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Supplementary Figure 4. Overlap of positive and negative correlations.

Supplemental Materials and Methods from TMPRSS2–ERG-Mediated Feed-Forward Regulation of Wild-Type ERG in Human Prostate Cancers

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.

Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth.

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target.

STAT3 is a master transcriptional regulator that plays an important role in the induction of both immune activation and immune tolerance in dendritic cells (DCs). The transcriptional targets of STAT3 in promoting DC activation are becoming increasingly understood; however, the mechanisms underpinning its role in causing DC suppression remain largely unknown. To determine the functional gene targets of STAT3, we compared the genome-wide binding of STAT3 using ChIP sequencing coupled with gene expression microarrays to determine STAT3-dependent gene regulation in DCs after histone deacetylase (HDAC) inhibition. HDAC inhibition boosted the ability of STAT3 to bind to distinct DNA targets and regulate gene expression. Among the top 500 STAT3 binding sites, the frequency of canonical motifs was significantly higher than that of noncanonical motifs. Functional analysis revealed that after treatment with an HDAC inhibitor, the upregulated STAT3 target genes were those that were primarily the negative regulators of proinflammatory cytokines and those in the IL-10 signaling pathway. The downregulated STAT3-dependent targets were those involved in immune effector processes and antigen processing/presentation. The expression and functional relevance of these genes were validated. Specifically, functional studies confirmed that the upregulation of IL-10Ra by STAT3 contributed to the suppressive function of DCs following HDAC inhibition.

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.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of tissue physiology and disease processes including cancer. In order to delineate genome-wide lncRNA expression, we curated 7,256 RNA-Seq libraries from tumors, normal tissues, and cell lines comprising over 43 terabases of sequence from 25 independent studies. We applied ab initio assembly methodology to this dataset, yielding a consensus human transcriptome of 91,013 expressed genes. Over 68% (58,648) of genes were classified as lncRNAs, of which 79% (48,952) were previously unannotated. About 1% (597) of the lncRNAs harbored ultraconserved elements and 7% (3,900) overlapped disease-associated single nucleotide polymorphisms (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 into normal biology and cancer pathogenesis, and be valuable for future biomarker development.

Accurate transcript structure and abundance inference from RNA sequencing (RNA-seq) data is foundational for molecular discovery. Here we present TACO, a computational method to reconstruct a consensus transcriptome from multiple RNA-seq data sets. TACO employs novel change-point detection to demarcate transcript start and end sites, leading to improved reconstruction accuracy compared with other tools in its class. The tool is available at http://tacorna.github.io and can be readily incorporated into RNA-seq analysis workflows.

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.

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.

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.

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.

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

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, LncRNAs have been associated with cancer. Here, the authors carry out a systematic review of lncRNAs in breast cancer and show that DSCAM-AS1 is highly expressed in oestrogen receptor positive tumours and enhances cancer through an interaction with hnRNPL; and is also associated with tamoxifen resistance.

Application of high-throughput transcriptome sequencing has spurred highly sensitive detection and discovery of gene fusions in cancer, but distinguishing potentially oncogenic fusions from random, “passenger” aberrations has proven challenging. Here we examine a distinctive group of gene fusions that involve genes present in the loci of chromosomal amplifications—a class of oncogenic aberrations that are widely prevalent in breast cancers. Integrative analysis of a panel of 14 breast cancer cell lines comparing gene fusions discovered by high-throughput transcriptome sequencing and genome-wide copy number aberrations assessed by array comparative genomic hybridization, led to the identification of 77 gene fusions, of which more than 60% were localized to amplicons including 17q12, 17q23, 20q13, chr8q, and others. Many of these fusions appeared to be recurrent or involved highly expressed oncogenic drivers, frequently fused with multiple different partners, but sometimes displaying loss of functional domains. As illustrative examples of the “amplicon-associated” gene fusions, we examined here a recurrent gene fusion involving the mediator of mammalian target of rapamycin signaling, RPS6KB1 kinase in BT-474, and the therapeutically important receptor tyrosine kinase EGFR in MDA-MB-468 breast cancer cell line. These gene fusions comprise a minor allelic fraction relative to the highly expressed full-length transcripts and encode chimera lacking the kinase domains, which do not impart dependence on the respective cells. Our study suggests that amplicon-associated gene fusions in breast cancer primarily represent a by-product of chromosomal amplifications, which constitutes a subset of passenger aberrations and should be factored accordingly during prioritization of gene fusion candidates.

