miR-32 promotes MYC-driven prostate cancer

Abstract

miR-32 is an androgen receptor (AR)-regulated microRNA, expression of which is increased in castration-resistant prostate cancer (PC). We have previously shown that overexpression of miR-32 in the prostate of transgenic mice potentiates proliferation in prostate epithelium. Here, we set out to determine whether increased expression of miR-32 influences growth or phenotype in prostate adenocarcinoma in vivo. We studied transgenic mice expressing MYC oncogene (hiMYC mice) to induce tumorigenesis in the mouse prostate and discovered that transgenic overexpression of miR-32 resulted in increased tumor burden as well as a more aggressive tumor phenotype in this model. Elevated expression of miR-32 increased proliferation as assessed by Ki-67 immunohistochemistry, increased nuclear density, and higher mitotic index in the tumors. By gene expression analysis of the tumorous prostate tissue, we confirmed earlier findings that miR-32 expression regulates prostate secretome by modulating expression levels of several PC-related target genes such as Spink1, Spink5, and Msmb. Further, we identified Pdk4 as a tumor-associated miR-32 target in the mouse prostate. Expression analysis of PDK4 in human PC reveals an inverse correlation with miR-32 expression and Gleason score, a decrease in castration-resistant and metastatic tumors compared to untreated primary PC, and an association of low PDK4 expression with a shorter recurrence-free survival of patients. Although decreased PDK4 expression induces the higher metabolic activity of PC cells, induced expression of PDK4 reduces both mitotic respiration and glycolysis rates as well as inhibits cell growth. In conclusion, we show that miR-32 promotes MYC-induced prostate adenocarcinoma and identifies PDK4 as a PC-relevant metabolic target of miR-32-3p.

Fig. 1. Transgenic miR-32 promotes tumor development in hiMYC model of prostate adenocarcinoma.

A Histology of prostate shows intraepithelial neoplasia at 1 month of age in both hiMYC and miR-32xhiMYC mice. Examples from HE-stained lateral lobe. B Prostate size at 6 months of age in wt, miR-32 transgenic, hiMYC, and miR-32xhiMYC mice. C Tumor burden quantified as a sum of tumor areas on whole slide images of tissue sections taken every 50 µm apart throughout the organ in prostates of hiMYC and miR-32xhiMYC mice at 6 months of age. D Example histology of tumors in 6-month-old hiMYC and miR-32xhiMYC mouse prostates. E Tumor nuclear density in most advanced tumors in 6-month-old hiMYC and miR-32xhiMYC mouse prostates. p values *<0.05, **<0.01.

Fig. 2. Proliferation is induced by transgenic miR-32 in prostate epithelium and tumors in hiMYC mice.

A Percentage of prostate epithelial nuclei positive for proliferation marker Ki-67 immunostaining in ventral, lateral, and dorsal lobes of wt, miR-32 transgenic, hiMYC, and miR-32xhiMYC mice at 1 month of age. B Percentage of nuclei positive for Ki-67 immunostaining in hiMYC and miR-32xhiMYC mice tumors at 6 months of age. – no staining, + weak to intermediate staining, ++ strong staining. C Example images of Ki-67 immunostaining in hiMYC and miR-32xhiMYC mouse prostate epithelium at 1 month and tumors at 6 months of age. D Percentage of prostate epithelial cells positive for mitotic marker pH3 immunostaining in hiMYC and miR-32xhiMYC mice at 1 month of age. E Percentage of cells positive for pH3 immunostaining in hiMYC and miR-32xhiMYC mice tumors at 6 months of age. p values *<0.05, **<0.01.

Fig. 3. Effects of transgenic miR-32 on gene expression in hiMYC-induced prostate cancer in mice.

Gene expression analysis was performed on miR-32xhiMYC compared to hiMYC mouse tumorous prostates at 6 months of age. A Protein class distribution, B biological processes, and C cellular component of genes differentially expressed in miR-32xhiMYC compared to hiMYC mice. D Venn diagram showing common genes between genes downregulated in mouse prostate by transgenic miR-32 expression from a previous study [29] and genes differentially expressed in tumorous mouse prostates of miR-32xhiMYC compared to hiMYC mice in this study. The identified commonly regulated genes are shown, as well as genes that are predicted or verified targets for miR-32 among the differentially expressed genes in miR-32xhiMYC compared to hiMYC mice in tumorous prostates. E RT-qPCR validation of genes downregulated by transgenic miR-32 expression. F RT-qPCR validation of genes upregulated by transgenic miR-32 expression.

Fig. 4. PDK4 is a prostate cancer-relevant target of miR-32.

A Correlation analysis of PDK4 expression defined by RNA-seq [31] and miR-32 expression defined by RT-qPCR in 15 human primary PC samples show an inverse correlation between the expression of these genes. B RNA expression analysis in prostate tissue samples of BPH, primary cancer (PC), and CRPC in the Tampere patient cohort showed decreased PDK4 expression in CRPC compared to PC. C RNA expression analysis in prostate tissue samples of normal, primary cancer, and metastases including both non-castrate and castration-resistant samples in the Taylor et al. [46] patient cohort showing decreased PDK4 expression in metastatic compared to PC samples. D RNA expression analysis in prostate tissue samples of primary PC samples in the Taylor et al. [46] patient cohort showing relatively decreased PDK4 expression in samples with higher Gleason grades. E Survival proportions of patients with primary PC in the Taylor et al. [46] data set between tumors of high and low PDK4 expression show decreased recurrence-free survival for patients with low PDK4-expressing tumors. F Luciferase assay in HeLa cells transfected with control (no-UTR, scramble-3′-UTR) and PDK4-3′UTR luciferase constructs and the indicated pre-miRNAs showing targeting of PDK4-3′-UTR construct targeting by pre-miR-32-3p. G Downregulation of PDK4 expression increases the relative metabolic activity as defined by Alamar Blue assay of 22Rv1 PC cells. H Immunofluorescence analysis of PC-3 cells transiently transfected with PDK4 (PDK4 oe) compared with cells transfected with the control plasmid (ctrl). Staining with α-PDK4 antibody shows no expression of endogenous PDK4 protein in PC-3 cells and positive PDK4 signal at 3 days after transfection. DAPI nuclear staining is shown for reference. Scale bar, 50 µm. I Oxygen consumption rate in PC-3 cells transfected with control (ctrl) or PDK4-expressing plasmids showing decreased basal and maximum mitochondrial respiration rate in PDK4 overexpressing cells. J Extracellular acidification rate in PC-3 cells transfected with control (ctrl) or PDK4-expressing plasmids showing decreased basal and maximum glycolytic rates. K PC-3 cells transiently transfected with PDK4 (PDK4 oe) show decreased growth compared to cells transfected with the control plasmid (ctrl). Error bars, standard deviation (F, G), SEM (I–K). p values *<0.05, **<0.01, ***<0.001.