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While the mutational and transcriptional landscapes of renal cell carcinoma (RCC) are well-known, the epigenome is poorly understood. We characterize the epigenome of clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC) by using ChIP-seq, ATAC-Seq, RNA-seq, and SNP arrays. We integrate 153 individual data sets from 42 patients and nominate 50 histology-specific master transcription factors (MTF) to define RCC histologic subtypes, including EPAS1 and ETS-1 in ccRCC, HNF1B in pRCC, and FOXI1 in chRCC. We confirm histology-specific MTFs via immunohistochemistry including a ccRCC-specific TF, BHLHE41. FOXI1 overexpression with knock-down of EPAS1 in the 786-O ccRCC cell line induces transcriptional upregulation of chRCC-specific genes, TFCP2L1, ATP6V0D2, KIT, and INSRR, implicating FOXI1 as a MTF for chRCC. Integrating RCC GWAS risk SNPs with H3K27ac ChIP-seq and ATAC-seq data reveals that risk-variants are significantly enriched in allelically-imbalanced peaks. This epigenomic atlas in primary human samples provides a resource for future investigation.

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. Here, we investigate the epigenomic basis of this resistance mechanism by profiling histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identify a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying cancer dependencies through epigenomic profiling., The molecular processes that lead to neuroendocrine prostate cancer after treating prostate adenocarcinoma (PRAD) are not well understood. Here the authors show that regulation by FOXA1 and changes in the epigenomic profile drive the transition from PRAD to a neuroendocrine phenotype.

The vast majority of disease-associated single-nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWASs) are located in the non-protein-coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here we describe a general pipeline in which candidate functional SNPs are first evaluated by fine mapping, epigenomic profiling, and epigenome editing, and then interrogated for causal function by using genome editing to create isogenic cell lines followed by phenotypic characterization. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top-scoring SNP. Epigenome editing confirmed that the rs339331 region possessed regulatory potential. By using transcription activator-like effector nuclease (TALEN)-mediated genome editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the 'T' risk allele increased transcription of the regulatory factor 6 (RFX6) gene, increased homeobox B13 (HOXB13) binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region compared to lines homozygous for the 'C' protective allele. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach that can be used to establish functional causality for noncoding sequence variants identified by GWASs.

Genome-wide association studies have identified several risk associations for ovarian carcinomas (OC) but not for mucinous ovarian carcinomas (MOC). Genotypes from OC cases and controls were imputed into the 1000 Genomes Project reference panel. Analysis of 1,644 MOC cases and 21,693 controls identified three novel risk associations: rs752590 at 2q13 (P = 3.3 × 10−8), rs711830 at 2q31.1 (P = 7.5 × 10−12) and rs688187 at 19q13.2 (P = 6.8 × 10−13). Expression Quantitative Trait Locus (eQTL) analysis in ovarian and colorectal tumors (which are histologically similar to MOC) identified significant eQTL associations for HOXD9 at 2q31.1 in ovarian (P = 4.95 × 10−4, FDR = 0.003) and colorectal (P = 0.01, FDR = 0.09) tumors, and for PAX8 at 2q13 in colorectal tumors (P = 0.03, FDR = 0.09). Chromosome conformation capture analysis identified interactions between the HOXD9 promoter and risk SNPs at 2q31.1. Overexpressing HOXD9 in MOC cells augmented the neoplastic phenotype. These findings provide the first evidence for MOC susceptibility variants and insights into the underlying biology of the disease.

Cancer-associated fibroblasts (CAFs) play an important role in tumor initiation and progression. The aim of this study is to explore the role of 2 CAF markers, fibroblast activated protein (FAP) and α-smooth muscle actin (αSMA), in patients with epithelial ovarian cancer (EOC) post-neoadjuvant chemotherapy. Sixty-six patients with the diagnosis of EOC treated with debulking surgery after neoadjuvant therapy were retrieved from the archives. Immunohistochemistry for FAP and αSMA antibodies were performed on paraffin-embedded tissue. Fisher's exact test was performed to test the association between FAP and αSMA expression and disease status. Kaplan-Meier method with log-rank test was used to check the survival difference between different FAP tumor/stroma expressions. FAP stroma (pos) . expression was strongly associated with higher recurrences rate [OR: 15.95; 95 % CI: 1.521-835.206; p = 0.0072]. Cases with combined FAP stroma (pos) and FAP tumor (neg) had higher death rate [OR: 4.845; 95 % CI: 1.53-16.61; p = 0.0046] and higher recurrence rate [OR: 5.12; 95 % CI: 0.91-54.42; p = 0.0487] compared to all the others. Cases with combined FAP stroma (neg) and FAP tumor (neg) were more likely to have lower recurrence rates [OR: 0.086; 95 % CI: 0.001-0.997; p = 0.0248]. αSMA was expressed by tumor-associated stroma in 95 % of cases and by tumor cells in 9 % of cases. No statistical power was found for αSMA and disease status. Our data indicate that FAP plays an important role in predicting tumor aggressiveness in patients with EOC post-neoadjuvant therapy, and its frequent expression in this malignancy implicates that FAP targeted therapy could be a very attractive strategy.

The SRC proto-oncogene is commonly overexpressed or activated during cancer development. Src family kinase inhibitors are approved for the treatment of certain leukemias, and are in clinical trials for the treatment of solid tumors. Src signaling is activated in endometriosis, a precursor of clear cell and endometrioid subtypes of epithelial ovarian cancers (OCs). We examined the expression of phosphorylated Src (Src-pY416) in 381 primary OC tissues. Thirty-six percent of OCs expressed Src-pY416. Src-pY416 expression was most common in endometriosis-associated OCs (EAOCs) (P=0.011), particularly in clear cell OCs where 58.5% of cases expressed Src-pY416. Src-pY416 expression was associated with shorter overall survival (log rank P=0.002). In vitro inhibition of Src signaling using 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) resulted in reduced anchorage-independent and -dependent growth, and in three-dimensional cell culture models PP2 disrupted aggregate formation in Src-pY416-positive but not in Src-pY416-negative cell lines. These data suggest that targeting active Src signaling could be a novel therapeutic opportunity for EAOCs, and support the further pre-clinical investigation of Src family kinase inhibitors for treating OCs expressing Src-pY416.