The microRNA -23b/-27b cluster suppresses the metastatic phenotype of castration-resistant prostate cancer cells.

MicroRNAs (miRs) are small, endogenous, non-coding RNAs that regulate the stability and/or translation of complementary mRNA targets. MiRs have emerged not only as critical modulators of normal physiologic processes, but their deregulation may significantly impact prostate and other cancers. The expression of miR-23b and miR-27b, which are encoded by the same miR cluster (miR-23b/-27b), are downregulated in metastatic, castration-resistant tumors compared to primary prostate cancer and benign tissue; however, their possible role in prostate cancer progression is unknown. We found that ectopic expression of miR-23b/-27b in two independent castration-resistant prostate cancer cell lines resulted in suppression of invasion and migration, as well as reduced survival in soft agar (a measure of anoikis). However, there was no effect of miR-23b/-27b on cell proliferation suggesting that these miRs function as metastasis (but not growth) suppressors in prostate cancer. Conversely, inhibition of miR-23b/-27b in the less aggressive androgen-dependent LNCaP prostate cancer cell line resulted in enhanced invasion and migration also without affecting proliferation. Mechanistically, we found that introduction of miR-23b/-27b in metastatic, castration-resistant prostate cancer cell lines resulted in a significant attenuation of Rac1 activity without affecting total Rac1 levels and caused increased levels of the tumor suppressor E-cadherin. Inhibition of these miRs had the opposite effect in androgen-dependent LNCaP cells. These results suggest that miR-23b/-27b are metastasis suppressors that might serve as novel biomarkers and therapeutic agents for castration-resistant disease

Abstract 1467: The miR-23b/-27b cluster decreases metastasis of aggressive prostate cancer

Abstract Metastasis is responsible for the vast majority of prostate cancer deaths. As such, it is essential to understand the mechanisms driving prostate cancer to this lethal stage. Deregulation of microRNAs is increasingly implicated in the progression and metastasis of prostate and other cancers. MiR-23b and miR-27b, two members of the same miR cluster (miR-23b/-27b), are down-regulated in human metastatic, castration resistant prostate cancer (CRPC) as compared to primary tumors and benign tissue. However, the role of the miR-23b/-27b cluster is not fully understood, particularly in metastatic disease. Interpretation of existing data is complicated by the analysis of the individual effects of miR-23b and miR-27b even though these miRs are co-transcribed and coordinately expressed. The primary goal of our study is to determine the effects of miR-23b/-27b on metastatic processes in vitro and in vivo. Ectopic expression of miR-23b/-27b in two aggressive prostate cancer cell lines decreases migration, invasion and anchorage-independent growth. Conversely, inhibition of miR-23b/-27b using specific antagomirs in relatively indolent prostate cancer cells promotes increased invasion and migration. E-cadherin protein and mRNA levels were inversely related to miR-23b/-27b levels. In contrast, manipulation of miR-23b/-27b levels had no effect on prostate cancer cell proliferation in any of the tested cell lines. These findings suggest that miR-23b/-27b is specifically linked to metastasis suppression. The Rho GTPase, Rac1, is hyperactive in CRPC cell lines and patient samples. Since Rac1 promotes invasion and migration, we investigated the effects of miR-23b/-27b expression and inhibition on Rac1 activity. Ectopic expression of miR-23b/-27b in aggressive prostate cancer cell lines significantly attenuates active Rac1 while having no effect on total Rac1 levels. Conversely, inhibition of miR-23b/-27b in less aggressive prostate cancer cells increased Rac1 activity but not Rac1 levels. We further examined the effects of miR-23b/-27b on prostate cancer metastasis in vivo. Metastatic prostate cancer cells expressing luciferase and miR-23b/-27b or a scrambled control, were injected into the ventral prostates of nude mice. Orthotopic tumor formation and metastases were assessed by bioluminescence imaging. Metastatic tumor burden was greatly decreased in the tumors derived from miR-23b/-27b expressing cells. Taken together, these data demonstrate that expression of miR-23b/-27b exerts metastasis-suppressing effects in vitro and in vivo. The miR-23b/-27b cluster may be a useful biomarker of poor prognosis in addition to having therapeutic potential in advanced, metastatic prostate cancer. Citation Format: Meghan A. Rice, Reema Ishteiwy, Thirupandiyur Udayakumar, Derek Dykxhoorn, Kerry L. Burnstein. The miR-23b/-27b cluster decreases metastasis of aggressive prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1467. doi:10.1158/1538-7445.AM2014-1467</jats:p

