High-throughput sequencing technologies, including RNA-seq, have made it possible to move beyond gene expression analysis to study transcriptional events including alternative splicing and gene fusions. Furthermore, recent studies in cancer have suggested the importance of identifying transcriptionally altered loci as biomarkers for improved prognosis and therapy. While many statistical methods have been proposed for identifying novel transcriptional events with RNA-seq, nearly all rely on contrasting known classes of samples, such as tumor and normal. Few tools exist for the unsupervised discovery of such events without class labels. In this paper, we present SigFuge for identifying genomic loci exhibiting differential transcription patterns across many RNA-seq samples. SigFuge combines clustering with hypothesis testing to identify genes exhibiting alternative splicing, or differences in isoform expression. We apply SigFuge to RNA-seq cohorts of 177 lung and 279 head and neck squamous cell carcinoma samples from the Cancer Genome Atlas, and identify several cases of differential isoform usage including CDKN2A, a tumor suppressor gene known to be inactivated in a majority of lung squamous cell tumors. By not restricting attention to known sample stratifications, SigFuge offers a novel approach to unsupervised screening of genetic loci across RNA-seq cohorts. SigFuge is available as an R package through Bioconductor.

MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR-RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with approximately 35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes.

Metaplastic breast cancer (MBC) is a rare special histologic type of triple-negative breast cancer, characterized by the presence of neoplastic cells showing differentiation towards squamous epithelium and/or mesenchymal elements. Here we sought to define whether histologically distinct subgroups of MBCs would be underpinned by distinct genomic and/or transcriptomic alterations. Microarray-based copy number profiling identified limited but significant differences between the distinct MBC subtypes studied here, despite the limited sample size (n = 17). In particular, we found that, compared to MBCs with chondroid or squamous cell metaplasia, MBCs with spindle cell differentiation less frequently harbored gain of 7q11.22-23 encompassing CLDN3 and CLDN4, consistent with their lower expression of claudins and their association with the claudin-low molecular classification. Microarray-based and RNA-sequencing-based gene expression profiling revealed that MBCs with spindle cell differentiation differ from MBCs with chondroid or squamous cell metaplasia on the expression of epithelial-to-mesenchymal transition-related genes, including down-regulation of CDH1 and EPCAM. In addition, RNA-sequencing revealed that the histologic patterns observed in MBCs are unlikely to be underpinned by a highly recurrent expressed fusion gene or a pathognomonic expressed mutation in cancer genes. Loss of PTEN expression or mutations affecting PIK3CA or TSC2 observed in 8/17 MBCs support the contention that PI3K pathway activation plays a role in the development of MBCs. Our data demonstrate that despite harboring largely similar patterns of gene copy number alterations, MBCs with spindle cell, chondroid and squamous differentiation are distinct at the transcriptomic level but are unlikely to be defined by specific pathognomonic genetic alterations.

Colorectal cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. and approximately 50% of patients develop metastatic disease (mCRC). Despite our understanding of long non-coding RNAs (lncRNAs) in primary colon cancer, their role in mCRC and treatment resistance remains poorly characterized. Therefore, through transcriptome sequencing of normal, primary, and distant mCRC tissues we find 148 differentially expressed RNAs Associated with Metastasis (RAMS). We prioritize RAMS11 due to its association with poor disease-free survival and promotion of aggressive phenotypes in vitro and in vivo. A FDA-approved drug high-throughput viability assay shows that elevated RAMS11 expression increases resistance to topoisomerase inhibitors. Subsequent experiments demonstrate RAMS11-dependent recruitment of Chromobox protein 4 (CBX4) transcriptionally activates Topoisomerase II alpha (TOP2α). Overall, recent clinical trials using topoisomerase inhibitors coupled with our findings of RAMS11-dependent regulation of TOP2α supports the potential use of RAMS11 as a biomarker and therapeutic target for mCRC.

