miR-200b Inhibits Prostate Cancer EMT, Growth and Metastasis

<div><p>miRNA regulate gene expression at post-transcriptional level and fine-tune the key biological processes, including cancer progression. Here, we demonstrate the involvement of miR-200b in the metastatic spread of prostate cancer. We identified miR-200b as a downstream target of androgen receptor and linked its expression to decreased tumorigenicity and metastatic capacity of the prostate cancer cells. Overexpression of miR-200b in PC-3 cells significantly inhibited their proliferation and the formation of subcutaneous tumors. Moreover, in an orthotopic model, miR-200b blocked spontaneous metastasis and angiogenesis by PC-3 cells. This decreased metastatic potential was likely due to the reversal of the epithelial-to-mesenchymal transition, as was evidenced by increased pan-epithelial marker E-cadherin and specific markers of prostate epithelium, cytokeratins 8 and 18. In contrast, mesenchymal markers, fibronectin and vimentin, were significantly downregulated by miR-200b. Our results suggest an important role for miR-200b in prostate cancer progression and indicate its potential utility for prostate cancer therapy.</p></div

AR activation alters the miRNA profile of PC3-AR cells.

<p>(A) Western blot to confirm inducible AR expression in PC3-AR cells. PC3-AR cells were treated 5 days with doxycycline to induce AR expression and with R1881 to induce AR activation and nuclear translocation. The comparison is to untreated control. Total cell lysates were used for analysis. (B). Heat map of miRNA expression in PC3-AR and control cells. Total RNA from cells in A was used for microarray analysis and each sample analyzed in triplicate. The statistical significance for expression changes shown has been determined using Student’s T-test. P values <0.05 were attributed statistical significance.</p

miRNA increased with AR expression and inhibitory effect in cancers with increased expression (p<0.05).

<p>miRNA increased with AR expression and inhibitory effect in cancers with increased expression (p<0.05).</p

miR-200b promotes differentiation of PC-3 cells.

<p>(A). CK 8 and CK 18 were analyzed by western blot of total protein lysates from PC3-ctrl and PC3 miR-200b cells. (B) Quantitative analysis of the experiment (A) with Image J software. The graph represents data average of two independent experiments. *, p≤0.05 and **, p<0.01 by Student’s T-test.</p

miR-200b suppresses angiogenesis in PCa.

<p>Sections from the tumors formed by control and miR-200b positive PC-3 tumors were stained for the endothelial marker CD31 and the number of microvessels per field determined in ten 10× fields. *, P<0.01.</p

Validation of select miRNA.

<p>(A). MiRNA expression was normalized to that of control cells (ctrl). PC3-AR and control cells were treated 5 days with doxycycline to induce AR expression and with R1881 to induce AR activation and nuclear translocation and total RNA collected for analysis. The comparison is to untreated control and RNU1A_1 non-coding RNA is used as an internal control. The statistical significance of observed differences compared to control is * p≤0.05, and **p≤0.01 as was determined by one-tailed Student’s T-test. The average values are calculated for two independent experiments performed in triplicate. (B) AR activation upregulates miR-200b. PC3-AR cells (grey bars) were treated 5 days with both doxycycline to induce AR expression and with R1881 to induce AR activation and nuclear translocation. The comparison is to untreated control. Flutamide was added where indicated to block AR activity. Control (ctrl) PC3-AR cells were left untreated. RNU1A_1 non-coding RNA was used as an internal control. *, p<0.05; **, p<0.01 as determined by Students T-test. The average values were calculated for two independent experiments performed in triplicate.</p

miR-200b is sufficient to decrease tumor growth.

<p>A) Forced expression of miR-200b in PC3 cells. PC3 cells were trasduced with a bicistronic lentiviral shuttle vector (pMIRNA1 pCDH-CMV-MCS-EF1-copRFP) encoding hsa-miR-200b and empty vector control (ctrl). Total RNA was isolated and miR- 200b expression measured using real-time RT-PCR. The values represent three independent experiments performed in triplicate. *, p≤0.01. (B) miR-200b reduced tumorigenesis by PC-3 cells. Parental PC3 cells, PC3 cells trasduced with miR control (ctrl) and miR-200b were subcutaneously injected into the rear hindquarters of athymic male mice (n = 5). Tumor weight at day 29 post-injection is shown. *, p≤0.05; **, p≤0.01. (C) The tumors maintained miR-200b expression. Relative miR-200b expression was measured by real-time RT-PCR in the tumors from panel (B). * p≤0.05. (D). miR 200b decreased tumor growth in an orthotopic model of prostate cancer. RFP-tagged PC3-ctrl and PC3-200b cells were implaned in the prostates of athymic male mice. The average fluorescence was measured 20 days post injection. (E) Fluorescence per animal was determined using whole body imaging, with Olympus OV100 system. *, p≤0.05. (F) Cell growth was measured by WST-1 assay. PC3-ctrl or PC3 miR-200b cells were seeded at 3000 cells per well in a 96-well plate. Absorbance was measured at indicated time points using a Biorad Model 680 microplate reader. The results represent the average of three independent experiments performed in triplicate. *, p≤0.05 by Student’s T-test. (G, H) Tumor sections were stained for Ki-67, to evaluate proliferation. Note a significant decrease in Ki-67–positive nuclei in the presence of miR-200b. *, p<0.001.</p

