Genomic hallmarks of localized, non-indolent prostate cancer.

Prostate tumours are highly variable in their response to therapies, but clinically available prognostic factors can explain only a fraction of this heterogeneity. Here we analysed 200 whole-genome sequences and 277 additional whole-exome sequences from localized, non-indolent prostate tumours with similar clinical risk profiles, and carried out RNA and methylation analyses in a subset. These tumours had a paucity of clinically actionable single nucleotide variants, unlike those seen in metastatic disease. Rather, a significant proportion of tumours harboured recurrent non-coding aberrations, large-scale genomic rearrangements, and alterations in which an inversion repressed transcription within its boundaries. Local hypermutation events were frequent, and correlated with specific genomic profiles. Numerous molecular aberrations were prognostic for disease recurrence, including several DNA methylation events, and a signature comprised of these aberrations outperformed well-described prognostic biomarkers. We suggest that intensified treatment of genomically aggressive localized prostate cancer may improve cure rates.

Reporters to mark and eliminate basal or luminal epithelial cells in culture and in vivo.

The contribution of basal and luminal cells to cancer progression and metastasis is poorly understood. We report generation of reporter systems driven by either keratin-14 (K14) or keratin-8 (K8) promoter that not only express a fluorescent protein but also an inducible suicide gene. Transgenic mice express the reporter genes in the right cell compartments of mammary gland epithelia and respond to treatment with toxins. In addition, we engineered the reporters into 4T1 metastatic mouse tumor cell line and demonstrate that K14+ cells, but not K14- or K8+, are both highly invasive in three-dimensional (3D) culture and metastatic in vivo. Treatment of cells in culture, or tumors in mice, with reporter-targeting toxin inhibited both invasive behavior and metastasis in vivo. RNA sequencing (RNA-seq), secretome, and epigenome analysis of K14+ and K14- cells led to the identification of amphoterin-induced protein 2 (Amigo2) as a new cell invasion driver whose expression correlated with decreased relapse-free survival in patients with TP53 wild-type (WT) breast cancer.

Evaluation and prognostic significance of ACAT1 as a marker of prostate cancer progression.

INTRODUCTION: Prostate cancer is the second leading cause of cancer-related death among men in North America. While a majority of prostate cancer cases remain indolent, subsets of patients develop aggressive cancers, which may lead to death. The current methods of detection include digital rectal examination and the serum PSA test. However, due to lack of specificity, neither of these approaches is able to accurately discriminate between indolent and aggressive cancer, which is why there is a need for additional prognostic factors. Previously, we identified enzymes of the ketogenic pathway, particularly ACAT1, to be elevated in aggressive prostate cancer. METHODS: In the current study, we assessed the diagnostic and prognostic potential of ACAT1 by analyzing its expression using immunohistochemistry on a tissue microarray consisting of 251 clinically localized prostate cancer patients who have undergone radical prostatectomy. RESULTS: Using quantitative digital imaging software, we found that ACAT1 expression was significantly greater in cancerous cores compared to adjacent benign cores (P < 0.0001), in Gleason score (GS) ≥8 cancers versus GS≤6 cancers (P < 0.0001), GS≥8 cancers versus GS7 cancers (P = 0.001), as well as pT3/pT4 versus pT2 cancers (P = 0.001). In addition, ACAT1 predicted biochemical recurrence in univariate (HR, 1.81, CI = 1.13-2.9, P = 0.0128), and multivariate models (HR, 1.69, CI = 1.01-2.81, P = 0.0431) including pre-operative PSA level, Gleason score and pathological stage. In univariate time-to-recurrence analysis, ACAT1 expression predicted recurrence in ERG negative cases (P = 0.0025), whereas ERG positive cases did not display any differences. DISCUSSION: Taken together, these findings indicate that ACAT1 expression could serve as a potential prognostic marker in prostate cancer, specifically in differentiating indolent and aggressive forms of cancer.

Transposable Elements Are Co-opted as Oncogenic Regulatory Elements by Lineage-Specific Transcription Factors in Prostate Cancer.