Automated RNA alignment algorithms often fail to recapture the essential conserved sites that are critical for function. To assist in the refinement of these algorithms, we manually curated a set of 148 alignments with a total of 9600 unique sequences, in which each alignment was backed by at least one crystal or NMR structure. These alignments included both naturally and artificially selected molecules. We used principles of isostericity to improve the alignments from an average of 83%–94% isosteric base pairs. We expect that this alignment collection will assist in a wide range of benchmarking efforts and provide new insight into evolutionary principles governing change in RNA structural motifs. The improved alignments have been contributed to the Rfam database.

Breast cancer is a heterogeneous disease, exhibiting a wide range of molecular aberrations and clinical outcomes. Here we employed paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers harbor an array of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving microtubule associated serine-threonine kinase (MAST) and Notch family genes. Both MAST and Notch family gene fusions exerted significant phenotypic effects in breast epithelial cells. Breast cancer lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and over-expression of MAST1 or MAST2 gene fusions had a proliferative effect both in vitro and in vivo. These findings illustrate that recurrent gene rearrangements play significant roles in subsets of carcinomas and suggest that transcriptome sequencing may serve to identify patients with rare, actionable gene fusions.

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.

Bone metastases typically develop in patients with advanced prostate cancer (PCa). Androgen deprivation is commonly used as treatment, but responses are short, and eventually the disease progresses to a castration-resistant form (CRPC). The fibroblast growth factor (FGF) axis is commonly altered during PCa progression. Our group and others have implicated the FGF axis in the pathogenesis of PCa progression in bone, identified it as a candidate target for therapy, and suggested that FGF receptor (FGFR)-1 mediates a positive feedback loop between PCa cells and bone cells. RNA sequencing studies of 183 human PCa samples indicated that the mean expression of FGFR-1 is the highest of all the FGFR family genes studied. Analyses of these FGFR-1 transcripts identified eight different protein-coding transcripts to be the most abundantly expressed with diverse human PCa tissue samples expressing different FGFR-1 isoforms. The overall goal of this project is to investigate implications of FGFR1/FGFR1 isoforms expression in the pathogenesis of PCa bone metastases. When we assessed the expression of the two best-characterized FGFR-1 isoforms (alpha (NM_023110.2) and beta (NM_023105.2), which represent the most abundant protein coding transcripts found in PCa, we observed that all patient-derived xenografts (PDXs) studied express significantly higher levels of FGFR-1 alpha than beta while PCa cell lines mostly express the beta isoform. Mining The Cancer Genome Atlas (TCGA) PCa data, we identified distinct patterns of gene expression associated with each FGFR-1 isoform (alpha and beta). Briefly, FGFR-1 beta (but not alpha) is significantly associated with pathways including the MAP-kinase signaling cascade, signaling by FGFR in disease and pathways in cancer. In vitro studies of FGF signaling activation in PCa cells expressing FGFR-1 isoforms alpha, beta or empty vector, confirmed these results. Male SCID mice injected intracardially with PC3-FGFR-1 alpha exhibited higher death rates compared to animals injected with either PC3-FGFR-1 beta or PC3-empty vector. Furthermore, higher number of metastases per mice as well as higher number of mice with bone metastases were observed in animals injected with PC3-FGFR-1 alpha. The fact that different prostate tumors express different FGFR-1 isoforms suggests that FGFR1 alpha and beta isoforms activate different genes or pathways in PCa cells and this may underlie, at least in part, PCa heterogeneity, pattern of progression, and differences in response to FGFR1 inhibitors. Further studies will warrant the understanding of the implications of FGFR-1 isoforms expression in the pathogenesis of PCa. Citation Format: Estefania Labanca, Jun Yang, Peter Shepherd, Justin Roberts, Michael Starbuck, Bradley Broom, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Expression of fibroblast growth factor receptor 1 isoforms and activation of different pathways in prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A026.

Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems—including the hammerhead ribozyme and the purine riboswitch—find additional elements, such as loop–loop interactions, required for function yet not phylogenetically conserved. Here we define a critical test of sufficiency: We embed a minimal, apparently sufficient motif for binding the amino acid tryptophan in a random-sequence background and ask whether we obtain functional molecules. After a negative result, we use a combination of three-dimensional structural modeling, selection, designed mutations, high-throughput sequencing, and bioinformatics to explore functional insufficiency. This reveals an essential unpaired G in a diverse structural context, varied sequence, and flexible distance from the invariant internal loop binding site identified previously. Addition of the new element yields a sufficient binding site by the insertion criterion, binding tryptophan in 22 out of 23 tries. Random insertion testing for site sufficiency seems likely to be broadly revealing.