miRNA decreased with AR expression and growth-promoting effect in cancers with increased expression (p<0.05).

<p>miRNA decreased with AR expression and growth-promoting effect in cancers with increased expression (p<0.05).</p

miR-200b reverses EMT and decreases invasion and metastasis by PC3 cells.

<p>(A) Markers affected by miR-200b in PC-3 cells. E-cadherin, Fibronectin, and Vimentin were detected in whole cell lysates from PC3 ctrl and PC3 miR-200b cells by western blot. (B) Quantitative analysis of the experiment shown in (A) performed with Image J software. *, p<0.05 and, **, p≤0.01 as determined by Student’s T-test. Two independent experiments were pooled together. (C) Western blot for ZEB1 and quantification performed as above (the average of two experiments is shown). (D) End-point PCR for ZEB1. (E) <i>In vitro</i> transwell invasion assay: the comparison of PC3-ctrl and PC3 miR-200b cells. 10% FBS was used as chemoattractant and the experiment was performed in duplicate. *, p<0.05 by Student’s T-test. (F) The <i>in vivo</i> spontaneous metastases by PC3-ctrl and PC3 miR-200b cells. The cells were implanted orthotopically in the ventral prostates of male nude mice. The mice were subjected to whole body imaging for RFP-positive masses using Surface image. At the end of experiment, the animals were sacrificed, peritoneal cavity opened and metastasis visualized by fluorescence imaging after the removal of a primary tumor inside the peritoneal cavity and on the frontal wall of the abdomen (Peritoneal cavity). Bright field (BF) and fluorescence (RFP, red fluorescent protein) are shown. (G) Quantification of the experiment shown in (F). The metastases were counted and total fluorescence per metastasis calculated (left). Gross metastatic burden was estimated as total fluorescence per mouse. Ctrl indicates control and 200b indicates miR-200b. **, p≤0.01 by Student’s T-test.</p

AR activation causes cell cycle arrest and senescence in PC3 cells.

<p>AR activation causes cell cycle arrest and senescence in PC3 cells. (<b>A</b>) <i>In situ</i> immunofluorescence with anti-AR antibody: note diffuse AR staining in control treated PC3-AR cells (left) and nuclear translocation on 3 day of DHT treatment (right). (<b>B</b>) Time-dependent inhibition of cell growth in vitro. PC-3 cells were cultured in Dox ± DHT; viable cells were measured using WST-1 reagent. Each time point represents mean ± standard deviation of three independent experiments. The difference between DHT treatment (red) and control EtOH (green) is statistically significant on day 5 (P<0.01). (<b>C</b>) Cell cycle analysis: the cells were grown in Dox, in the absence (D) and in the presence of DHT (DD) for 3 and 5 days; after 7 days the cells were passed (P1) and incubated for another 5 days. Cell cycle distribution was analyzed by flow cytometry. Note increased cell number in Go/G1 phase accompanied by decreased in S and G2/M populations in DHT-treated cells (DD) compared to Dox alone (D). (<b>D</b>) PC3-AR cells were cultured in Dox and treated with DHT ± Flutamide (Fl, 20 µM). After five days senescence was measured using SA-βGal assay. Note increased βGal positivity upon AR activation and the lack of senescence in the presence of Fl. (<b>E</b>) Senescent cells were counted on the digital images of 5 random fields using Image Tool 3.00 software (UTHSCSA); means of three independent experiments with S.D.M. are shown. (<b>F</b>) RWPE-1 cells were transfected with control lentivirus or lentiviral vector encoding AR. AR expression was measured in whole cell lysates by Western blot. GAPDH antibody was used to assess loading. (<b>G, H</b>) RWPE-AR and vector transfected (RWPE-C) cells were cultured in DHT, DHT and Flutamide (Fl) or with equal volume of EtOH added as control. Senescence was assessed with SA-βGal assay (I) and quantified as in (E).</p