ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells

Abstract

Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function.

Keywords: ADT; AR; Apoptosis; Cancer; FKBP51; ID4; Migration; bHLH; p21.

Figure 1

Stable Overexpression of ID4 with pCMV-ID4 in PC3 cells. Expression of ID4 was evaluated with real time PCR (A) and Western Blot (B). C: Morphology of the PC3-CMV and PC3+ID4 cells in culture. D and E: Analysis of E-cadherin by Western blot and Immuno-cytochemistry. F: Proliferation rate of PC3-CMV and PC3+ID4 cells expressed as absorption at 570 nm (means ± SEM, n = 3; ***, P < .001). G: Ki-67 expression by Western Blot. H: Percent of cells undergoing apoptosis by flow cytometry (***: P < 0.001). I: Western blot analysis of BAX, and p21. GAPDH was used as loading control.

Figure 2

ID4 regulates Migration. A: ID4 significantly inhibited migration in PC3+ID4 cells compared to PC3–CMV and PC3+EGF cells (positive control). Each bar represents Mean ± SEM (n = 3). (*** p < 0.001 compared to PC3-CMV). B: Representative images of PC3+EGF, PC3 CMV and PC3+ID4 cells after migration of cells through Transwell (×10).

Figure 3

ID4 regulates expression of AR. A and B: Quantitative real time RT-PCR of AR and FKBP51 respectively (mean± SEM, n=3 in triplicate, normalized to GAPDH followed by fold change over PC3-CMV cells ***: P < 0.001). C: Western blot analysis of AR and FKBP51 in PC3– CMV and PC3+ID4 cells (±R1881). GAPDH was used as loading control. D: AR immuno-cytochemistry (red). The cells were counterstained with DAPI (blue) to reveal the nucleus (×200). E: Enrichment of Pol II and AR, on FKBP51 promoter in PC3-CMV, PC3+ID4 cells. The data is expressed (mean±SEM, n=3 in triplicate) as % of input (“a”, and “b” corresponding to Pol II, and AR respectively, p<0.001 compared with PC3 - CMV cells. F: The AR transcriptional activity as determined by PSA AR response element driven luciferase reported plasmid. The data is normalized to Renilla luciferase. The mutant ARR3 luciferase reporter plasmid was used as a negative control (mean ± SEM ***: P < 0.001 as compared to respective ARR3 luciferase, a: p<0.001 as compared to PSA luciferase activity in PC3-CMV, c: p<0.01 as compared to PC3+ID4, n=3).

Figure 4

table overexpression of ID4 in PC3 cells suppresses tumor growth in vivo. Male nude mice were injected with PC3–CMV and PC3+ID4 cells and evaluated for tumor volumes and tumor weights. A: Representative xenograft images with numbers of mice with similar tumor (n) are shown. B: Volumes of the tumors were measured weekly (expressed as mm³, means±SEM, n = 3/group, ***, p < .001, and *p<0.01 between PC3–CMV and PC3+ID4). The mice were sacrificed at 6 weeks. C, D: Relative volumes and weights (means±SEM, n = 3) of the tumors after excision (***, P < .001, between PC3– CMV and PC3+ID4 tumors). E: TUNEL assay demonstrated increased apoptosis in in PC3+ID4 mice xenograft (Brown staining) as compared to the PC3–CMV cells. The TUNEL positive cells were counted in five fields (at 400×) on three different tissue samples.