Background Although analysis pipelines have been developed to use RNA-seq to identify long non-coding RNAs (lncRNAs), inference of their biological and pathological relevance remains a challenge. As a result, most transcriptome studies of autoimmune disease have only assessed protein-coding transcripts. Results We used RNA-seq data from 99 lesional psoriatic, 27 uninvolved psoriatic, and 90 normal skin biopsies, and applied computational approaches to identify and characterize expressed lncRNAs. We detect 2,942 previously annotated and 1,080 novel lncRNAs which are expected to be skin specific. Notably, over 40% of the novel lncRNAs are differentially expressed and the proportions of differentially expressed transcripts among protein-coding mRNAs and previously-annotated lncRNAs are lower in psoriasis lesions versus uninvolved or normal skin. We find that many lncRNAs, in particular those that are differentially expressed, are co-expressed with genes involved in immune related functions, and that novel lncRNAs are enriched for localization in the epidermal differentiation complex. We also identify distinct tissue-specific expression patterns and epigenetic profiles for novel lncRNAs, some of which are shown to be regulated by cytokine treatment in cultured human keratinocytes. Conclusions Together, our results implicate many lncRNAs in the immunopathogenesis of psoriasis, and our results provide a resource for lncRNA studies in other autoimmune diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0570-4) contains supplementary material, which is available to authorized users.

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.

Background: In a prior transcriptomic analysis focused on lncRNA interrogation using some of these data, we discovered tens of thousands of novel lncRNAs (Iyer and Niknafs et al, Nature Genetics, 2015). Building on this analysis, we recently developed a markedly improved bioinformatics tool for novel gene/isoform discovery from massive RNA-seq datasets named TACO (Niknafs et al, Nature Methods, In press, tacorna.github.io). TACO produces high-fidelity transcript structure predictions from large RNA-seq datasets. We now set out to comprehensively leverage both TACO and the publicly available RNA-seq data for discovery of novel transcriptional cancer associations. Additionally, in order to widely disseminate these findings in an accessible manner, we have built a web-tool that provides the scientific community access to these data and analyses. Methods: We have downloaded, curated, and processed 23,623 RNA-seq samples largely from the TCGA, ICGC, GTEx, and CCLE, comprising 37 tissue types and over 35 cancer types. RNA-seq data processing was performed using STAR, Cufflinks, Kallisto, and TACO. The web tool for visualization and access to these data and analyses was built using a JavaScript-based server infrastructure (Noje.js) and a relational PostgreSQL database. Results: Generation of a consensus transcriptome from this large-scale RNA-seq dataset via TACO resulted in the discovery of tens of thousands of novel transcriptional elements, including intergenic non-coding RNAs and novel splice isoforms of known genes. Such an expansive RNA-seq cohort that includes many normal tissue samples enabled statistically powerful cancer association expression analyses that revealed a myriad of novel cancer genes, especially in tissues for which there was previously little-to-no normal tissue RNA-seq data (e.g., brain and pancreas). Many of these novel transcriptional elements discovered using TACO were also found to be cancer associated. We have built a web-tool to facilitate further analysis and discovery using these data and analyses by the scientific community. The web-tool provides a powerful and intuitive interface for researchers with little-to-no bioinformatics expertise to leverage large-scale RNA-seq datasets. Conclusion: Here we present the largest reported compendium of RNA-seq data, and reveal many novel cancer gene associations. Using a new, powerful gene discovery tool, TACO, we identify a multitude of novel transcriptional elements that are also cancer associated. Despite the abundance of publicly available RNA-seq data, necessary computing resources, data storage, and bioinformatics expertise are barriers to usability of these data by scientific community. Our RNA-seq expression web-tool bridges this gap, and enables users to powerfully interrogate cancer expression across dozens of tumor and tissue types. Citation Format: Yashar Niknafs, Nicholas Molen, Balaji Pandian, Matthew Iyer, Arul Chinnaiyan. Bridging the gap between NGS data and its usability: cancer gene discovery through massive-scale transcriptomic analyses and development of a powerful web-tool for dissemination of these findings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3038. doi:10.1158/1538-7445.AM2017-3038

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.

BackgroundImproved 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.MethodsProstate 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.Findings1008 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

Bone is the most common site of prostate cancer (PCa) progression to a therapy-resistant, lethal phenotype. We found that blockade of fibroblast growth factor receptors (FGFRs) with the receptor tyrosine kinase inhibitor dovitinib has clinical activity in a subset of men with castration-resistant PCa and bone metastases. Our integrated analyses suggest that FGF signaling mediates a positive feedback loop between PCa cells and bone cells and that blockade of FGFR1 in osteoblasts partially mediates the antitumor activity of dovitinib by improving bone quality and by blocking PCa cell-bone cell interaction. These findings account for clinical observations such as reductions in lesion size and intensity on bone scans, lymph node size, and tumor-specific symptoms without proportional declines in serum prostate-specific antigen concentration. Our findings suggest that targeting FGFR has therapeutic activity in advanced PCa and provide direction for the development of therapies with FGFR inhibitors.