Transposable elements hold regulatory functions that impact cell fate determination by controlling gene expression. However, little is known about the transcriptional machinery engaged at transposable elements in pluripotent and mature versus oncogenic cell states. Through positional analysis over repetitive DNA sequences of H3K27ac chromatin immunoprecipitation sequencing data from 32 normal cell states, we report pluripotent/stem and mature cell state-specific "regulatory transposable elements." Pluripotent/stem elements are binding sites for pluripotency factors (e.g., NANOG, SOX2, OCT4). Mature cell elements are docking sites for lineage-specific transcription factors, including AR and FOXA1 in prostate epithelium. Expanding the analysis to prostate tumors, we identify a subset of regulatory transposable elements shared with pluripotent/stem cells, including Tigger3a. Using chromatin editing technology, we show how such elements promote prostate cancer growth by regulating AR transcriptional activity. Collectively, our results suggest that oncogenesis arises from lineage-specific transcription factors hijacking pluripotent/stem cell regulatory transposable elements. SIGNIFICANCE: We show that oncogenesis relies on co-opting transposable elements from pluripotent stem cells as regulatory elements altering the recruitment of lineage-specific transcription factors. We further discover how co-option is dependent on active chromatin states with important implications for developing treatment options against drivers of oncogenesis across the repetitive DNA. This article is featured in Selected Articles from This Issue, p. 2293.

Association of tissue promoter methylation levels of APC, TGFß2, HOXD3 and RASSF1A with prostate cancer progression.

Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer (PCa). We correlated quantitative promoter methylation levels of APC, TGFß2 and RASSF1A in 219 radical prostatectomies diagnosed between 1998 and 2001 with clinicopathological follow-up data available including Gleason Pattern (GP), Gleason Score (GS) and pathological stage and explored their potential in predicting biochemical recurrence using univariate and multivariate analyses. We observed that the average methylation levels of APC increased significantly from GS ≤ 6 to GS7, and pT2 to pT3a, and that of TGFß2 increased from GS ≤ 6 to GS7, but not for RASSF1A. PCa samples were also stratified into high methylation (HM) and low methylation (LM) groups based on the PMR scores of all cases analyzed for each marker. The HM frequency of APC was greater in pT3a than pT2, and in GS ≥ 8 than GS ≤ 6. The HM frequency also increased significantly from GP3 to GP4 for APC, TGFß2 and RASSF1A. APC methylation level was a significant predictor of biochemical recurrence in univariate analysis (p-value = 0.028). Finally, we combined methylation data of these three genes with the previously reported novel methylation biomarker HOXD3. Quantitative methylation assessment of a multiplex panel of markers, consisting of APC, HOXD3 and TGFß2, outperforms any single marker for the prediction of biochemical recurrence (p-value = 0.017). Our study demonstrated that quantitative increase in promoter methylation levels of APC, HOXD3 and TGFß2 are associated with PCa progression.

Reorganization of the 3D Genome Pinpoints Noncoding Drivers of Primary Prostate Tumors.

Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However, the impact of this instability on the noncoding genome and its three-dimensional organization to aid progression is unclear. Using primary benign and tumor tissue, we find a high concordance in higher-order three-dimensional genome organization. This concordance argues for constraints to the topology of prostate tumor genomes. Nonetheless, we identified changes in focal chromatin interactions, typical of loops bridging noncoding cis-regulatory elements, and showed how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events resulted in opposing differential expression of genes found at antipodes of rearrangements. Collectively, these results argue that changes to focal chromatin interactions, as opposed to higher-order genome organization, allow for aberrant gene regulation and are repeatedly mediated by structural variants in primary prostate cancer. SIGNIFICANCE: This work showcases how the noncoding genome can be hijacked by focal insults to its three-dimensional organization that contribute to prostate cancer oncogenesis.

Transcriptome-Wide Off-Target Effects of Steric-Blocking Oligonucleotides.

Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to RNA transcripts and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization driven, whereas differential expression events are more common and driven by other mechanisms (including spurious experimental variation). We further evaluated the performance of in silico screens for off-target splicing events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate (FDR) of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR of 90%. Despite these large improvements in performance, this approach does not detect the majority of events at an FDR <99%. Our results suggest that in silico methods are currently of limited use for predicting the off-target effects of SBOs, and experimental screening by RNA-seq should be the preferred approach.

