The miR-361-3p increases enzalutamide (Enz) sensitivity via targeting the ARv7 and MKNK2 to better suppress the Enz-resistant prostate cancer

Abstract

The androgen receptor splicing variant 7 (ARv7) that lacks the ligand-binding domain is increasingly considered as a key player leading to enzalutamide (Enz) resistance in patients with prostate cancer (PCa). However, the detailed mechanisms of how ARv7 expression is regulated and whether it also needs other factors to induce maximal Enz resistance remain unclear. Here, we identified a microRNA, miR-361-3p, whose expression is lower in patients with recurrent PCa, could function via binding to the 3'UTR of ARv7, but not the wild type of AR, to suppress its expression to increase the Enz sensitivity. Importantly, we found that miR-361-3p could also bind to the 3'UTR of MAP kinase-interacting serine/threonine kinase 2 (MKNK2) to suppress its expression to further increase the Enz sensitivity. In turn, the increased Enz can then function via a feedback mechanism through altering the HIF-2α/VEGFA signaling to suppress the expression of miR-361-3p under hypoxia conditions. Preclinical studies using an in vivo mouse model with orthotopically xenografted CWR22Rv1 cells demonstrated that combining the Enz with the small molecule miR-361-3p would result in better suppression of the Enz-resistant PCa tumor progression. Together, these preclinical studies demonstrate that miR-361-3p can function via suppressing the expression of ARv7 and MKNK2 to maximally increase the Enz sensitivity, and targeting these newly identified Enz/miR-361-3p/ARv7 and/or Enz/miR-361-3p/MKNK2 signals with small molecules may help in the development of novel therapies to better suppress the CRPC in patients that already have developed the Enz resistance.

Fig. 1. The miRNA-361-3p suppressed androgen receptor splicing variant 7 (ARv7) expression and prostate cancer (PCa) formation.

a ARv7 has its unique 3′UTR compared to the 3′UTR of fAR. b The selected tumor suppressor miRNA has binding sites to the ARv7 3′UTR, and is differentially expressed in recurrent tumors. c Western blot analysis shows that adding miR-361-3p suppresses ARv7 protein expression in CWR22Rv1 cells. The effect of adding miRNA is shown on the left. d Western blot analysis shows that adding miR-361-3p suppresses ARv7 protein expression in C4-2 Enz-R cells. The effect of adding miRNA is shown on the left. e Expression of miR-361-3p in CWR22Rv1, C4-2 Enz-R cells, and normal prostate RWPE-1 cells. f Expression of miR-361-3p in CWR22Rv1, C4-2 Enz-R cells, and normal prostate RWPE-1 cells after transfecting miR-361. g TCGA dataset analyses indicate that miR-361-3p expression is lower in PCa with more advanced pathological stages (left) and higher Gleason scores (right). h Analyses of 17 PCa samples and their matched para-cancer tissues surgically collected by us indicate that miR-361-3p expression is lower in PCa. Representative results are shown as mean ± SD. *P < 0.05.

Fig. 2. Validation of androgen receptor splicing variant 7 (ARv7) as miR-361-3p target.

a Sequence alignment of miR-361-3p to ARv7 3′UTR. b RIP analysis in CWR22Rv1 cells (22Rv1) reveals recruitment of ARv7 mRNA to microRNA ribonucleoprotein complex following immunoprecipitation against Ago2. IgG immunoprecipitation is used as a negative control. c Synthetic sense or antisense ARv7 3′UTR with biotin is mixed with the total RNA followed by detection of miR-361-3p in the pulldown. d The wild-type and mutated ARv7 3′UTR via deleting the miRNA-binding sites is separately used as control for (e) and (f). e, f Luciferase reporter assay reveals that expressing miR-361-3p suppresses ARv7 3′UTR luciferase activity in 22Rv1 cells (e) and C4-2 cells (f). The assays are repeated three times with each assay being performed in triplicate wells and similar results being obtained each time. Representative results are shown as mean ± SD. *P < 0.05.

Fig. 3. The miR-361-3p alters the Enz sensitivity of the CRPC cell growth through suppressing androgen receptor splicing variant 7 (ARv7).

a Representative dishes and quantification results are shown for clongenic formation assays of CWR22Rv1 (22Rv1) cells. b Expressing miR-361-3p in 22Rv1 cells increases Enz sensitivity. c The miR-361-3p-suppressed Enz-mediated 22Rv1 cell growth is partially reversed after expressing the ARv7, left is western blot, right is MTT assay. d Expressing miR-361-3p decreases the cell growth in C4-2 Enz-R cells. e The miR-361-3p-suppressed Enz-mediated C4-2 Enz-R cell growth is partially reversed after expressing the ARv7, left is western blot, right is MTT assay. The final concentration of Enz in cell media is 10 μM. The values of the Y axis represent the ratios of cell vitality treated with Enz to DMSO (as control) in each group. Representative results are shown as mean ± SD. *P < 0.05, **P < 0.01.