Androgen receptor (AR) signaling continues to play a dominant role in all stages of prostate cancer, including castration-resistant prostate cancers (CRPC) that have developed resistance to second generation AR antagonists such as enzalutamide. In this study, we identified a long noncoding RNA (lncRNA), NXTAR (LOC105373241) that is located convergent with the AR gene and is repressed in human prostate tumors and cell lines. NXTAR bound upstream of the AR promoter and promoted EZH2 recruitment, causing significant loss of AR (and AR-V7) expression. Paradoxically, AR bound the NXTAR promoter, and inhibition of AR by the ACK1/TNK2 small molecule inhibitor (R)-9b excluded AR from the NXTAR promoter. The histone acetyltransferase GCN5 bound and deposited H3K14 acetylation marks, enhancing NXTAR expression. Application of an oligonucleotide derived from NXTAR exon 5 (NXTAR-N5) suppressed AR/AR-V7 expression and prostate cancer cell proliferation, indicating the translational relevance of the negative regulation of AR. In addition, pharmacologic restoration of NXTAR using (R)-9b abrogated enzalutamide-resistant prostate xenograft tumor growth. Overall, this study uncovers a positive feedback loop, wherein NXTAR acts as a novel prostate tumor-suppressing lncRNA by inhibiting AR/AR-V7 expression, which in turn upregulates NXTAR levels, compromising enzalutamide-resistant prostate cancer. The restoration of NXTAR could serve as a new therapeutic modality for patients who have acquired resistance to second generation AR antagonists. SIGNIFICANCE: This study identifies NXTAR as a tumor suppressive lncRNA that can epigenetically downregulate AR/AR-V7 expression and provides a therapeutic strategy to reinstate NXTAR expression for treating recurrent CRPC.

Late-stage relapse (LSR) in patients with breast cancer (BC) occurs more than five years and up to 10 years after initial treatment and has less than 30% 5-year relative survival rate. Long non-coding RNAs (lncRNAs) play important roles in BC yet have not been studied in LSR BC. Here, we identify 1127 lncRNAs differentially expressed in LSR BC via transcriptome sequencing and analysis of 72 early-stage and 24 LSR BC patient tumors. Decreasing expression of the most up-regulated lncRNA, LINC00355, in BC and MCF7 long-term estrogen deprived cell lines decreases cellular invasion and proliferation. Subsequent mechanistic studies show that LINC00355 binds to MENIN and changes occupancy at the CDKN1B promoter to decrease p27Kip. In summary, this is a key study discovering lncRNAs in LSR BC and LINC00355 association with epigenetic regulation and proliferation in BC.

Colorectal cancer (CRC) is the most common gastrointestinal malignancy and a leading cause of cancer deaths in the United States. More than half of CRC patients develop metastatic disease (mCRC) with an average 5-year survival rate of 13%. Circular RNAs (circRNAs) have recently emerged as important tumorigenesis regulators; however, their role in mCRC progression remains poorly characterized. Further, little is known about their cell-type specificity to elucidate their functions in the tumor microenvironment (TME). To address this, we performed total RNA sequencing (RNA-seq) on 30 matched normal, primary and metastatic samples from 14 mCRC patients. Additionally, five CRC cell lines were sequenced to construct a circRNA catalog in CRC. We detected 47 869 circRNAs, with 51% previously unannotated in CRC and 14% novel candidates when compared to existing circRNA databases. We identified 362 circRNAs differentially expressed in primary and/or metastatic tissues, termed circular RNAs associated with metastasis (CRAMS). We performed cell-type deconvolution using published single-cell RNA-seq datasets and applied a non-negative least squares statistical model to estimate cell-type specific circRNA expression. This predicted 667 circRNAs as exclusively expressed in a single cell type. Collectively, this serves as a valuable resource, TMECircDB (accessible at https://www.maherlab.com/tmecircdb-overview), for functional characterization of circRNAs in mCRC, specifically in the TME.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal form of prostate cancer. Although long-noncoding RNAs (lncRNAs) have been implicated in mCRPC, past studies have relied on bulk sequencing methods with low depth and lack of single-cell resolution. Hence, we performed a lncRNA-focused analysis of single-cell RNA-sequencing data (n = 14) from mCRPC biopsies followed by integration with bulk multi-omic datasets. This yielded 389 cell-enriched lncRNAs in prostate cancer cells and the tumor microenvironment (TME). These lncRNAs demonstrated enrichment with regulatory elements and exhibited alterations during prostate cancer progression. Prostate-lncRNAs were correlated with AR mutational status and response to treatment with enzalutamide, while TME-lncRNAs were associated with RB1 deletions and poor prognosis. Finally, lncRNAs identified between prostate adenocarcinomas and neuroendocrine tumors exhibited distinct expression and methylation profiles. Our findings demonstrate the ability of single-cell analysis to refine our understanding of lncRNAs in mCRPC and serve as a resource for future mechanistic studies.

miR-24, upregulated during terminal differentiation of multiple lineages, inhibits cell-cycle progression. Antagonizing miR-24 restores postmitotic cell proliferation and enhances fibroblast proliferation, whereas overexpressing miR-24 increases the G1 compartment. The 248 mRNAs downregulated upon miR-24 overexpression are highly enriched for DNA repair and cell-cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance (MYC, E2F2, CCNB1, and CDC2) or inhibit (p27Kip1 and VHL) cell-cycle progression. miR-24 directly regulates MYC and E2F2 and some genes that they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2, but not MYC, and cell proliferation, inhibited by miR-24 overexpression, is rescued by miR-24-insensitive E2F2. Therefore, E2F2 is a critical miR-24 target. The E2F2 3'UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2, MYC, AURKB, CCNA2, CDC2, CDK4, and FEN1 by recognizing seedless but highly complementary sequences.

