Housheng Hansen He

ERG induces androgen receptor-mediated regulation of SOX9 in prostate cancer

Fusion of the androgen receptor-regulated (AR-regulated) TMPRSS2 gene with ERG in prostate cancer (PCa) causes androgen-stimulated overexpression of ERG, an ETS transcription factor, but critical downstream effectors of ERG-mediating PCa development remain to be established. Expression of the SOX9 transcription factor correlated with TMPRSS2:ERG fusion in 3 independent PCa cohorts, and ERG-dependent expression of SOX9 was confirmed by RNAi in the fusion-positive VCaP cell line. SOX9 has been shown to mediate ductal morphogenesis in fetal prostate and maintain stem/progenitor cell pools in multiple adult tissues, and has also been linked to PCa and other cancers. SOX9 overexpression resulted in neoplasia in murine prostate and stimulated tumor invasion, similarly to ERG. Moreover, SOX9 depletion in VCaP cells markedly impaired invasion and growth in vitro and in vivo, establishing SOX9 as a critical downstream effector of ERG. Finally, we found that ERG regulated SOX9 indirectly by opening a cryptic AR-regulated enhancer in the SOX9 gene. Together, these results demonstrate that ERG redirects AR to a set of genes including SOX9 that are not normally androgen stimulated, and identify SOX9 as a critical downstream effector of ERG in TMPRSS2:ERG fusion-positive PCa.

MiR-221 promotes the development of androgen independence in prostate cancer cells via downregulation of HECTD2 and RAB1A

Hormone-sensitive prostate cancer typically progresses to castration resistant prostate cancer (CRPC) after the androgen deprivation therapy. We investigated the impact of microRNAs (miRs) in the transition of prostate cancer to CRPC. MiR-221/-222 was highly expressed in bone metastatic CRPC tumor specimens. We previously demonstrated that transient overexpression of miR-221/-222 in LNCaP promoted the development of the CRPC phenotype. In current study, we show that stably overexpressing miR-221 confers androgen independent (AI) cell growth in LNCaP by rescuing LNCaP cells from growth arrest at G1 phase due to the lack of androgen. Overexpressing of miR-221 in LNCaP reduced the transcription of a subgroup of androgen-responsive genes without affecting the androgen receptor (AR) or AR-androgen integrity. By performing systematic biochemical and bioinformatical analyses, we identified two miR-221 targets, HECTD2 and RAB1A, which could mediate the development of CRPC phenotype in multiple prostate cancer cell lines. Downregulation of HECTD2 significantly affected the androgen-induced and AR-mediated transcription, and downregulation of HECTD2 or RAB1A enhances AI cell growth. As a result of the elevated expression of miR-221, expression of many cell cycle genes was altered and pathways promoting epithelial to mesenchymal transition/tumor metastasis were activated. We hypothesize that a major biological consequence of upregulation of miR-221 is reprogramming of AR signaling, which in turn may mediate the transition to the CRPC phenotype.

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.

Modulation of long noncoding RNAs by risk SNPs underlying genetic predispositions to prostate cancer

Long noncoding RNAs (lncRNAs) represent an attractive class of candidates to mediate cancer risk. Through integrative analysis of the lncRNA transcriptome with genomic data and SNP data from prostate cancer genome-wide association studies (GWAS), we identified 45 candidate lncRNAs associated with risk to prostate cancer. We further evaluated the mechanism underlying the top hit, PCAT1, and found that a risk-associated variant at rs7463708 increases binding of ONECUT2, a novel androgen receptor (AR)-interacting transcription factor, at a distal enhancer that loops to the PCAT1 promoter, resulting in upregulation of PCAT1 upon prolonged androgen treatment. In addition, PCAT1 interacts with AR and LSD1 and is required for their recruitment to the enhancers of GNMT and DHCR24, two androgen late-response genes implicated in prostate cancer development and progression. PCAT1 promotes prostate cancer cell proliferation and tumor growth in vitro and in vivo. These findings suggest that modulating lncRNA expression is an important mechanism for risk-associated SNPs in promoting prostate transformation.