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.

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.

CXCR4 is a chemokine receptor that mediates invasion and metastasis. CXCR4 expression is transcriptionally regulated in cancer cells and is associated with aggressive prostate cancer phenotypes. Previously, we and others have shown that the transcription factor ERG regulates CXCR4 expression in prostate cancer cells and that androgens modulate CXCR4 expression via increasing ERG expression. Herein, the molecular mechanisms of ERG-mediated CXCR4 promoter activation, phosphorylation of ERG by intracellular kinases and subsequent CXCR4 expression, as well as the status of ERG and CXCR4 in human prostate cancer specimens were investigated. Using multiple molecular strategies, it was demonstrated that (i) ERG expressed in TMPRSS2-ERG fusion positive VCaP cells selectively binds to specific ERG/Ets bindings sites in the CXCR4 promoter; (ii) distal binding sites mediate promoter activation; (iii) exogenously expressed ERG promotes CXCR4 expression; (iv) ERG is phosphorylated at Serine-81 and -215, by both IKK and Akt kinases, and Akt mediates CXCR4 expression; (v) ERG-induced CXCR4 drives CXCL12-dependent adhesion to fibronectin; and (vi) ERG and CXCR4 were coexpressed in human prostate cancer tissue, consistent with ERG-mediated transcriptional activation of CXCR4. These data demonstrate that ERG activates CXCR4 expression by binding to specific ERG/Ets responsive elements and via intracellular kinases that phosphorylate ERG at discrete serine residues. Implications: These findings provide a mechanistic link between TMPRSS2-ERG translocations and intracellular kinase-mediated phosphorylation of ERG on enhanced metastasis of tumor cells via CXCR4 expression and function in prostate cancer cells. Mol Cancer Res; 11(11); 1349–61. ©2013 AACR.

Castrate-resistant progression and bone metastases are hallmarks of advanced prostate cancer (PCa). The fibroblast growth factor (FGF)/FGF receptor (FGFR) complex mediates tumor-stromal interactions and is commonly altered in PCa. FGFR1, FGFR2, FGFR3, and FGFR4 genes encode alternatively spliced variants of FGFRs that vary in the extracellular ligand-binding and intracellular kinase domains. A published study from our group implicated FGFR1 as a therapy target for PCa bone metastases (STM 2014; 6:252ra122). Further, our studies of FGFR1 transcripts by RNA sequencing of 183 human PCas identified eight different protein coding transcripts as the most abundantly expressed with different human PCas expressing different FGFR1 isoforms (Abstract #3913 AACR 2015). These results suggest that different FGFR1 isoforms in PCa cells may partially underlie the biological heterogeneity of PCa. The studies presented here focus in two of the best-characterized isoforms: FGFR1alpha (R1alpha), with 3 Ig-like domains, and 822 aa in length; and FGFR1beta (R1beta), with only 2 Ig-like domains and 733 aa in length. We assessed whether these isoforms induce activation of the same pathways using PC3 cells transiently transfected with empty vector (EV), R1alpha (NM_023110.2) or R1beta (NM_023105.2) and treated with vehicle, FGF2 or FGF9. By Western blot analysis we found that total FGFR1 expression (relative to a loading control) was similar in cells transfected with R1beta or R1alpha. Levels of p-FGFR1 were high in untreated cells transfected with R1alpha, but no further induction was observed after treatment with FGF2 or FGF9. However, p-FGFR1 expression was almost undetectable in untreated cells expressing R1beta and was slightly induced by FGF2 but not by FGF9. p-PLCγ expression was found only in cells expressing R1alpha. We subsequently stably transfected these isoforms in PC3 cells and discovered that while no significant difference in R1alpha and R1beta transcript levels was detected, the levels of R1alpha protein were higher than those of R1beta suggesting that these isoforms may undergo different translational regulation, We also performed in vivo studies by subcutaneous injection of R1alpha or R1beta expressing PC3 cells in immunocompromised mice and weekly monitored tumor volume. We found that PC3 cells expressing R1alpha developed significantly larger tumors than PC3 cells expressing R1beta. We are now in the process of analyzing at the molecular level these tissue specimens. In conclusion, R1alpha and R1beta isoforms trigger different biological effects in PCa cells. Because different isoforms are expressed in different prostate tumors, the expression of these isoforms could be associated with the typical PCa heterogeneity and could explain differences in therapy responses to FGFR1 blockade. These results warrant further studies to fully understand the biological implications of FGFR1 isoforms in the pathogenesis of PCa. Citation Format: Estefania Labanca, Xinhai Wan, Jun Yang, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Alpha and beta isoforms of fibroblast growth factor receptor 1 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1871.