PROM1 is the gene encoding prominin-1 or CD133, an important cell surface marker for the isolation of both normal and cancer stem cells. PROM1 transcripts initiate at a range of transcription start sites (TSS) associated with distinct tissue and cancer expression profiles. Using high resolution Cap Analysis of Gene Expression (CAGE) sequencing we characterize TSS utilization across a broad range of normal and developmental tissues. We identify a novel proximal promoter (P6) within CD133(+) melanoma cell lines and stem cells. Additional exon array sampling finds P6 to be active in populations enriched for mesenchyme, neural stem cells and within CD133(+) enriched Ewing sarcomas. The P6 promoter is enriched with respect to previously characterized PROM1 promoters for a HMGI/Y (HMGA1) family transcription factor binding site motif and exhibits different epigenetic modifications relative to the canonical promoter region of PROM1.

The second wave of next generation sequencing technologies, referred to as single-molecule sequencing (SMS), carries the promise of profiling samples directly without employing polymerase chain reaction steps used by amplification-based sequencing (AS) methods. To examine the merits of both technologies, we examine mRNA sequencing results from single-molecule and amplification-based sequencing in a set of human cancer cell lines and tissues. We observe a characteristic coverage bias towards high abundance transcripts in amplification-based sequencing. A larger fraction of AS reads cover highly expressed genes, such as those associated with translational processes and housekeeping genes, resulting in relatively lower coverage of genes at low and mid-level abundance. In contrast, the coverage of high abundance transcripts plateaus off using SMS. Consequently, SMS is able to sequence lower- abundance transcripts more thoroughly, including some that are undetected by AS methods; however, these include many more mapping artifacts. A better understanding of the technical and analytical factors introducing platform specific biases in high throughput transcriptome sequencing applications will be critical in cross platform meta-analytic studies.

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

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.

RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER+) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1-6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective.

Chromosomal rearrangements often result in active regulatory regions juxtaposed upstream of an oncogene to generate an expressed gene fusion. Repeated activation of a common downstream partner-with differing upstream regions across a patient cohort-suggests a conserved oncogenic role. Analysis of 9,638 patients across 32 solid tumor types revealed an annotated long noncoding RNA (lncRNA), Breast Cancer Anti-Estrogen Resistance 4 (BCAR4), was the most prevalent, uncharacterized, downstream gene fusion partner occurring in 11 cancers. Its oncogenic role was confirmed using multiple cell lines with endogenous BCAR4 gene fusions. Furthermore, overexpressing clinically prevalent BCAR4 gene fusions in untransformed cell lines was sufficient to induce an oncogenic phenotype. We show that the minimum common region to all gene fusions harbors an open reading frame that is necessary to drive proliferation.

High-grade serous ovarian cancer (HGSC) is the most lethal histotype of ovarian cancer and the majority of cases present with metastasis and late-stage disease. Over the last few decades, the overall survival for patients has not significantly improved, and there are limited targeted treatment options. We aimed to better characterize the distinctions between primary and metastatic tumors based on short- or long-term survival. We characterized 39 matched primary and metastatic tumors by whole exome and RNA sequencing. Of these, 23 were short-term (ST) survivors (overall survival (OS) < 3.5 years) and 16 were long-term (LT) survivors (OS > 5 years). We compared somatic mutations, copy number alterations, mutational burden, differential gene expression, immune cell infiltration, and gene fusion predictions between the primary and metastatic tumors and between ST and LT survivor cohorts. There were few differences in RNA expression between paired primary and metastatic tumors, but significant differences between the transcriptomes of LT and ST survivors in both their primary and metastatic tumors. These findings will improve the understanding of the genetic variation in HGSC that exist between patients with different prognoses and better inform treatments by identifying new targets for drug development.

