Tumor-Stromal Interactions Influence Radiation Sensitivity in Epithelial- versus Mesenchymal-Like Prostate Cancer Cells

HS-27a human bone stromal cells, in 2D or 3D coultures, induced cellular plasticity in human prostate cancer ARCaPE and ARCaPM cells in an EMT model. Cocultured ARCaPE or ARCaPM cells with HS-27a, developed increased colony forming capacity and growth advantage, with ARCaPE exhibiting the most significant increases in presence of bone or prostate stroma cells. Prostate (Pt-N or Pt-C) or bone (HS-27a) stromal cells induced significant resistance to radiation treatment in ARCaPE cells compared to ARCaPM cells. However pretreatment with anti-E-cadherin antibody (SHEP8-7) or anti-alpha v integrin blocking antibody (CNT095) significantly decreased stromal cell-induced radiation resistance in both ARCaPE- and ARCaPM-cocultured cells. Taken together the data suggest that mesenchymal-like cancer cells reverting to epithelial-like cells in the bone microenvironment through interaction with bone marrow stromal cells and reexpress E-cadherin. These cell adhesion molecules such as E-cadherin and integrin alpha v in cancer cells induce cell survival signals and mediate resistance to cancer treatments such as radiation

miR-17* Suppresses Tumorigenicity of Prostate Cancer by Inhibiting Mitochondrial Antioxidant Enzymes

Aberrant micro RNA (miRNA) expression has been implicated in the pathogenesis of cancer. Recent studies have shown that the miR-17-92 cluster is overexpressed in many types of cancer. The oncogenic function of mature miRNAs encoded by the miR-17–92 cluster has been identified from the 5′ arm of six precursors. However, the function of the miRNAs produced from the 3′ arm of these precursors remains unknown. The present study demonstrates that miR-17* is able to suppress critical primary mitochondrial antioxidant enzymes, such as manganese superoxide dismutase (MnSOD), glutathione peroxidase-2 (GPX2) and thioredoxin reductase-2 (TrxR2). Transfection of miR-17* into prostate cancer PC-3 cells significantly reduces levels of the three antioxidant proteins and activity of the luciferase reporter under the control of miR-17* binding sequences located in the 3′-untranslated regions of the three target genes. Disulfiram (DSF), a dithiolcarbomate drug shown to have an anticancer effect, induces the level of mature miR-17* and cell death in PCa cells, which can be attenuated by transfection of antisense miR-17*. Increasing miR-17* level in PC-3 cells by a Tet-on based conditional expression system markedly suppresses its tumorigencity. These results suggest that miR-17* may suppress tumorigenicity of prostate cancer through inhibition of mitochondrial antioxidant enzymes

Suppression of tumorigenicity of PC-3 by expression of miR-17*.

<p>A, has-miR-17* was cloned in a Tet-on lentiviral vector and stably transected into PC-3 cells. The clone was tested by RFP screening under Dox- inductive conditions and then confirmed by measuring the expression of the three target genes using Western blots with β-actin normalization. B and C, the generated clone was injected into male nude mice to determine its tumorigenicity. The vehicle control was included. The number of days needed for tumor size to reach 500 mm³ is shown in (B) and calculated tumor growth rates in (C). D, total RNA and proteins were isolated from the tumor tissues and the level of miR-17* and corresponding activities of the three antioxidant proteins were quantified. Three samples (n = 3) were used in testing the generated miR-17* inducible clone (A). Nine vehicle control animals (n = 9) with or without DOX treatment and eighteen miR-17* expressed animals (n = 18) with or without DOX treatment were used to test the effect of miR-17* on tumor growth (B), (C), (D). * (p<0.05) and ** (p<0.01) indicate significances as compared to without DOX control (A) and (C).</p

Identification of three mitochondrial antioxidant proteins as miR-17* targets.

<p>A, the levels of miR-17 and miR-17* expressed in PCa and control cell lines were measured by RT-PCR. The ratio of miR-17 to miR-17* in each cell line is presented. B and C, transfection of miR-17* in PC-3 cells to validate its function in repressing the expression of antioxidant proteins and diminishing TNF-mediated MnSOD induction. D, the repressive effect of miR-17* on the antioxidant proteins is estimated by quantitative luciferase reporter assay. RNU24 and β-actin were used as internal controls to normalize miRNA levels (A), protein levels (B and C). Images were normalized with the internal controls and then normalized by PrEC (A), by control miRNA (B), and by no TNF treatment (C). β-gal activity was used to normalize luciferase reporter activities (D). Three samples (n = 3) were used in the experiments and fold changes in Western blots are indicated. * (p<0.05) and ** (p<0.01) indicate significances as compared to the controls: PrEC (A), control miRNA (B) and (D), and untreated samples (C).</p

Cytotoxicity of miR-17* in PCa cells.