Background: We have previously performed a bioinformatics-based analysis on 7,256 RNA sequencing libraries from tumors, normal tissues, and cell lines to delineate the landscape of long noncoding RNAs (lncRNAs) in the human transcriptome. This analysis identified 58,648 lncRNAs, including over 45,000 novel transcripts (Iyer and Niknafs et al, Nature Genetics, 2015). We now interrogate this lncRNA compendium to identify top candidate estrogen receptor (ER)-associated lncRNAs in breast cancer and characterize their association with disease progression. Methods: To identify and prioritize lncRNAs that were differentially expressed in cancer vs normal tissue, and in ER-positive vs ER-negative disease, we performed differential expression testing using a previously described non-parametric tool for RNA-seq count data called Sample Set Enrichment Analysis (SSEA) on over 1000 RNA sequencing libraries including breast cancer and normal tissue samples from the The Cancer Genome Atlas (TCGA) project. The effect of the top prioritized lncRNA on cancer phenotypes was studied via proliferation, colony formation, invasion and tamoxifen resistance assays in MCF7 and T47D cell lines, and via mouse xenograft studies in vivo. The mechanism by which this lncRNA promotes tumor progression was investigated by identifying its top protein interactors and its subdomains responsible for function, and then studying the effects of disrupting function of this lncRNA on cancer phenotypes. Finally, in a “guilt-by-association” study, we developed a signature of 150 protein coding genes most strongly associated with our lncRNA of interest, and investigated the association of this signature with tumor grade, recurrence, and metastases using Oncomine analyses. Results: Differential expression analyses on over 1000 TCGA RNA-seq samples nominated Breast Cancer Associated Transcript (BRCAT431) as the top overexpressed ER-regulated lncRNA in breast cancer. In vitro experiments demonstrate that shRNA-mediated knockdown of BRCAT431 resulted in significantly decreased proliferation, colony formation, and invasion (by >50% in most assays). Tamoxifen resistance was associated with significantly increased BRCAT431 levels in both MCF7 and T47D cells, and knockdown of BRCAT431 reversed tamoxifen resistance. In vivo xenograft studies demonstrate that knockdown of BRCAT431 also significantly decreased xenograft growth and tumor invasion by >50%. RNA pulldown followed by mass spectrometry identified the RNA binding protein hnRNPL as a key protein interacting with BRCAT431. Deletion studies identified a 27 base-pair region of BRCAT431 necessary for its interaction with hnRNPL, and loss of this region abrogated BRCAT431-induced invasion. Finally, guilt-by-association studies demonstrate a strong association between BRCAT431 overexpression and tumor grade, recurrence, and metastases. Conclusion: In this study, we develop the largest reported compendium of breast cancer lncRNAs. We prioritize BRCAT431 as the top lncRNA upregulated in ER-positive breast cancers, and demonstrate that it confers aggressive oncogenic phenotypes in vitro and in vivo. We identify a novel mechanism by which this lncRNA functions through interaction with hnRNPL. Our results suggest that by promoting tamoxifen resistance, this lncRNA increases the clinical risk of recurrence and metastases in breast cancer. Citation Format: Yashar S. Niknafs, Sumin Han, Teng Ma, Chao Zhang, Matthew K. Iyer, Rohit Malik, Anton Poliakov, Corey Speers, Daniel Hayes, James Rae, Felix Y. Feng, Arul M. Chinnaiyan. Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B19.

Long non-coding RNA (lncRNAs) have been shown to play an important role is a variety of cellular processes including cancer initiation and progression. Recently, we described a landscape of lncRNA using RNA-Seq data from more than 5000 cancer samples from 18 tissue types. Using novel bioinformatics based approach we were able to discover almost 50,000 novel transcripts. This list includes a subset of lncRNAs that contain ultra-conserved elements (UCE) termed “Highly Conserved Long Intergenic Non-Coding RNAs (HICLINCs)”. This provides a new opportunity for exploring the functions of highly conserved lncRNAs using genomic approaches. Among them, we focus on an lncRNA which we have termed HiClinc-1 (Highly Conserved Intergenic long-noncoding RNA-1), that is specifically expressed only in testis in benign tissue, and re-expressed in a broad range of cancer types. We described that HiClinc-1 exists in the messenger Ribonucleoprotein (mRNP) complex and binds to Insulin-Like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1), one of the component of mRNP complex in both human and zebrafish. HiClinc-1 regulates cell proliferation both in vivo and in vitro through the regulation of IGF2BP1's downstream target Insulin-Like Growth Factor 2 (IGF2). HiClinc-1 also enhanced tumor growth in mouse xenograft model. Notably, Human-HiClinc-1 injection in zebrafish embryos accelerated the onset and growth of NRAS K61-induced melanoma. Similarly, HiClinc-1 knockout background showed delayed onset of NRAS K61-induced melanoma. Taken together, we identified a novel Cancer-Testis lncRNA, which is highly conserved through human to zebrafish that can be a potential therapeutic target for cancer treatment. Citation Format: Yasuyuki Hosono, John Prensner, Matthew Iyer, Yashar Niknafs, Rohit Malik, Anton Poliakov, Xuhong Cao, Jordan Shavit, Rohit Mehra, Weibin Zhou, Arul M. Chinnaiyan. HiClinc-1, a highly conserved Cancer-Testis lncRNA, regulates cell proliferation and tumor onset. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR10.