Snail1 is a zinc finger transcriptional repressor whose pathological expression has been linked to cancer cell epithelial-mesenchymal transition (EMT) programs and the induction of tissue-invasive activity, but pro-oncogenic events capable of regulating Snail1 activity remain largely uncharacterized. Herein, we demonstrate that p53 loss-of-function or mutation promotes cancer cell EMT by de-repressing Snail1 protein expression and activity. In the absence of wild-type p53 function, Snail1-dependent EMT is activated in colon, breast, and lung carcinoma cells as a consequence of a decrease in miRNA-34 levels, which suppress Snail1 activity by binding to highly conserved 3' untranslated regions in Snail1 itself as well as those of key Snail1 regulatory molecules, including β-catenin, LEF1, and Axin2. Although p53 activity can impact cell cycle regulation, apoptosis, and DNA repair pathways, the EMT and invasion programs initiated by p53 loss of function or mutation are completely dependent on Snail1 expression. These results identify a new link between p53, miR-34, and Snail1 in the regulation of cancer cell EMT programs.

Cell plasticity regulated by the balance between the mesenchymal to epithelial transition (MET) and the opposite program, EMT, is critical in the metastatic cascade. Several transcription factors (TFs) are known to regulate EMT, though the mechanisms of MET remain unclear. We demonstrate a novel function of two TFs, OVOL1 and OVOL2, as critical inducers of MET in human cancers. Our findings indicate that the OVOL-TFs control MET through a regulatory feedback loop with EMT-inducing TF ZEB1, and the regulation of mRNA splicing by inducing Epithelial Splicing Regulatory Protein 1 (ESRP1). Using mouse prostate tumor models we show that expression of OVOL-TFs in mesenchymal prostate cancer cells attenuates their metastatic potential. The role of OVOL-TFs as inducers of MET is further supported by expression analyses in 917 cancer cell lines, suggesting their role as crucial regulators of epithelial-mesenchymal cell plasticity in cancer.

Protein-DNA interaction constitutes a basic mechanism for the genetic regulation of target gene expression. Deciphering this mechanism has been a daunting task due to the difficulty in characterizing protein-bound DNA on a large scale. A powerful technique has recently emerged that couples chromatin immunoprecipitation (ChIP) with next-generation sequencing, (ChIP-Seq). This technique provides a direct survey of the cistrom of transcription factors and other chromatin-associated proteins. In order to realize the full potential of this technique, increasingly sophisticated statistical algorithms have been developed to analyze the massive amount of data generated by this method.

We have encountered pancreatic tumors with unique histologic features, which do not conform to any of the known tumors of the pancreas or other anatomical sites. We aimed to define their clinicopathologic features and whether they are characterized by recurrent molecular signatures. Eight cases were identified; studied histologically and by immunohistochemistry. Selected cases were also subjected to whole-exome sequencing (WES; n = 4), RNA-sequencing (n = 6), Archer FusionPlex assay (n = 5), methylation profiling using the Illumina MethylationEPIC (850k) array platform (n = 6), and TERT promoter sequencing (n = 5). Six neoplasms occurred in females. The mean age was 43 years (range: 26-75). Five occurred in the head/neck of the pancreas. All patients were treated surgically; none received neoadjuvant/adjuvant therapy. All patients are free of disease after 53 months of median follow-up (range: 8-94). The tumors were well-circumscribed, and the median size was 1.8 cm (range: 1.3-5.8). Microscopically, the unencapsulated tumors had a geographic pattern of epithelioid cell nests alternating with spindle cell fascicles. Some areas showed dense fibrosis, in which enmeshed tumor cells imparted a slit-like pattern. The predominant epithelioid cells had scant cytoplasm and round-oval nuclei with open chromatin. The spindle cells displayed irregular, hyperchromatic nuclei. Mitoses were rare. No lymph node metastases were identified. All tumors were positive for vimentin, CD99 and cytokeratin (patchy), while negative for markers of solid pseudopapillary neoplasm, neuroendocrine, acinar, myogenic/rhabdoid, vascular, melanocytic, or lymphoid differentiation, gastrointestinal stromal tumor as well as MUC4. Whole-exome sequencing revealed no recurrent somatic mutations or amplifications/homozygous deletions in any known oncogenes or tumor suppressor genes. RNA-sequencing and the Archer FusionPlex assay did not detect any recurrent likely pathogenic gene fusions. Single sample gene set enrichment analysis revealed that these tumors display a likely mesenchymal transcriptomic program. Unsupervised analysis (t-SNE) of their methylation profiles against a set of different mesenchymal neoplasms demonstrated a distinct methylation pattern. Here, we describe pancreatic neoplasms with unique morphologic/immunophenotypic features and a distinct methylation pattern, along with a lack of abnormalities in any of key genetic drivers, supporting that these neoplasms represent a novel entity with an indolent clinical course. Given their mesenchymal transcriptomic features, we propose the designation of "sclerosing epithelioid mesenchymal neoplasm" of the pancreas.