MDM2 regulates the stability of AR, AR-V7, and TM4SF3 proteins in prostate cancer

Abstract

Androgen receptor (AR) and its constitutively active splice variant, AR Variant 7 (AR-V7), regulate genes essential for the development and progression of prostate cancer. Degradation of AR and AR-V7 by the ubiquitination proteasomal pathway is important for the regulation of both their protein stability. Our published results demonstrate that the interaction of TM4SF3 with either AR or AR-V7 leads to mutual stabilization due to a reduction in their ubiquitination and proteasomal degradation. These results led us to search for a common E3 ligase for AR, AR-V7, and TM4SF3. Depletion by siRNA of several E3 ligases identified MDM2 as the common E3 ligase. MDM2 inhibition by siRNA depletion or using a pharmacological inhibitor (MDM2i) of its E3 ligase activity led to elevated levels of endogenous AR, AR-V7, and TM4SF3 in prostate cancer cells. MDM2 knockdown in PC-3 cells, which do not express AR, also increased TM4SF3, demonstrating that MDM2 affects the TM4SF3 protein independent of AR. We further demonstrate that MDM2i treatment reduced the ubiquitination of AR and TM4SF3, suggesting that MDM2 can induce the ubiquitination of these proteins. Increased AR and AR-V7 protein levels induced by MDM2i treatment resulted in the expected increased expression of AR-regulated genes and enhanced proliferation and migration of both LNCaP and Enzalutamide-resistant CWR-22Rv1 prostate cancer cells. Thus, our study expands the known roles of MDM2 in prostate cancer to include its potential involvement in the important mutual stabilization that TM4SF3 exhibits when interacting with either AR or AR-V7.

Keywords: AR; AR-V7; MDM2; TM4SF3; prostate cancer.

Figure 1

MDM2 regulates AR and TM4SF3 protein levels. LNCaP cells were grown in 2% DCC for 48 h before (A) transfection with indicated siRNAs, (B) treatment with MDM2i, or transfection with siMDM2, (C) treatment with vehicle (0) or 2.5 and 5 μM MDM2i, or (D) treatment with vehicle (−), 10 μM MG132 or 5 μM MDM2i. After 48 h, or transfection or treatment, cells were lysed with M-PER, and Western blotting was performed to measure AR and TM4SF3 protein levels. (E, F) HEK-293 cells grown in 2% DCC were co-transfected with FLAG-AR (E) or TM4SF3-FLAG (F) and either empty or HA-Ubiquitin and treated with either vehicle (−) or 5 μM MDM2i for 24 h. After 24 h, cells were treated with 10 μM MG132 for another 6 h before performing a denatured cell lysis and IP with anti-FLAG antibody. Following the IP and Western blot, ubiquitination was detected by blotting the proteins with anti-HA antibodies. AR or TM4SF3, and HA-Ubiquitin were detected in input by using indicated antibodies. In all Western blots, β-actin was used as a loading control and is shown here as a representative of three independent experiments. Note that the vehicle is DMSO.

Figure 2

MDM2 regulates AR-V7 protein stability. CWR-22Rv1 cells grown in 2% DCC for 48 h were (A) transfected with siRNA, (B) treated with vehicle (DMSO), 5 μM MDM2i, or transfected with siMDM2, or (C) treated with 0 (vehicle), 2.5, or 5 μM MDM2i. (D) R49F cells grown in 2% DCC for 48 h were treated with 0 (vehicle), 2.5, or 5 μM MDM2i. After 48 h of transfection or treatment, cells were lysed with M-PER, and Western blotting was performed to detect AR, AR-V7, and TM4SF3 as indicated. In all Western blots, β-actin was used as a loading control.

Figure 3

MDM2 inhibition promotes the growth of prostate cancer cells in androgen-free conditions. (A) LNCaP, (B) CWR-22Rv1, and (C) PC-3 cells grown in 2% DCC for 48 h were treated with vehicle (ethanol) or 10 nM R1881, and either DMSO (−) or 5 μM MDM2i (+). After 0, 2, or 4 days, cell proliferation was measured using an MTT proliferation assay. (D) LNCaP and (E) CWR-22Rv1 cells grown in 2% DCC for 48 h were treated with 0, 2.5, and 5 μM MDM2i for 48 h and monitored for migration, represented in bar graph after normalizing for proliferation. Bar graphs represent averages of three independent experiments plus s.d. A Student’s t-test was performed to show statistical significance ( P < 0.05), as indicated by the asterisks, (A, B, C) in the number of cells on day 2 or day 4 as compared to day 0 or (D, E) in cell migration with different concentrations of MDM2i as compared to vehicle.

Figure 4

MDM2 inhibition increases the expression of AR and AR-V7 target genes in androgen-free conditions. (A, C) LNCaP and (B, D) CWR-22Rv1 cells grown in 2% DCC for 48 h were treated with (A, B) 0 (vehicle), 2.5, or 5 μM MDM2i or (C, D) ethanol (−) or R1881 (+) and DMSO (−) or 5 μM MDM2i (+) for 48 h before mRNA extraction and qRT-PCR to measure the expression of AR and AR-V7 target genes (as indicated in the text). Bar graphs represent averages of three independent experiments plus s.d. A Student’s t-test was performed to show statistical significance (P < 0.05), as indicated by the asterisks, in gene expression in the presence of MDM2i (+) as compared to vehicle (−).