The Transition from Quiescent to Activated States in Human Hematopoietic Stem Cells Is Governed by Dynamic 3D Genome Reorganization.

Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.

DNA methylation of HOXD3 as a marker of prostate cancer progression.

DNA methylation in gene promoters causes gene silencing and is a common event in cancer development and progression. The ability of aberrant methylation events to serve as diagnostic and prognostic markers is being appreciated for many cancers, including prostate cancer. Using quantitative MethyLight technology, we evaluated the relationship between HOXD3 methylation and clinicopathological parameters including biochemical recurrence, pathological stage, Gleason score (GS), and Gleason pattern in a series of 232 radical prostatectomies performed between 1998 and 2001. HOXD3 methylation was significantly greater in GS 7 cancers vs GS < or = 6 cancers (P-value <0.001) as well as pT3/pT4 vs pT2 cancers (P-value <0.001). The proportion of cases with high methylation in GS 7 vs < or = GS 6 and pT3/pT4 vs pT2 were also significantly different (P-values=0.002 and 0.005, respectively). There were also significant increases in methylation from Gleason pattern 2-3 and from pattern 3 to 4/5 (paired t-test P-values=0.01 and <0.001, respectively), whereas methylation from lymph node metastases was decreased when compared with matched tumor tissue (P-value=0.029). HOXD3 methylation was associated with biochemical recurrence in univariate analysis (P-value=0.043) and showed evidence for interaction with pathological stage as a predictor variable in Cox regression analysis (P-value=0.028). The results indicate that HOXD3 methylation distinguishes low-grade prostate cancers from intermediate and high-grade ones and may also have prognostic value when considered together with pathological stage.

ATP7B variant c.1934T > G p.Met645Arg causes Wilson disease by promoting exon 6 skipping.

Wilson disease is a recessive genetic disorder caused by pathogenic loss-of-function variants in the ATP7B gene. It is characterized by disrupted copper homeostasis resulting in liver disease and/or neurological abnormalities. The variant NM_000053.3:c.1934T > G (Met645Arg) has been reported as compound heterozygous, and is highly prevalent among Wilson disease patients of Spanish descent. Accordingly, it is classified as pathogenic by leading molecular diagnostic centers. However, functional studies suggest that the amino acid change does not alter protein function, leading one ClinVar submitter to question its pathogenicity. Here, we used a minigene system and gene-edited HepG2 cells to demonstrate that c.1934T > G causes ~70% skipping of exon 6. Exon 6 skipping results in frameshift and stop-gain, leading to loss of ATP7B function. The elucidation of the mechanistic effect for this variant resolves any doubt about its pathogenicity and enables the development of genetic medicines for restoring correct splicing.

Noncoding mutations target cis-regulatory elements of the FOXA1 plexus in prostate cancer.

Prostate cancer is the second most commonly diagnosed malignancy among men worldwide. Recurrently mutated in primary and metastatic prostate tumors, FOXA1 encodes a pioneer transcription factor involved in disease onset and progression through both androgen receptor-dependent and androgen receptor-independent mechanisms. Despite its oncogenic properties however, the regulation of FOXA1 expression remains unknown. Here, we identify a set of six cis-regulatory elements in the FOXA1 regulatory plexus harboring somatic single-nucleotide variants in primary prostate tumors. We find that deletion and repression of these cis-regulatory elements significantly decreases FOXA1 expression and prostate cancer cell growth. Six of the ten single-nucleotide variants mapping to FOXA1 regulatory plexus significantly alter the transactivation potential of cis-regulatory elements by modulating the binding of transcription factors. Collectively, our results identify cis-regulatory elements within the FOXA1 plexus mutated in primary prostate tumors as potential targets for therapeutic intervention.

Cistrome Partitioning Reveals Convergence of Somatic Mutations and Risk Variants on Master Transcription Regulators in Primary Prostate Tumors.