<p>A, the PCa cells were treated with DSF at the indicated concentrations for colony survival analysis. The formed colonies were counted and plotted in a log scale. B, the PC-3 cells were transfected with miR-17* and control miRNAs prior to the DSF treatment. The effects of miR-17* and antisense miR-17* on colony survival were determined. C and D, miR-17* was co-transfected with constructs for expression of the three antioxidant proteins. The overexpressed antioxidant proteins were confirmed by Western blots with β-actin normalization and fold changes are indicated (D). Protective effects of the transfected antioxidant enzymes on the cells against miR-17* toxicity were determined by a trypan blue exclusion assay (C). Three samples (n = 3) were used in the experiments. * (p<0.05) and ** (p<0.01) indicate significances as compared to control miRNA samples (B) and compared to vehicle control samples (C), (D).</p

MiR-185 and 342 inhibition of SREBP mRNA and protein and expression patterns of miR-185 and 342 in prostate cancer cells.

<p><b>A,</b> Both miR-185 and 342 inhibited mRNA expression of SREBP-1, SREBP-2, FASN, HMGCR and AR in LNCaP and C4-2B prostate cancer cells determined by qRT-PCR. -: non-transfected; NC: miR-negative control. The relative mRNA expression (fold) was assigned as 1.0 in non-transfected cells. Data were normalized to 18S rRNA and represent the mean ± SD of three independent duplicate experiments. **, <i>P</i> < 0.005 significant differences from NC. <b>B,</b> MiR-185 and 342 inhibited precursor (125 kDa) and mature (68 kDa) forms of SREBP-1 and SREBP-2, FASN, HMGCR and AR expression in LNCaP and C4-2B cells assayed by Western blot. β2-microglobulin (β2M) was used as a loading control. <b>C,</b> MiR-185 and 342 inhibitors (antisense oligonucleotides against miR-185 and 342) increased SREBP-1, SREBP-2, FASN, HMGCR and AR expression in LNCaP and C4-2B cells determined by qRT-PCR. The relative mRNA expression (fold) was assigned as 1.0 in non-transfected cells. Data were normalized to 18S rRNA and represent the mean ± SD of three independent duplicate experiments. **, <i>P</i> < 0.005 significant differences from NC. <b>D,</b> Expression of intrinsic miR-185 and 342 in RWPE-1, LNCaP and C4-2B cells. qRT-PCR results showed that relative expression of miR-185 and 342 was significantly decreased in prostate cancer cells compared with normal/non-cancerous RWPE-1. Lower expression of both miRNAs was observed in aggressive C4-2B compared with LNCaP cells. The relative miRNA expression (fold) was assigned as 1.0 in RWPE-1 cells. **, <i>P</i> < 0.005 significant differences from RWPE-1. Data were normalized to RNU6B control and represent the mean ± SD of three independent experiments performed in quadruplicate.</p

Induction of miR-17* in PCa cells by DSF.

<p>A, PCa cells were treated with DSF at indicated concentrations. The levels of the three antioxidant proteins were measured by Western blots. B, mRNA levels of the three antioxidant genes were quantified by RT-PCR. C, the levels of miR-17 and miR-17* in the DSF-treated cells were quantified by RT-PCR. The miR-17* levels were confirmed by Northern blots. D, the effect of DSF-induced miR-17* on the reporter responses was determined. E, after transfected anti-miR-17*, PC-3 cells were treated with DSF. The effect of anti-miR-17* on restoring antioxidant proteins was quantified by Western blots. β-actin was used to normalize the levels of proteins (A), (E), and the levels of mRNA (B). The fold changes are indicated. RNU24 was used to normalize the levels of miR-17 and miR-17* (C). β-gal activity was used to normalize luciferase reporter activities (D). Three samples (n = 3) were used in the experiments (with the exception of Northern blot). * (p<0.05) and ** (p<0.01) indicate significances as compared to no DSF treatment (A), (C), (D), and compared to no DSF and no miRNA transfected samples (E).</p