable pathway that is active in the KRAS-Dep but not in the KRAS-Ind phenotype, and so defining new potential druggable targets for treating KRAS dependent lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-35. doi:1538-7445.AM2012-LB-35