Fig. 4. The miR-361-3p influences Enz sensitivity via targeting the MAP kinase-interacting serine/threonine kinase 2 (MKNK2) 3′UTR and altering MKNK2 expression.

a TCGA database indicates that the higher MKNK2 level in human prostate cancer (PCa) tissues is correlated with lower overall survival rates. b The expression of MKNK2 is negatively correlated with the miR-361-3p level. c Sequence alliance of miR-361-3p to MKNK2 3′UTR. d The structure of luciferase reporter. e RNA immunoprecipitation (RIP) analysis of CWR22Rv1 (22Rv1) cells reveals recruitment of MKNK2 mRNA to microRNA ribonucleoprotein complex following immunoprecipitation against Ago2. IgG immunoprecipitation is used as a negative control. f Luciferase reporter assay reveals that adding miR-361-3p suppresses MKNK2 3′UTR luciferase activity in 22RV1 cells. The mutated MKNK2 3′UTR via deleting the miRNA- binding sites is used as control. g Western blot analysis shows that adding miR-361-3p suppresses MKNK2 and downstream gene p-eIF4G protein expression in CWR22Rv1 cells. h Suppression of androgen receptor splicing variant 7 (ARv7)/MKNK2 expression by shRNA could not change MKNK2 (left) or ARv7 (right) protein expressions. i Cell-proliferation assay shows that adding miR-361-3p increases Enz sensitivity in CWR22Rv1 cells, which can also be partially reversed by adding MKNK2. Representative results are shown as mean ± SD. *P < 0.05.

Fig. 5. Mechanism dissection of how Enz influences miR-361-3p expression.

a Quantitative real-time PCR analysis shows that miR-361-3p expression in C4-2 Enz-R cells is lower than in their parental Enz-sensitive C4-2 cells. b Western blot analysis shows that androgen receptor splicing variant 7 (ARv7) protein expression increased in C4-2 Enz-R cells compared to parental Enz-sensitive C4-2 cells. c, d Quantitative real-time PCR analysis shows that Enz decreases miR-361-3p expression in (c) C4-2 and (d) LNCaP parental cells after 72 h, but not at shorter times (24 or 48 h). e C4-2 cells treated with Enz for 24 h have decreased miR-361-3p expression under hypoxia (left); meanwhile, the expression of hypoxia-responsive genes is as expected (right). f Rescue assays show that decreased miR-361-3p expression in C4-2 cells could be reversed when hypoxia is antagonized by HIF-2α inhibitor TC-S 7009 (left). Expression of hypoxia-responsive genes is indeed reduced after treating with the inhibitor (right). g LNCaP cells treated with Enz for 24 h have decreased miR-361-3p expression under hypoxia (left); meanwhile, the expression of hypoxia-responsive genes is as expected (right). h Rescue assays show that decreased miR-361-3p expression in LNCaP cells could be reversed when hypoxia is antagonized by HIF-2α inhibitor TC-S 7009 (left). Expression of hypoxia-responsive genes is indeed reduced after treating with the inhibitor (right). i Hypoxia-responsive genes are increased in C4-2 Enz-R cells. Representative results are shown as mean ± SD. *P < 0.05.

Fig. 6. The miR-361-3p suppressed Enz-resistant prostate cancer (PCa) tumors in vivo.

CWR22Rv1 cells with or without expression of miR-361-3p were labeled with luciferase and injected orthotopically into nude mice. After 1 week of implantation, each set of mice was randomly assigned into two experimental groups (treated with either DMSO or 35 mg/kg Enz by i.p. injection 3×/per week for 2 weeks). a One week after implantation, tumors were formed and visualized by IVIS image. Representative IVIS images of each group are shown. b, c Tumor sizes (b) and weights (c) in the four groups after sacrifice and tumor removal. ^*P < 0.05. d, e IHC staining shows androgen receptor splicing variant 7 (ARv7) (d) and MAP kinase-interacting serine/threonine kinase 2 (MKNK2) (e) protein expression in four groups. We omitted the primary antibody in the control group. Representative IHC images of each group are shown. Magnification is ×400. Representative results are shown as mean ± SD. *P < 0.05, NS not significant.