There are remarkable disparities among patients of different races with prostate cancer; however, the mechanism underlying this difference remains unclear. Here, we present a comprehensive landscape of the transcriptome profiles of 14 primary prostate cancers and their paired normal counterparts from the Chinese population using RNA-seq, revealing tremendous diversity across prostate cancer transcriptomes with respect to gene fusions, long noncoding RNAs (long ncRNA), alternative splicing and somatic mutations. Three of the 14 tumors (21.4%) harbored a TMPRSS2-ERG fusion, and the low prevalence of this fusion in Chinese patients was further confirmed in an additional tumor set (10/54=18.5%). Notably, two novel gene fusions, CTAGE5-KHDRBS3 (20/54=37%) and USP9Y-TTTY15 (19/54=35.2%), occurred frequently in our patient cohort. Further systematic transcriptional profiling identified numerous long ncRNAs that were differentially expressed in the tumors. An analysis of the correlation between expression of long ncRNA and genes suggested that long ncRNAs may have functions beyond transcriptional regulation. This study yielded new insights into the pathogenesis of prostate cancer in the Chinese population.

Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.

Summary: Next generation sequencing (NGS) technologies have enabled de novo gene fusion discovery that could reveal candidates with therapeutic significance in cancer. Here we present an open-source software package, ChimeraScan, for the discovery of chimeric transcription between two independent transcripts in high-throughput transcriptome sequencing data. Availability: http://chimerascan.googlecode.com Contact: cmaher@dom.wustl.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Recurrent gene fusions involving ETS family genes are a distinguishing feature of human prostate cancers, with TMPRSS2–ERG fusions representing the most common subtype. The TMPRSS2–ERG fusion transcript and its splice variants are well characterized in prostate cancers; however, not much is known about the levels and regulation of wild-type ERG. By employing an integrative approach, we show that the TMPRSS2–ERG gene fusion product binds to the ERG locus and drives the overexpression of wild-type ERG in prostate cancers. Knockdown of TMPRSS2–ERG in VCaP cells resulted in the downregulation of wild-type ERG transcription, whereas stable overexpression of TMPRSS2–ERG in the gene fusion-negative PC3 cells was associated with the upregulation of wild-type ERG transcript. Further, androgen signaling-mediated upregulation of TMPRSS2–ERG resulted in the concomitant upregulation of wild-type ERG transcription in VCaP cells. The loss of wild-type ERG expression was associated with a decrease in the invasive potential of VCaP cells. Importantly, 38% of clinically localized prostate cancers and 27% of metastatic prostate cancers harboring the TMPRSS2–ERG gene fusions exhibited overexpression of wild-type ERG. Taken together, these results provide novel insights into the regulation of ERG in human prostate cancers. Cancer Res; 71(16); 5387–92. ©2011 AACR.

High-throughput sequencing of polyA+ RNA (RNA-Seq) in human cancer shows remarkable potential to identify both novel markers of disease and uncharacterized aspects of tumor biology, particularly non-coding RNA (ncRNA) species. We employed RNA-Seq on a cohort of 102 prostate tissues and cells lines and performed ab initio transcriptome assembly to discover unannotated ncRNAs. We nominated 121 such Prostate Cancer Associated Transcripts (PCATs) with cancer-specific expression patterns. Among these, we characterized PCAT-1 as a novel prostate-specific regulator of cell proliferation and target of the Polycomb Repressive Complex 2 (PRC2). We further found that high PCAT-1 and PRC2 expression stratified patient tissues into molecular subtypes distinguished by expression signatures of PCAT-1-repressed target genes. Taken together, the findings presented herein identify PCAT-1 as a novel transcriptional repressor implicated in 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.

