The microRNA-23b/-27b cluster suppresses prostate cancer metastasis via Huntingtin-interacting protein 1-related

Abstract

Deregulation of microRNAs (miRs) contributes to progression and metastasis of prostate and other cancers. miR-23b and -27b, encoded in the same miR cluster (miR-23b/-27b), are downregulated in human metastatic prostate cancer compared with primary tumors and benign tissue. Expression of miR-23b/-27b decreases prostate cancer cell migration, invasion and results in anoikis resistance. Conversely, antagomiR-mediated miR-23b and -27b silencing produces the opposite result in a more indolent prostate cancer cell line. However, neither miR-23b/-27b expression or inhibition impacts prostate cancer cell proliferation suggesting that miR-23b/-27b selectively suppresses metastasis. To examine the effects of miR-23b/-27b on prostate cancer metastasis in vivo, orthotopic prostate xenografts were established using aggressive prostate cancer cells transduced with miR-23b/-27b or non-targeting control miRNA. Although primary tumor formation was similar between miR-23b/-27b-transduced cells and controls, miR-23b/-27b expression in prostate cancer cells decreased seminal vesicle invasion and distant metastases. Gene-expression profiling identified the endocytic adaptor, Huntingtin-interacting protein 1-related (HIP1R) as being downregulated by miR-23b/-27b. Increased HIP1R expression in prostate cancer cells inversely phenocopied the effects of miR-23b/-27b overexpression on migration, invasion and anchorage-independent growth. HIP1R rescued miR-23b/-27b-mediated repression of migration in prostate cancer cells. HIP1R mRNA levels were decreased in seminal vesicle tissue from mice bearing miR-23b/-27b-transduced prostate cancer cell xenografts compared with scrambled controls, suggesting HIP1R is a key functional target of miR-23b/-27b. In addition, depletion of HIP1R led to a more rounded, less mesenchymal-like cell morphology, consistent with decreased metastatic properties. Together, these data demonstrate that the miR-23b/-27b cluster functions as a metastasis-suppressor by decreasing HIP1R levels in pre-clinical models of prostate cancer.

Figure 1

MiR-23b/-27b decreases local invasion and distal metastasis in an in vivo preclinical model of prostate cancer. (a) PC3-ML Luc cells transduced with miR-23b/-27b or scrambled control were tested in Matrigel invasion assays as described in Materials and methods section. (b) PC3-ML Luc cells expressing miR-23b/-27b or scrambled control were injected into the prostate ventral lobes of nude mice. IVIS imaging was performed at day 40; shown are the representative images of mice from both groups (scrambled n = 15, miR-23b/-27b n = 14). Post mortem ex vivo imaging was performed on isolated prostate (c) and seminal vesicles (d). (e) Distal metastasis was defined as all bioluminescence signal remaining post mortem after removal of prostates and seminal vesicles. (f) Individual metastases were identified as distinct photon regions of interest (ROIs; ± s.e.m.) using Living Image software on IVIS images of mice after removal of prostates and seminal vesicles. (g) Representative images of hematoxylin and eosin immunostaining of formalin-fixed tissues. *P<0.05, **P<0.01, ***P<0.001, NS, not significant.

Figure 2

HIP1R is downregulated by miR-23b/-27b in prostate cancer cells. PC3-ML and ALVA31 cells were transduced with miR-23b/-27b or scrambled control, and LNCaP cells were transfected with antagomiRs to both miR-23b and -27b or control antagomiRs. (a) RNA was isolated and reverse transcriptase quantitative PCR (RT-qPCR) was performed. Values are of HIP1R mRNA levels normalized to β-actin mRNA control. (b) Protein lysates were collected, and western blot analysis was performed for HIP1R and actin. Blot shown is representative of three independent experiments. (c) PC3-ML and ALVA31 cells, transduced as described above, and LNCaP cells were additionally transfected with luciferase reporter constructs containing either the wild-type 3′-UTR of HIP1R or an empty vector (EV) control. Forty-eight hours following transfection, luciferase assays were performed on cell lysates. Mean ratios of relative luciferase units (± s.e.m.) of three independent experiments performed in triplicate are shown. Relative luciferase units were obtained by normalization to Renilla luciferase. ***P<0.001.

