XMRV accelerates cellular proliferation, transformational activity, and invasiveness of prostate cancer cells by downregulating p27(Kip1)

Abstract

Background: Xenotropic murine leukemia virus-related retrovirus (XMRV) is a recently discovered gammaretrovirus that was originally detected in prostate tumors. However, a causal relationship between XMRV and prostate cancer remains controversial due to conflicting reports on its etiologic occurrence. Even though gammaretroviruses are known to induce cancer in animals, a mechanism for XMRV-induced carcinogenesis remains unknown. Several mechanisms including insertional mutagenesis, proinflammatory effects, oncogenic viral proteins, immune suppression, and altered epithelial/stromal interactions have been proposed for a role of XMRV in prostate cancer. However, biochemical data supporting any of these mechanisms are lacking. Therefore, our aim was to evaluate a potential role of XMRV in prostate carcinogenesis.

Methods: Growth kinetics of prostate cancer cells are conducted by MTT assay. In vitro transformation and invasion was carried out by soft agar colony formation, and Matrigel cell invasion assay, respectively. p27(Kip1) expression was determined by Western blot and MMP activation was evaluated by gelatin-zymography. Up-regulation of miR221 and miR222 expression was examined by real-time PCR.

Results: We demonstrate that XMRV infection can accelerate cellular proliferation, enhance transformation, and increase invasiveness of slow growing prostate cancer cells. The molecular basis of these viral induced activities is mediated by the downregulation of cyclin/cyclin dependent kinase inhibitor p27(Kip1) . Downstream analyses illustrated that XMRV infection upregulates miR221 and miR222 expression that target p27(Kip1) mRNA.

Conclusions: We propose that downregulation of p27(Kip1) by XMRV infection facilitates transition of G1 to S, thereby accelerates growth of prostate cancer cells. Our findings implicate that if XMRV is present in humans, then under appropriate cellular microenvironment it may serve as a cofactor to promote cancer progression in the prostate.

Fig. 1. XMRV infection analysis

(A) Western blot analysis of cell lysates from uninfected (−) and XMRV infected (+) cells. We detected XMRV p30 protein by using goat polyclonal anti-Rauscher MLV p30 Gag. The XMRV p30 capsid was detected in the infected cells but not in the uninfected cells. (B) RT-PCR analysis of RNA isolated from uninfected (−) and XMRV infected (+) LNCaP cells using XMRV Env specific primers. The Env amplicon was detected in the XMRV infected cells but not in the uninfected cells. The RT-PCR assay was conducted to exclude possible contamination of mouse DNA in our experiments.

Fig. 2. Effect of XMRV infection on proliferation and colony forming ability of prostate cancer cells

(A–B) Cellular proliferation was determined by MTT assay by measuring the OD₅₇₀ nm. 5×10⁴ cells per well were seeded in a 24-well plate and proliferation was then measured at different time intervals. (A) The virally infected LNCaP cells outgrew the uninfected cells by day nine. Data presented are mean value of three independent experiments that are performed in triplicates. The results are expressed as mean ± S.E. for three separate experiments. * p < 0.01 is for XMRV-infected cells compared with uninfected cells. (B) XMRV infection in PC-3 cells have minimal or no effect on cellular proliferation. (C–D) Colony formation was determined by soft agar assay by seeding 2×10⁴ cells in 12-well culture dishes and feeding with growth media once a week for 2–3 weeks. Colonies were stained overnight at 37°C overnight and counted under microscope. (C) Representative frames depicting number and size of colonies in uninfected and XMRV infected LNCaP cells. The insets in each panel represent the magnification at which the frames were visualized. (D) Comparative analysis of relative number of colonies formed by uninfected and infected cells. Data represents average colony counts from three independent experiments with triplicates for each sample. * p < 0.005 is for XMRV-infected cells compared with uninfected cells.

Fig. 3. XMRV infection increases the invasiveness of prostate cancer cells

Cell invasion properties of uninfected and infected cells were determined by Matrigel invasion assay. 5×10⁴ cells in were seeded in the upper chamber of a 24-well invasion chamber system. Using serum as the chemo-attractant in the lower compartment, invasion was measured by the ability of cells to migrate through the intermediate membrane. Assays were performed in triplicates with each experiment repeated three times. Invasion assay of LNCaP (A–B) and PC-3 (C–D) cells. (A and C) Representative frames showing number of cells invaded through the membrane barrier. (B and D) Comparative analysis of relative cell numbers that invaded through the membrane. The results are expressed as mean ± S.E. for three separate experiments. * p < 0.001 and ** p < 0.001 are for XMRV-infected cells compared with uninfected cells.

Fig. 4. MMP activities of prostate cancer cells are increased by XMRV infection

Impact of XMRV infection on the zymogen activity of MMP-2 and MMP-9 in LNCaP (A–B) and PC-3 (C–D) cells. (A and C) Cell lysates were analyzed by Gelatin zymography and MMP activation was detected by commassie blue staining. (B and D) Densitometric measurement for quantitative analysis of relative MMP-2 and MMP-9 activities. The results are expressed as mean ± S.E. for three separate experiments. * p < 0.01 is for XMRV-infected cells compared with uninfected cells.

Fig. 5. XMRV infection downregulates p27^Kip1 in LNCaP cells

(A) To determine the p27^Kip1 proteins levels, cell lysates from uninfected and infected cells were analyzed by western blot. (B) XMRV induced decrease in p27^Kip1 level was almost 50% as determined by densitometry. (C–D) Evaluation of p27^Kip1 protein expression in LNCaP cells infected by ampho-MLV. In contrast to XMRV, Ampho-MLV had little/no impact on p27^Kip1. The results are mean ± S.E. for three separate experiments. * p < 0.005 is for XMRV-infected cells compared with uninfected cells.

Fig. 6. Downregulation p27^Kip1 induces G1→S transition in LNCaP cells and is mediated by upregulation of miR-221 and miR-222

(A) G1→S cell cycle transition was evaluated by propidium iodide staining by determining DNA content by flow cytometry. Three independent experiments were performed in triplicates. (B) XMRV induced downregulation of p27^Kip1 is mediated by upregulation of miR-221 and miR-222 expression. For this experiment, total RNA isolated from cells were used in real time PCR analysis and relative fold expression of miR-221 and miR-222 were determined with reference to the expression of 5S ribosomal RNA. The results are expressed as mean ± S.E. for three separate experiments with triplicates.

Fig. 7. Androgen influences XMRV-induced molecular alterations in prostate cancer cells

Androgen independent DU145 cells were infected with XMRV and infection was confirmed by detection of p30 protein (B). (A) Cellular proliferation of androgen dependent DU145 cells was determined by MTT assay as described in Fig. 2. (B) p27^Kip1 levels was determined by western blot. (C) Densitometry analysis of p27^Kip1 levels. (D) Zymogen activation of MMPs and (E) densitometry analysis was carried out as described in Fig. 4.

Fig. 8. A model depicting a plausible role of XMRV in prostate cancer progression

(A) In eukaryotic cells, p27^Kip1 plays a critical role in cellular proliferation as it binds to the cyclin/CDK complexes and arrests cell cycle progression. (B) In XMRV infected cells, we hypothesize that by upregulating miR-221 and miR-222, XMRV downregulates p27^Kip1 expression, thereby propels G1→S transition of the cell cycle. Subsequently, by upregulating the MMP activities that degrade ECM and basement membranes, XMRV enhances invasiveness of prostate cancer cells. Enhanced cellular proliferation and invasiveness of prostate cancer cells by XMRV infection may play a role in prostate cancer progression.