Dendritic cells play a vital role in the induction of either activation or tolerance of an immune response. However the molecular pathways regulating these opposite responses to immune stimuli remain poorly understood. STAT3 is a master transcriptional regulator of immune response including those mediated by DCs. STAT3 can either positively or negatively regulate DC responses, but the mechanisms are unknown. STAT3 is post-translationally modified by either acetylation of phosphorylation. While much is understood about transcriptional targets of phosphorylated STAT3, the gene targets and the functional impact of acetylated STAT3 remain unclear. We aimed to answer the gene targets of acetylated-STAT3 and test the hypothesis that acetylation of STAT3 plays a key role on negative regulation of DCs. To determine the transcriptional targets of acetyl-STAT3, we first performed genome-wide binding analysis of acetyl-STAT3 by ChIP-Seq. We additionally performed gene expression microarrays on these samples and coupled gene expression to the acetyl-STAT3 binding datasets then analyzed with Gene Set Enrichment Analysis (GSEA). Inhibition of histone deacetylases (HDACi) acetylates STAT3, therefore, we treated DC with either HDACi or diluent and performed ChIP-Seq and analyzed with Genomic Regions Enrichment Annotation Tool (GREAT). The analysis revealed 3598 binding sites in 4605 gene loci as potential targets of acety-STAT3. Theses binding sites were mostly proximal but some were also distal up to over 100 kb from transcription start site. Gene expression array showed 1701 genes up-regulated and 1668 genes down-regulated. Proximal binding of acety-STAT-3 showed more effective transcription function than distal binding. Detailed analysis was performed utilizing TRANSFAC to analyze canonical GAS motif (TTCnnnGAA) and the non-canonical motif (single-nucleotide variants) of binding for acetyl-STAT3. In top 500 binding peaks, canonical motif sites bound by acetyl-STAT3 were 349 versus 87 in control (p We next validated the expression of some of the up-regulated (IL-10Ra, Cdk6, E2F2, Rb1, Mef2c, and E2F2) and down-regulated genes (Map3k, Fcgr3, Slfn2, and Prdm1) that are not known to be direct targets of phospo-STAT3 and further confirmed that the acetyl-STAT-3 binding peaks at these gene loci were associated with H3K4me3 marks We next performed analysis for functional pathways utilizing 2 different bioinformatics tools: IPA (Ingenuity Pathway Analysis) and MSigDB conjugated with and displayed by GREAT. IPA predicted that the most up-regulated pathways were those involved in Th2 differentiation and negative regulation of cytokine production and the most down-regulated pathways involved in antigen processing and presentation. MSigDB predicted that IL-10 signaling was among the most significantly altered pathway in the acetylated STAT-3 group. We next validated with ChIP-qPCR the genes involved in IL-10 and its signaling. We found the acetyl-STAT3 binding peaks at IL-10Ra and Ido1 gene loci were associated with H3K4me3 marks, demonstrating open configuration and increased transcription. We next determined its biological relevance. We found that DCs with increased acetylated-STAT3 showed enhanced response to IL-10 and showed reduced responses to TLR (LPS) stimulation and reduction in activation of T cells. Further functional studies showed that IL-10 treatment of DCs with acetyl-STAT3 caused greater expression in total STAT3 and IL-10 demonstrating a positive feed forward mechanism for sustaining tolerogeneic functions of DCs. These data thus collectively demonstrate (a) acetylation of STAT3 targets different genes than phosphorylation of STAT3 and (b) acetylated STAT3 acts in a feedforward mechanism to enhance DC tolerance by increasing IL-10Ra and thus enhancing sensitivity to IL-10. Thus, we identified a novel acetylated-STAT3 dependent regulatory pathway in DCs. Disclosures No relevant conflicts of interest to declare.

Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration resistant prostate cancer (CRPC). Substantial prior work has focused on targeting AR directly; however, the identification and therapeutic targeting of co-activators of AR signaling remains an underexplored area of potential clinical significance. Here we demonstrate that the MLL (mixed-lineage leukemia) complex, a well-known contributor in MLL-fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via its critical subunit, menin. Small molecule inhibition of the menin-MLL interaction blocks AR signaling and inhibits the growth of castration resistant tumors in vivo. Furthermore, we find that menin is up-regulated in castration resistant prostate cancer and high expression correlates with poor overall survival. Taken together, our study identifies the MLL complex as a critical co-activator of AR that can be targeted in advanced prostate cancer. Citation Format: Rohit Malik, Amjad P. Khan, Irfan A. Asangani, Marcin Cieślik, John R. Prensner, Xiaoju Wang, Matthew K. Iyer, Xia Jiang, Dmitry Borkin, June Escara-Wilke, Rachell Stender, Yi-Mi Wu, Xuhong Cao, Felix Y. Feng, Jolanta Grembecka, Tomasz Cierpicki, Arul M. Chinnaiyan. Targeting the MLL complex in castration resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3636. doi:10.1158/1538-7445.AM2015-3636