MiR-23b/

<p>-<b>27b significantly increases E-cadherin expression in castration –resistant cell lines.</b> ALVA31 or PC3-ML cells expressing miR-23b/-27b or transduced with scrambled control and LNCaP cells transfected with antagomiR-23b/-27b or control antagomiR were subjected to western blotting for E-cadherin and actin. Representative blots are shown of a total of 2–6 experiments.</p

MiR-23b/

<p>-<b>27b significantly decreases Rac1 activity but not total Rac1 levels in castration-resistant prostate cancer cell lines.</b> Rac1 activity assays were performed on ALVA31 (A) and PC3-ML (B) cells expressing miR-23b/-27b or transduced with scrambled control. GTP-bound Rac1 was separated from GDP-Rac1 using a pull-down assay as described in Materials and Methods. Complexes containing GTP-Rac1 (active Rac1) were collected, denatured and resolved by SDS-PAGE followed by Western blotting with anti-Rac1 antibodies. Total Rac1 (GDP-and GTP-bound) represents 5% of the original cell lysate. Actin was used as a loading control. Quantification of three independent experiments is shown with error bars representing SEM (*P<0.05), (**P<0.01).</p

MiR-23b/

<p>-<b>27b expression decreases the invasiveness of castration-resistant prostate cancer cells while inhibition of miR-23b/</b>-<b>27b in androgen-dependent prostate cancer cells increases invasiveness.</b> A, Matrigel invasion assays were performed on ALVA31 cells (A) and PC3-ML cells (B) expressing miR-23b/-27b or scrambled control-transduced cells. Upper panels show representative regions of the chamber filters with crystal violet-stained cells. The fold change (±SEM) represents the number of invaded cells per chamber divided by controls from three independent experiments performed in triplicate for A and the means (±SD) of one independent experiment done in triplicate for B (***P<0.001). C, LNCaP cells were transfected with 50 nM control antagomiR or antagomiR-23b/-27b. Matrigel invasion assays were performed 72 hours post transfection as explained above. The means (±SD) of two independent experiments performed in triplicate are shown (***P<0.001).</p

MiR-23b/

<p>-<b>27b does not regulate prostate cancer cell proliferation.</b> A and B, Proliferation of cells expressing miR-23b/-27b was compared to that of scrambled control-transduced cells. The mean cell number (±SEM) of a total of three independent experiments performed in triplicate is shown for ALVA31 cells and the mean cell number (±SD) of a representative experiment performed in triplicate is shown for PC3-ML cells. C, Proliferation of LNCaP cells transfected with antagomiR-23b/-27b or control antagomiR was assessed. The mean cell number (±SD) from two independent experiments performed in triplicate is shown.</p

MiR-23b/

<p>-<b>27b decreases anchorage independent growth of castration-resistant prostate cancer cell lines.</b> Soft agar assays were performed on ALVA31 and PC3-ML cells expressing miR-23b/-27b or transduced with scrambled control (**P<0.01). Mean colony number (±SEM) per plate from two independent experiments performed in triplicate for each cell line is shown (**P<0.01).</p

MiR-23b/

<p>-<b>27b expression decreases castration-resistant prostate cancer cell migration while inhibition of miR-23b/</b>-<b>27b increases migration of androgen-dependent prostate cancer cells.</b> Scratch assays were performed on ALVA31 cells expressing miR-23b/-27b or scrambled control (A) and LNCaP cells transfected with 50 nM antagomiR-23b-27b or control antagomiR (B). Images of the cleared zones (representative images shown on the right) were taken before and after a 4 hr incubation and the cleared areas measured using Image J software. The mean percentage of gap closure (±SD) (due to cell migration) is shown for two independent experiments from 7 scratches (A) and 6 scratches (B) (***P<0.001).</p