Thousands of noncoding somatic single-nucleotide variants (SNVs) of unknown function are reported in tumors. Partitioning the genome according to cistromes reveals the enrichment of somatic SNVs in prostate tumors as opposed to adjacent normal tissue cistromes of master transcription regulators, including AR, FOXA1, and HOXB13. This parallels enrichment of prostate cancer genetic predispositions over these transcription regulators' tumor cistromes, exemplified at the 8q24 locus harboring both risk variants and somatic SNVs in cis-regulatory elements upregulating MYC expression. However, Massively Parallel Reporter Assays reveal that few SNVs can alter the transactivation potential of individual cis-regulatory elements. Instead, similar to inherited risk variants, SNVs accumulate in cistromes of master transcription regulators required for prostate cancer development.

The Proteogenomic Landscape of Curable Prostate Cancer.

DNA sequencing has identified recurrent mutations that drive the aggressiveness of prostate cancers. Surprisingly, the influence of genomic, epigenomic, and transcriptomic dysregulation on the tumor proteome remains poorly understood. We profiled the genomes, epigenomes, transcriptomes, and proteomes of 76 localized, intermediate-risk prostate cancers. We discovered that the genomic subtypes of prostate cancer converge on five proteomic subtypes, with distinct clinical trajectories. ETS fusions, the most common alteration in prostate tumors, affect different genes and pathways in the proteome and transcriptome. Globally, mRNA abundance changes explain only ∼10% of protein abundance variability. As a result, prognostic biomarkers combining genomic or epigenomic features with proteomic ones significantly outperform biomarkers comprised of a single data type.

Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors.

The aim of the present study was to explore DNA methylation aberrations in association with cribriform architecture and intraductal carcinoma (IDC) of the prostate, as there is robust evidence that these morphological features are associated with aggressive disease and have significant clinical implications. Herein, the associations of a panel of seven known prognostic DNA methylation biomarkers with cribriform and IDC features were examined in a series of 91 Gleason pattern (GP) 4 tumors derived from Gleason score 7 radical prostatectomies. Gene specific DNA methylation was compared between cribriform and/or IDC positive vs. negative cases, and in association with clinicopathological features, using Chi square and Mann-Whitney U tests. DNA methylation of the adenomatous polyposis coli, Ras association domain family member 1 and T-box 15 genes was significantly elevated in GP4 tumors with cribriform and/or IDC features compared with negative cases (P=0.045, P=0.007 and P=0.013, respectively). To the best of our knowledge, this provides the first evidence for an association between cribriform and/or IDC and methylation biomarkers, and warrants further investigation of additional DNA methylation events in association with various architectural patterns in prostate cancer.

TMPRSS2-ERG fusion co-opts master transcription factors and activates NOTCH signaling in primary prostate cancer.

TMPRSS2-ERG (T2E) structural rearrangements typify ∼50% of prostate tumors and result in overexpression of the ERG transcription factor. Using chromatin, genomic and expression data, we show distinct cis-regulatory landscapes between T2E-positive and non-T2E primary prostate tumors, which include clusters of regulatory elements (COREs). This difference is mediated by ERG co-option of HOXB13 and FOXA1, implementing a T2E-specific transcriptional profile. We also report a T2E-specific CORE on the structurally rearranged ERG locus arising from spreading of the TMPRSS2 locus pre-existing CORE, assisting in its overexpression. Finally, we show that the T2E-specific cis-regulatory landscape underlies a vulnerability against the NOTCH pathway. Indeed, NOTCH pathway inhibition antagonizes the growth and invasion of T2E-positive prostate cancer cells. Taken together, our work shows that overexpressed ERG co-opts master transcription factors to deploy a unique cis-regulatory landscape, inducing a druggable dependency on NOTCH signaling in T2E-positive prostate tumors.

Epigenome-Wide DNA Methylation Profiling Identifies Differential Methylation Biomarkers in High-Grade Bladder Cancer.

