The RNA-binding protein hnRNPA2 regulates ß-catenin protein expression and is overexpressed in prostate cancer.

INTRODUCTION: The RNA-binding protein hnRNPA2 (HNRNPA2B1) is upregulated in cancer, where it controls alternative pre-mRNA splicing of cancer-relevant genes. Cytoplasmic hnRNPA2 is reported in aggressive cancers, but is functionally uncharacterized. We explored the role of hnRNPA2 in prostate cancer (PCa). METHODS: hnRNPA2 function/localization/expression in PCa was determined using biochemical approaches (colony forming/proliferation/luciferase reporter assays/flow cytometry/immunohistocytochemistry). Binding of hnRNPA2 within cancer-relevant 3'-UTR mRNAs was identified by bioinformatics. RESULTS: RNAi-mediated knockdown of hnRNPA2 reduced colony forming and proliferation, while hnRNPA2 overexpression increased proliferation of PCa cells. Nuclear hnRNPA2 is overexpressed in high-grade clinical PCa, and is also observed in the cytoplasm in some cases. Ectopic expression of a predominantly cytoplasmic variant hnRNPA2-ΔRGG also increased PCa cell proliferation, suggesting that cytoplasmic hnRNPA2 may also be functionally relevant in PCa. Consistent with its known cytoplasmic roles, hnRNPA2 was associated with 3'-UTR mRNAs of several cancer-relevant mRNAs including ß-catenin (CTNNB1). Both wild-type hnRNPA2 and hnRNPA2-ΔRGG act on CTNNB1 3'-UTR mRNA, increasing endogenous CTNNB1 mRNA expression and ß-catenin protein expression and nuclear localization. CONCLUSION: Nuclear and cytoplasmic hnRNPA2 are present in PCa and appear to be functionally important. Cytoplasmic hnRNPA2 may affect the cancer cell phenotype through 3'-UTR mRNA-mediated regulation of ß-catenin expression and other cancer-relevant genes.

Next-generation sequencing of advanced prostate cancer treated with androgen-deprivation therapy.

BACKGROUND: Androgen-deprivation therapy (ADT) is standard treatment for locally advanced or metastatic prostate cancer (PCa). Many patients develop castration resistance (castration-resistant PCa [CRPC]) after approximately 2-3 yr, with a poor prognosis. The molecular mechanisms underlying CRPC progression are unclear. OBJECTIVE: To undertake quantitative tumour transcriptome profiling prior to and following ADT to identify functionally important androgen-regulated pathways or genes that may be reactivated in CRPC. DESIGN, SETTING, AND PARTICIPANTS: RNA sequencing (RNA-seq) was performed on tumour-rich, targeted prostatic biopsies from seven patients with locally advanced or metastatic PCa before and approximately 22 wk after ADT initiation. Differentially regulated genes were identified in treatment pairs and further investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on cell lines and immunohistochemistry on a separate CRPC patient cohort. Functional assays were used to determine the effect of pathway modulation on cell phenotypes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We searched for gene expression changes affecting key cell signalling pathways that may be targeted as proof of principle in a CRPC in vitro cell line model. RESULTS AND LIMITATIONS: We identified ADT-regulated signalling pathways, including the Wnt/ß-catenin signalling pathway, and observed overexpression of ß-catenin in a subset of CRPC by immunohistochemistry. We validated 6 of 12 (50%) pathway members by qRT-PCR on LNCaP/LNCaP-AI cell RNAs, of which 4 (67%) demonstrated expression changes consistent with RNA-seq data. We show that the tankyrase inhibitor XAV939 (which promotes ß-catenin degradation) reduced androgen-independent LNCaP-AI cell line growth compared with androgen-responsive LNCaP cells via an accumulation of cell proportions in the G0/G1 phase and reduction in the S and G2/M phases. Our biopsy protocol did not account for tumour heterogeneity, and pathway inhibition was limited to pharmacologic approaches. CONCLUSIONS: RNA-seq of paired PCa samples revealed ADT-regulated signalling pathways. Proof-of-principle inhibition of the Wnt/ß-catenin signalling pathway specifically delays androgen-independent PCa cell cycle progression and proliferation and warrants further investigation as a potential target for therapy for CRPC.