Bone metastases dominate the clinical picture of men with advanced prostate cancer (PCa). The fibroblast growth factor (FGF)/FGF receptor (FGFR) complex is commonly altered during PCa progression. Four highly conserved genes (FGFR1, FGFR2, FGFR3, and FGFR4) encode a repertoire of alternatively spliced variants of FGFRs that vary in the extracellular ligand-binding and intracellular kinase domains. A previous study from our group implicated FGFR1 as a therapy target for PCa bone metastases (Sci Transl Med 2014; 6:252ra122). One of the most important mechanisms by which FGFRs determine specificity for different FGFs is by alternative exon usage of the immunoglobulin-like (Ig-like) motif of the extracellular domain. We analyzed FGFR1 transcripts in 183 human PCa samples and in PCa patient-derived xenografts (PDXs) assessed by RNA sequencing. We identified eight different protein coding transcript to be the most abundantly expressed, namely ENST00000326324; ENST00000356207; ENST00000397103 (with a predicted protein length of 731 to 733 aa) and ENST00000397091; ENST00000397108; ENST00000397113; ENST00000425967; ENST00000532791 (with a predicted protein length of 820 to 853aa). Probably reflecting FGFR1alpha/beta isoforms: FGFR1alpha, containing three Ig-like domains and FGFR1beta, containing only two Ig-like domains. Interestingly, different PCa tissue samples expressed different isoforms. We subsequently assessed, by real-time reverse transcription polymerase chain reaction, the expression of FGFR1alpha and beta in three PCa cell lines (PC3, DU145 and C4-2B) and seven PCa PDXs (MDA PCa 2b, MDA PCa 118b, MDA PCa 155-12; MDA PCa 146-10; MDA PCa 146-12; MDA PCa 150-3 and MDA PCa 183) derived from primary PCa, bone metastases and brain metastases and reflecting the typical adenocarcinoma as well as, adenocarcinomas with neuroendrocrine differentiation and small cell carcinomas of PCa. We found that all PDXs express primarily FGFR1alpha isoform while PCa cell lines express FGFR1beta. We stably transfected PC3 cells with FGFR1alpha (NM_023110.2) and FGFR1beta (NM_023105.2) isoforms. After clones were established, we placed the cells for 10 days in culture and found that tissue culture plates containing PC3 expressing FGFR1alpha had significantly more cells compared with dishes containing PC3 expressing FGF1beta isoform as assessed by direct cell counting in a phase contrast microscope. Furthermore, PC3 cells transfected with FGFR1 alpha display a more adhesive phenotype than cells transfected with FGFR1beta or empty vector as assessed by cell attachment in a 24-well plate. We conclude that FGFR1 isoforms are involved in the pathogenesis of PCa and can be used to develop markers of response to FGFR1 blockade. These results warrant further studies to fully understand the biological implications of FGFR1 isoforms in the pathogenesis of PCa. Citation Format: Estefania Labanca, Xinhai Wan, Jun Yang, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Fibroblast growth factor receptor 1 isoforms in prostate cancer bone metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3913. doi:10.1158/1538-7445.AM2015-3913

Long non-coding RNAs (lncRNAs) are emerging as important players in cancer. The emergence of high-throughput RNA sequencing (RNA-Seq) technology provides a revolutionary means for systematic discovery of transcribed elements. However, efforts to catalog lncRNAs have thus far failed to appreciate the transcriptional robustness and complexity in the human genome because they were confined to select cell lines or small numbers of mostly normal tissue. In order to delineate genome-wide lncRNA expression, we curated 7,256 RNA-Seq libraries comprising over 43 terabases of sequence from 25 independent studies including tumors, normal tissues, and cell lines. The data were processed using ab initio transcriptome meta-assembly methodologies to develop a consensus of 91,013 expressed genes. Over 68% (58,648) of genes were classified as lncRNAs, of which 79% (48,952) were previously unannotated. About 1% (597) of the lncRNAs harbor ultraconserved elements and 7% (3,900) overlap disease-associated single nucleotide polymorphisms (SNPs). To prioritize lineage-specific, disease-associated lncRNA expression, we developed a non-parametric method for differential expression testing called Sample Set Enrichment Analysis (SSEA), and nominated 7,942 lineage- or cancer-associated lncRNA genes. In order to interrogate potential functionality for these lncRNAs, gene set enrichment testing was performed on genes correlated with each lncRNA. By uncovering this expansive landscape of cancer-associated lncRNAs, we provide the scientific community a powerful starting point to begin investigating their biological relevance. We anticipate that the lncRNAs identified by this study, as well as the computational tools developed herein, will provide a foundation for lncRNA genomics, biomarker development, and the delineation of cancer disease mechanisms. Citation Format: Yashar S. Niknafs, Matthew K. Iyer, Arul M. Chinnaiyan. The landscape of long non-coding RNAs in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2015-2992

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>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 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.

A transcriptome can be readily assembled from RNA-Seq data in the absence of a reference genome.