Transcriptional landscape of the human cell cycle

Significance Our study provided a comprehensive view of the transcriptional landscape across the cell cycle. We revealed lag between transcription and steady-state RNA expression at the cell-cycle level and characterized a large amount of active transcription during early mitosis. In addition, our analysis identified thousands of enhancer RNAs and related transcription factors that are highly correlated with cell-cycle–regulated transcription but not with steady-state expression, thus highlighting the importance of transcriptional and epigenetic dynamics during cell-cycle progression. Steady-state gene expression across the cell cycle has been studied extensively. However, transcriptional gene regulation and the dynamics of histone modification at different cell-cycle stages are largely unknown. By applying a combination of global nuclear run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and histone-modification Chip sequencing (ChIP-seq), we depicted a comprehensive transcriptional landscape at the G0/G1, G1/S, and M phases of breast cancer MCF-7 cells. Importantly, GRO-seq and RNA-seq analysis identified different cell-cycle–regulated genes, suggesting a lag between transcription and steady-state expression during the cell cycle. Interestingly, we identified genes actively transcribed at early M phase that are longer in length and have low expression and are accompanied by a global increase in active histone 3 lysine 4 methylation (H3K4me2) and histone 3 lysine 27 acetylation (H3K27ac) modifications. In addition, we identified 2,440 cell-cycle–regulated enhancer RNAs (eRNAs) that are strongly associated with differential active transcription but not with stable expression levels across the cell cycle. Motif analysis of dynamic eRNAs predicted Kruppel-like factor 4 (KLF4) as a key regulator of G1/S transition, and this identification was validated experimentally. Taken together, our combined analysis characterized the transcriptional and histone-modification profile of the human cell cycle and identified dynamic transcriptional signatures across the cell cycle.

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Significance Alternative RNA splicing and the spliceosome machinery have been implicated in cancer progression. A genome-wide CRISPR screen identified the RNA processing factor heterogeneous nuclear ribonucleoprotein L (HNRNPL) as required for prostate cancer growth by regulating alternative RNA splicing and circular RNA formation. HNRNPL and its RNA clients are overexpressed during prostate cancer progression, supporting their potential role as therapeutic targets. Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP–RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.

LSD1-Mediated Epigenetic Reprogramming Drives CENPE Expression and Prostate Cancer Progression

Androgen receptor (AR) signaling is a key driver of prostate cancer, and androgen-deprivation therapy (ADT) is a standard treatment for patients with advanced and metastatic disease. However, patients receiving ADT eventually develop incurable castration-resistant prostate cancer (CRPC). Here, we report that the chromatin modifier LSD1, an important regulator of AR transcriptional activity, undergoes epigenetic reprogramming in CRPC. LSD1 reprogramming in this setting activated a subset of cell-cycle genes, including CENPE, a centromere binding protein and mitotic kinesin. CENPE was regulated by the co-binding of LSD1 and AR to its promoter, which was associated with loss of RB1 in CRPC. Notably, genetic deletion or pharmacological inhibition of CENPE significantly decreases tumor growth. Our findings show how LSD1-mediated epigenetic reprogramming drives CRPC, and they offer a mechanistic rationale for its therapeutic targeting in this disease. Cancer Res; 77(20); 5479-90. ©2017 AACR.

Variant Set Enrichment: an R package to identify disease-associated functional genomic regions

BackgroundGenetic predispositions to diseases populate the noncoding regions of the human genome. Delineating their functional basis can inform on the mechanisms contributing to disease development. However, this remains a challenge due to the poor characterization of the noncoding genome. Here, we propose an R package that can pinpoint which genomic features are etiologically important based on the genetic predispositions.ResultsVariant Set Enrichment (VSE) is an R package to calculate the enrichment of a set of disease-associated variants across functionally annotated genomic regions, consequently highlighting the mechanisms important in the etiology of the disease studied.ConclusionsVSE is implemented as an R package and can easily be implemented in any system with R.

Reactivation of androgen receptor-regulated lipid biosynthesis drives the progression of castration-resistant prostate cancer

Androgen receptor (AR) is a transcriptional activator that, in prostate cells, stimulates gene expression required for various cellular functions, including metabolisms and proliferation. AR signaling is also essential for the development of hormone-dependent prostate cancer (PCa) and its activity can be blocked by androgen-deprivation therapies (ADTs). Although PCa patients initially respond well to ADTs, the cancer inevitably relapses and progresses to lethal castration-resistant prostate cancer (CRPC). Although AR activity is generally restored in CRPC despite the castrate level of androgens, it is unclear whether AR signaling is significantly reprogrammed. In this study, we examined the AR cistrome in a PCa cell line-derived CRPC model using integrated bioinformatical analyses. Significantly, we found that the AR cistrome is largely retained in the CRPC stage. In particular, AR-mediated lipid biosynthesis is highly conserved and reactivated during the progression to CRPC, and increased level of lipid synthesis is associated with poor prognosis. The restoration of lipid biosynthetic pathways is partially due to the increased expression of AR splice variants. Blocking lipid/cholesterol synthesis in AR variants-expressing CRPC cell line and xenograft models markedly reduces tumor growth through inhibition of mTOR pathway. Silencing the expression of a fatty acid elongase, ELOVL7, also leads to the regression of CRPC xenograft tumors. These results demonstrate the importance of reactivation of AR-regulated lipid biosynthetic pathways in driving CRPC progression, and suggest that ADTs may be therapeutically enhanced by blocking lipid biosynthetic pathways.

Heterochromatin protein 1α mediates development and aggressiveness of neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management.Significance: Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(10); 2691-704. ©2018 AACR.