Widespread and Functional RNA Circularization in Localized Prostate Cancer.

The cancer transcriptome is remarkably complex, including low-abundance transcripts, many not polyadenylated. To fully characterize the transcriptome of localized prostate cancer, we performed ultra-deep total RNA-seq on 144 tumors with rich clinical annotation. This revealed a linear transcriptomic subtype associated with the aggressive intraductal carcinoma sub-histology and a fusion profile that differentiates localized from metastatic disease. Analysis of back-splicing events showed widespread RNA circularization, with the average tumor expressing 7,232 circular RNAs (circRNAs). The degree of circRNA production was correlated to disease progression in multiple patient cohorts. Loss-of-function screening identified 11.3% of highly abundant circRNAs as essential for cell proliferation; for ∼90% of these, their parental linear transcripts were not essential. Individual circRNAs can have distinct functions, with circCSNK1G3 promoting cell growth by interacting with miR-181. These data advocate for adoption of ultra-deep RNA-seq without poly-A selection to interrogate both linear and circular transcriptomes.

MYC Protein Interactome Profiling Reveals Functionally Distinct Regions that Cooperate to Drive Tumorigenesis.

Transforming members of the MYC family (MYC, MYCL1, and MYCN) encode transcription factors containing six highly conserved regions, termed MYC homology boxes (MBs). By conducting proteomic profiling of the MB interactomes, we demonstrate that half of the MYC interactors require one or more MBs for binding. Comprehensive phenotypic analyses reveal that two MBs, MB0 and MBII, are universally required for transformation. MBII mediates interactions with acetyltransferase-containing complexes, enabling histone acetylation, and is essential for MYC-dependent tumor initiation. By contrast, MB0 mediates interactions with transcription elongation factors via direct binding to the general transcription factor TFIIF. MB0 is dispensable for tumor initiation but is a major accelerator of tumor growth. Notably, the full transforming activity of MYC can be restored by co-expression of the non-transforming MB0 and MBII deletion proteins, indicating that these two regions confer separate molecular functions, both of which are required for oncogenic MYC activity.

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.

BPG: Seamless, automated and interactive visualization of scientific data.

BACKGROUND: We introduce BPG, a framework for generating publication-quality, highly-customizable plots in the R statistical environment. RESULTS: This open-source package includes multiple methods of displaying high-dimensional datasets and facilitates generation of complex multi-panel figures, making it suitable for complex datasets. A web-based interactive tool allows online figure customization, from which R code can be downloaded for integration with computational pipelines. CONCLUSION: BPG provides a new approach for linking interactive and scripted data visualization and is available at http://labs.oicr.on.ca/boutros-lab/software/bpg or via CRAN at https://cran.r-project.org/web/packages/BoutrosLab.plotting.general.

Comparison of pre-processing methods for Infinium HumanMethylation450 BeadChip array.

MOTIVATION: Microarrays are widely used to quantify DNA methylation because they are economical, require only small quantities of input DNA and focus on well-characterized regions of the genome. However, pre-processing of methylation microarray data is challenging because of confounding factors that include background fluorescence, dye bias and the impact of germline polymorphisms. Therefore, we present valuable insights and a framework for those seeking the most optimal pre-processing method through a data-driven approach. RESULTS: Here, we show that Dasen is the optimal pre-processing methodology for the Infinium HumanMethylation450 BeadChip array in prostate cancer, a frequently employed platform for tumour methylome profiling in both the TCGA and ICGC consortia. We evaluated the impact of 11 pre-processing methods on batch effects, replicate variabilities, sensitivities and sample-to-sample correlations across 809 independent prostate cancer samples, including 150 reported for the first time in this study. Overall, Dasen is the most effective for removing artefacts and detecting biological differences associated with tumour aggressivity. Relative to the raw dataset, it shows a reduction in replicate variances of 67% and 76% for ß- and M-values, respectively. Our study provides a unique pre-processing benchmark for the community with an emphasis on biological implications. AVAILABILITY AND IMPLEMENTATION: All software used in this study are publicly available as detailed in the article. CONTACT: paul.boutros@oicr.on.ca. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The telomere length landscape of prostate cancer.

Replicative immortality is a hallmark of cancer, and can be achieved through telomere lengthening and maintenance. Although the role of telomere length in cancer has been well studied, its association to genomic features is less well known. Here, we report the telomere lengths of 392 localized prostate cancer tumours and characterize their relationship to genomic, transcriptomic and proteomic features. Shorter tumour telomere lengths are associated with elevated genomic instability, including single-nucleotide variants, indels and structural variants. Genes involved in cell proliferation and signaling are correlated with tumour telomere length at all levels of the central dogma. Telomere length is also associated with multiple clinical features of a tumour. Longer telomere lengths in non-tumour samples are associated with a lower rate of biochemical relapse. In summary, we describe the multi-level integration of telomere length, genomics, transcriptomics and proteomics in localized prostate cancer.

