Novel tumor suppressor microRNA at frequently deleted chromosomal region 8p21 regulates epidermal growth factor receptor in prostate cancer

Abstract

Genomic loss of chromosome (chr) 8p21 region, containing prostate-specific NKX3.1 gene, is a frequent alteration of the prostate cancer (PCa) oncogenome. We propose a novel, paradigm shifting hypothesis that this frequently deleted locus is also associated with a cluster of microRNA genes- miR-3622a/b- that are lost in PCa and play an important mechanistic role in progression and metastasis. In this study, we demonstrate the role of miR-3622b in prostate cancer. Expression analyses in a cohort of PCa clinical specimens and cell lines show that miR-3622b expression is frequently lost in prostate cancer. Low miR-3622b expression was found to be associated with tumor progression and poor biochemical recurrence-free survival. Further, our analyses suggest that miR-3622b expression is a promising prostate cancer diagnostic biomarker that exhibits 100% specificity and 66% sensitivity. Restoration of miR-3622b expression in PCa cell lines led to reduced cellular viability, proliferation, invasiveness, migration and increased apoptosis. miR-3622b overexpression in vivo induced regression of established prostate tumor xenografts pointing to its therapeutic potential. Further, we found that miR-3622b directly represses Epidermal Growth Factor Receptor (EGFR). In conclusion, our study suggests that miR-3622b plays a tumor suppressive role and is frequently downregulated in prostate cancer, leading to EGFR upregulation. Importantly, miR-3622b has associated diagnostic, prognostic and therapeutic potential. Considering the association of chr8p21 loss with poor prognosis, our findings are highly significant and support a novel concept that associates a long standing observation of frequent loss of a chromosomal region with a novel miRNA in prostate cancer.

Keywords: EGFR; chr8p21; miR-3622b; prostate cancer; tumor suppressor.

Figure 1. MicroRNA-3622b located in frequently deleted chr8p21 region is under expressed in prostate cancer

A. Schematic representation of chr8p21 region highlighting the location of miR-3622a/b cluster. miR-3622b is located at position 27701673-27701767 on the antisense strand. B. CNAs at miR-3622b locus in the TCGA cohort of prostate adenocarcinomas. C. miR-3622b expression levels in PCa specimens relative to matched adjacent normals as assessed by real-time PCR. D. Relative miR-3622b expression levels in immortalized non-malignant cell line (BPH1) and PCa cell lines as assessed by RT-PCR. Data were normalized to RNU48 control. (*P< .05).

Figure 2. Low miR-3622b expression is associated with biochemical recurrence in prostate cancer

A. Correlation of miR-3622b expression with clinicopathological characteristics of prostate cancer patient cohort. P-values based on a Chi square test. B. Kaplan-Meier survival analysis for recurrence-free survival (RFS) of PCa patients, stratified based on miR-3622b levels. P-value based on a log rank test. C. Kaplan-Meier analyses for overall survival of PCa patients, stratified based on miR-3622b levels. P-value based on a log rank test.

Figure 3. Potential diagnostic utility of miR-3622b expression in prostate cancer

A. Correlation of miR-3622b expression with age-adjusted serum PSA levels in PCa patients. (* P< .05). B. ROC curve analysis showing the ability of miR-3622b expression to discriminate between malignant and non-malignant cases. C. Diagnostic utility measures of miR-3622a as a prostate cancer biomarker. (* P< .05).

Figure 4. miR-3622b overexpression suppresses tumorigenicity in vitro in prostate cancer cell lines

To assess the functional significance of miR-3622b, miR-3622b or a control miR (miR-CON) was overexpressed in PCa cell lines (PC3, LNCaP, Du145) by transient transfections followed by functional assays 72 hrs post-transfection (* P< .05). A. Cell viability assays B. Colony formation assays C. Transwell migration and invasion assays in PC3/ LNCaP/ Du145 cells transfected with mock/ miR-CON/ miR-3622b.

Figure 5. miR-3622b overexpression induces apoptosis in prostate cancer cell lines

Flow cytometric analysis of Annexin-V-FITC-7-AAD stained A. LNCaP, B. Du145 and C. PC3 transfected with miR-CON/ miR-3622b.

Figure 6. Intratumoral delivery of miR-3622b leads to tumor regression in PCa xenografts

PC3 cells were subcutaneously injected into nude mice and maintained until solid, palpable tumors (Day 30), following which control miRNA/ miR-3622b mimics were injected intratumorally at periodic intervals (indicated) and tumor growth was monitored. (* P< .05). A. Tumor volumes following miR-CON/miR-3622b administration at the indicated time points. B. Representative images of mice from the two groups on day 54 are shown. C. Relative miR-3622b expression in PCa xenografts as assessed by real-time PCR.

Figure 7. miR-3622b targets Epidermal Growth Factor Receptor in prostate cancer

A. Immunoblots of endogenous EGFR and BMI1 in PC3 (left panels), LNCaP (middle panels) and Du145 cells (right panels) transfected with mock/miR-CON/miR-3622b. GAPDH was used a loading control. B. Schematic representation of the EGFR 3'-UTR showing the putative miR-3622b binding sites. Respective mutant EGFR sites are represented below. C. Luciferase reporter assays with the indicated wt and mutant EGFR 3' UTR constructs or control luciferase construct co-transfected with miR-CON/ miR-3622b in PC3 cells. Firefly luciferase values were normalized to Renilla luciferase activity and plotted as relative luciferase activity (* P< .05). D. LNCaP cells were transfected with three sets of siRNAs (siRNA-A, siRNA-B, siRNA-C) against EGFR or a nonspecific (NS) control siRNA/mock transfected for 72 h (*P< .05) followed by functional assays. Left panel: Real time PCR analyses of relative EGFR expression. Data were normalized to GAPDH control. siRNA-A and siRNA-B were effective in knocking down EGFR expression by ~70% as compared to NS and were used in subsequent experiments. Right panel: Immunoblot analyses for EGFR protein expression after NS/EGFR siRNA transfections. GAPDH was used as a loading control. E. Cellular viability assay, F. Transwell migration and invasion assay after NS/EGFR siRNA- A and siRNA-B transfections. G. Apoptosis assay upon NS siRNA (left panel) or EGFR siRNA-A (middle panel) or siRNA-B (right panel) transfections.