Figure 3

HIP1R mRNA is significantly upregulated in human metastatic prostate cancer compared with primary site disease in multiple data sets. Prostate cancer mRNA analyses from Oncomine were surveyed for HIP1R in primary prostate cancer as compared with sites of metastasis. HIP1R levels, graphed as log2 median-centered ratio, are listed for three separate significant studies: Grasso Prostate Statistics, primary site (59 samples), metastasis (35 samples). Chandran Prostate Statistics, primary site (10 samples), metastasis (21 samples). Yu Prostate Statistics, primary site (64 samples), metastasis (24 samples). **P<0.01, ***P<0.001.

Figure 4

HIP1R increases invasion and migration, but not proliferation of the relatively indolent prostate cancer cell line LNCaP. (a) Boyden Chamber (b) Matrigel Invasion and (c) proliferation assays were performed as described on LNCaP cells transfected with pIRES-HIP1R or pIRES-empty vector (EV). Representative images are shown for Boyden chamber assays. For all, the mean cell number (± s.d.) of a representative experiment (n = 3) performed in triplicate is shown. *P<0.05, **P<0.01.

Figure 5

Depletion of HIP1R decreases invasion, migration, and anchorage-independent growth of aggressive prostate cancer cells. PC3-ML and ALVA-31 cells were stably transduced with either a short-hairpin against HIP1R (shHIP1R) or GFP as a control (shGFP). (a) Cellular lysates were collected and HIP1R and actin were detected by western blotting. (b) Boyden Chamber, (c) Matrigel Invasion and (d) proliferation assays were also performed for these cells. The mean cell number (± s.d.) of a representative experiment (n = 3) performed in triplicate is shown for b–d. (e) Anchorage-independent growth was analyzed for cells depleted of HIP1R using a soft agar assay. Results of three experiments were averaged, and graphed relative to shGFP control (±s.e.m.). *P<0.05, ***P<0.001.

Figure 6

HIP1R rescues miR-23b/-27b-mediated suppression of migration in aggressive prostate cancer cells. (a) PC3-ML and (b) ALVA31 cells transduced with either miR-23b/-27b or scrambled control were transfected with pIRES-HIP1R or a pIRES-empty vector (EV) control. Forty-eight hours following transfection, cells were serum-starved overnight then seeded in Boyden Chambers for migration assays. The mean cell number (± s.d.) of a representative experiment (n = 3) performed in triplicate is shown. *P<0.05, **P<0.01, NS, not significant.

Figure 7

HIP1R knockdown in aggressive prostate cancer cells results in decreased cell elongation. (a) PC3-ML and (b) ALVA31 cells transduced with shHIP1R or shGFP were grown for 5 days in supplemented media, then stained for Phalloidin immunofluorescence. Cell shape was measured by determining long axis versus short axis and graphed (± s.e.m.), n≥30 cells per condition. Mann–Whitney's test was used to analyze cell axes ratios. **P<0.01, ***P<0.001.

Figure 8

Decreased HIP1R mRNA is linked to miR-23b/-27b-mediated anti-invasive actions in vivo. Seminal vesicle tissue from mice bearing PC3-ML Luc orthotopic prostate xenografts expressing miR-23b/-27b or a scrambled control were homogenized in Trizol and assayed for mRNA. (a) RT-qPCR analysis was performed using Taqman probes to miR-23b and -27b. Samples were normalized to SNU6, and controlled to vehicle tissue. (b) HIP1R human-specific Taqman probes were used for RT-qPCR analysis, and normalized to GAPDH mRNA. Mann–Whitney non-parametric test was used to determine significance. *P<0.05, **P<0.01, ***P<0.001.