The DNA methylation landscape of advanced prostate cancer.

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.

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.

Molecular landmarks of tumor hypoxia across cancer types.

Many primary-tumor subregions have low levels of molecular oxygen, termed hypoxia. Hypoxic tumors are at elevated risk for local failure and distant metastasis, but the molecular hallmarks of tumor hypoxia remain poorly defined. To fill this gap, we quantified hypoxia in 8,006 tumors across 19 tumor types. In ten tumor types, hypoxia was associated with elevated genomic instability. In all 19 tumor types, hypoxic tumors exhibited characteristic driver-mutation signatures. We observed widespread hypoxia-associated dysregulation of microRNAs (miRNAs) across cancers and functionally validated miR-133a-3p as a hypoxia-modulated miRNA. In localized prostate cancer, hypoxia was associated with elevated rates of chromothripsis, allelic loss of PTEN and shorter telomeres. These associations are particularly enriched in polyclonal tumors, representing a constellation of features resembling tumor nimbosus, an aggressive cellular phenotype. Overall, this work establishes that tumor hypoxia may drive aggressive molecular features across cancers and shape the clinical trajectory of individual tumors.

Genome-wide germline correlates of the epigenetic landscape of prostate cancer.

Oncogenesis is driven by germline, environmental and stochastic factors. It is unknown how these interact to produce the molecular phenotypes of tumors. We therefore quantified the influence of germline polymorphisms on the somatic epigenome of 589 localized prostate tumors. Predisposition risk loci influence a tumor's epigenome, uncovering a mechanism for cancer susceptibility. We identified and validated 1,178 loci associated with altered methylation in tumoral but not nonmalignant tissue. These tumor methylation quantitative trait loci influence chromatin structure, as well as RNA and protein abundance. One prominent tumor methylation quantitative trait locus is associated with AKT1 expression and is predictive of relapse after definitive local therapy in both discovery and validation cohorts. These data reveal intricate crosstalk between the germ line and the epigenome of primary tumors, which may help identify germline biomarkers of aggressive disease to aid patient triage and optimize the use of more invasive or expensive diagnostic assays.

Sex Differences in Cancer Driver Genes and Biomarkers.

Cancer differs significantly between men and women; even after adjusting for known epidemiologic risk factors, the sexes differ in incidence, outcome, and response to therapy. These differences occur in many but not all tumor types, and their origins remain largely unknown. Here, we compare somatic mutation profiles between tumors arising in men and in women. We discovered large differences in mutation density and sex biases in the frequency of mutation of specific genes; these differences may be associated with sex biases in DNA mismatch repair genes or microsatellite instability. Sex-biased genes include well-known drivers of cancer such as ß-catenin and BAP1 Sex influenced biomarkers of patient outcome, where different genes were associated with tumor aggression in each sex. These data call for increased study and consideration of the molecular role of sex in cancer etiology, progression, treatment, and personalized therapy.Significance: This study provides a comprehensive catalog of sex differences in somatic alterations, including in cancer driver genes, which influence prognostic biomarkers that predict patient outcome after definitive local therapy. Cancer Res; 78(19); 5527-37. ©2018 AACR.

MYC Interacts with the G9a Histone Methyltransferase to Drive Transcriptional Repression and Tumorigenesis.

MYC is an oncogenic driver that regulates transcriptional activation and repression. Surprisingly, mechanisms by which MYC promotes malignant transformation remain unclear. We demonstrate that MYC interacts with the G9a H3K9-methyltransferase complex to control transcriptional repression. Inhibiting G9a hinders MYC chromatin binding at MYC-repressed genes and de-represses gene expression. By identifying the MYC box II region as essential for MYC-G9a interaction, a long-standing missing link between MYC transformation and gene repression is unveiled. Across breast cancer cell lines, the anti-proliferative response to G9a pharmacological inhibition correlates with MYC sensitivity and gene signatures. Consistently, genetically depleting G9a in vivo suppresses MYC-dependent tumor growth. These findings unveil G9a as an epigenetic regulator of MYC transcriptional repression and a therapeutic vulnerability in MYC-driven cancers.

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology.

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.

Germline BRCA2 mutations drive prostate cancers with distinct evolutionary trajectories.