Epigenetic changes, including CpG island hypermethylation, occur frequently in bladder cancer (BC) and may be exploited for BC detection and distinction between high-grade (HG) and low-grade (LG) disease. Genome-wide methylation analysis was performed using Agilent Human CpG Island Microarrays to determine epigenetic differences between LG and HG cases. Pathway enrichment analysis and functional annotation determined that the most frequently methylated pathways in HG BC were enriched for anterior/posterior pattern specification, embryonic skeletal system development, neuron fate commitment, DNA binding, and transcription factor activity. We identified 990 probes comprising a 32-gene panel that completely distinguished LG from HG based on methylation. Selected genes from this panel, EOMES, GP5, PAX6, TCF4, and ZSCAN12, were selected for quantitative polymerase chain reaction-based validation by MethyLight in an independent series (n=84) of normal bladder samples and LG and HG cases. GP5 and ZSCAN12, two novel methylated genes in BC, were significantly hypermethylated in HG versus LG BC (P≤.03). We validated our data in a second independent cohort of LG and HG BC cases (n=42) from The Cancer Genome Atlas (TCGA). Probes representing our 32-gene panel were significantly differentially methylated in LG versus HG tumors (P≤.04). These results indicate the ability to distinguish normal tissue from cancer, as well as LG from HG, based on methylation and reveal important pathways dysregulated in HG BC. Our findings were corroborated using publicly available data sets from TCGA. Ultimately, the creation of a methylation panel, including GP5 and ZSCAN12, able to distinguish between disease phenotypes will improve disease management and patient outcomes.

Pre-neoplastic epigenetic disruption of transcriptional enhancers in chronic inflammation.

Chronic periodontitis (CP) is a chronic inflammatory disease independently associated with higher incidence of oral cavity squamous cell carcinoma (OSCC). However, the molecular mechanism responsible for this increased incidence is unknown. Here we profiled the DNA methylome of CP patients and healthy controls and compared to a large set of OSCC samples from TCGA. We observed a significant overlap between the altered DNA methylation patterns in CP and in OSCC, suggesting an emergence of a pre-neoplastic epigenome in CP. Remarkably, the hypermethylated CpGs in CP were significantly enriched for enhancer elements. This aberrant enhancer methylation is functional and able to disrupt enhancer activity by preventing the binding of chromatin looping factors. This study provides new insights on the molecular mechanisms linking chronic inflammation and tumor predisposition, highlighting the role of epigenetic disruption of transcriptional enhancers.

Dynamic interplay between locus-specific DNA methylation and hydroxymethylation regulates distinct biological pathways in prostate carcinogenesis.

BACKGROUND: Despite the significant global loss of DNA hydroxymethylation marks in prostate cancer tissues, the locus-specific role of hydroxymethylation in prostate tumorigenesis is unknown. We characterized hydroxymethylation and methylation marks by performing whole-genome next-generation sequencing in representative normal and prostate cancer-derived cell lines in order to determine functional pathways and key genes regulated by these epigenomic modifications in cancer. RESULTS: Our cell line model shows disruption of hydroxymethylation distribution in cancer, with global loss and highly specific gain in promoter and CpG island regions. Significantly, we observed locus-specific retention of hydroxymethylation marks in specific intronic and intergenic regions which may play a novel role in the regulation of gene expression in critical functional pathways, such as BARD1 signaling and steroid hormone receptor signaling in cancer. We confirm a modest correlation of hydroxymethylation with expression in intragenic regions in prostate cancer, while identifying an original role for intergenic hydroxymethylation in differentially expressed regulatory pathways in cancer. We also demonstrate a successful strategy for the identification and validation of key candidate genes from differentially regulated biological pathways in prostate cancer. CONCLUSIONS: Our results indicate a distinct function for aberrant hydroxymethylation within each genomic feature in cancer, suggesting a specific and complex role for the deregulation of hydroxymethylation in tumorigenesis, similar to methylation. Subsequently, our characterization of key cellular pathways exhibiting dynamic enrichment patterns for methylation and hydroxymethylation marks may allow us to identify differentially epigenetically modified target genes implicated in prostate cancer tumorigenesis.

Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer.

Sustained expression of the estrogen receptor-α (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program. Somatic copy number alterations involving the ESR1 gene occur in approximately 1% of ESR1-positive breast cancers, suggesting that other mechanisms underlie the persistent expression of ESR1. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating ESR1 in 7% of ESR1-positive breast cancers. These mutations regulate ESR1 expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk-associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.

Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.