Germline mutations in the BRCA2 tumour suppressor are associated with both an increased lifetime risk of developing prostate cancer (PCa) and increased risk of aggressive disease. To understand this aggression, here we profile the genomes and methylomes of localized PCa from 14 carriers of deleterious germline BRCA2 mutations (BRCA2-mutant PCa). We show that BRCA2-mutant PCa harbour increased genomic instability and a mutational profile that more closely resembles metastastic than localized disease. BRCA2-mutant PCa shows genomic and epigenomic dysregulation of the MED12L/MED12 axis, which is frequently dysregulated in metastatic castration-resistant prostate cancer (mCRPC). This dysregulation is enriched in BRCA2-mutant PCa harbouring intraductal carcinoma (IDC). Microdissection and sequencing of IDC and juxtaposed adjacent non-IDC invasive carcinoma in 10 patients demonstrates a common ancestor to both histopathologies. Overall we show that localized castration-sensitive BRCA2-mutant tumours are uniquely aggressive, due to de novo aberration in genes usually associated with metastatic disease, justifying aggressive initial treatment.

Germline Mutations in the Kallikrein 6 Region and Predisposition for Aggressive Prostate Cancer.

Background: There is a need for markers that can specifically identify individuals at increased risk of harboring aggressive forms of prostate cancer (PCa). Methods: We surveyed the Kallikrein ( KLK ) region ( KLK 1-15) for single-nucleotide polymorphisms (SNPs) associated with aggressive PCa (Gleason Score ≥ 8) in 1858 PCa patients. Discovery cohorts (Swiss arm of the European Randomized Study of Screening for PCa, n = 379; Toronto, Canada, n = 540) and a validation cohort (Prostate, Lung, Colorectal and Ovarian [PLCO] screening trial, n = 939) were analyzed. Fine-mapping within the KLK region was carried out by genotyping and imputation in the discovery cohort, whereas PLCO data were provided through database of Genotypes and Phenotypes ( dbGaP ). The influence of SNPs of interest on biochemical-free survival was evaluated in a cohort of localized PCa from the International Cancer Genome Consortium (ICGC; n = 130) analyzed with next-generation sequencing. Single- and multi-SNP association studies, as well as haplotype analyses, were performed. All statistical tests were two-sided. Results: Several SNPs in very strong linkage disequilibrium in the KLK 6 region and located within the same haplotype (rs113640578, rs79324425, rs11666929, rs28384475, rs3810287), identified individuals at increased risk of aggressive PCa in both discovery (odds ratio [OR] = 3.51-3.64, 95% confidence interval [CI] = 2.01 to 6.36, P = 1.0x10 -5 -8.4x10 -6 ) and validation (OR = 1.89-1.96, 95% CI = 0.99 to 3.71, P = .04-.05) cohorts. The overall test of haplotype association was highly statistically significant in each cohort ( P = 3.5x10 -4 and .006, respectively) and in the three data sets combined ( P = 2.3x10 -5 ). These germline SNPs independently predicted relapse in the ICGC cohort (hazard ratio = 3.15, 95% CI = 1.57 to 6.34, P = .001). Conclusions: Our fine-mapping study has identified novel loci in the KLK 6 region strongly associated with aggressive PCa.

A Progesterone-CXCR4 Axis Controls Mammary Progenitor Cell Fate in the Adult Gland.

Progesterone drives mammary stem and progenitor cell dynamics through paracrine mechanisms that are currently not well understood. Here, we demonstrate that CXCR4, the receptor for stromal-derived factor 1 (SDF-1; CXC12), is a crucial instructor of hormone-induced mammary stem and progenitor cell function. Progesterone elicits specific changes in the transcriptome of basal and luminal mammary epithelial populations, where CXCL12 and CXCR4 represent a putative ligand-receptor pair. In situ, CXCL12 localizes to progesterone-receptor-positive luminal cells, whereas CXCR4 is induced in both basal and luminal compartments in a progesterone-dependent manner. Pharmacological inhibition of CXCR4 signaling abrogates progesterone-directed expansion of basal (CD24+CD49fhi) and luminal (CD24+CD49flo) subsets. This is accompanied by a marked reduction in CD49b+SCA-1- luminal progenitors, their functional capacity, and lobuloalveologenesis. These findings uncover CXCL12 and CXCR4 as novel paracrine effectors of hormone signaling in the adult mammary gland, and present a new avenue for potentially targeting progenitor cell growth and malignant transformation in breast cancer.

Spatial genomic heterogeneity within localized, multifocal prostate cancer.

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.