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. 2019 Mar 15;79(6):1124-1137.
doi: 10.1158/0008-5472.CAN-18-1753. Epub 2019 Jan 9.

Constant Degradation of the Androgen Receptor by MDM2 Conserves Prostate Cancer Stem Cell Integrity

Premkumar Vummidi Giridhar #  1 , Karin Williams #  2 , Andrew P VonHandorf #  3 , Paul L Deford  3 , Susan Kasper  4
Affiliations

Constant Degradation of the Androgen Receptor by MDM2 Conserves Prostate Cancer Stem Cell Integrity

Premkumar Vummidi Giridhar et al. Cancer Res. .

Abstract

Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR-) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR+ tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR- CSCs in addition to the bulk of AR+ prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance.Significance: These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/6/1124/F1.large.jpg.

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Conflict of interest statement

Conflict of interest statement: The authors declare no potential conflicts of interest.

Figures

Figure 1.
Figure 1.. AR expression in CSC-like PCa cells is modulated by the proteasome.
(A, B) AR expression and transcriptional activity are only induced following transfection with high concentrations of pSVARo plasmid. HPET cells (A) and HuSLCs (B) were transfected with increasing concentrations of pSVARo plasmid (expressing full-length, wt human AR). AR protein levels were analyzed by Western blot and semi-quantified by Densitometry. Cells were transfected with ARR2PB-luc (24,49) and Renilla luciferase reporter genes. Luciferase activity was determined using the Promega Dual-Luciferase® Reporter Assay System kit and protocol (Promega, cat.#: E1910). (C, D) Proteasomal inhibition initiates AR expression and transcriptional activity. HPET cells (C) and HuSLCs (D) were treated with increasing concentrations of MG132 with/without addition of 10−8 M DHT and dual luciferase activity was determined as above. Abbreviations: wt, wild type; DHT, dihydrotestosterone. Data are expressed as mean ± SEM; n = 4. *, p<0.01; **, p<0.001; ***, p<0.0001.
Figure 2.
Figure 2.. CSC-like HPET cells and HuSLCs express full-length AR, but not AR-Vs.
(A) RT-qPCR analysis of endogenous HPET and HuSLC AR-fl following treatment with DHT with/without OHF. HPET cells (upper panel) were treated with 2 μM MG132 to induce endogenous AR, or 30 μg pSVARo to express exogenous AR-fl, and treated with 10−8 M DHT with/without 10−5 M OHF. Vehicle control, 95% ethanol. HuSLCs (lower panel) were treated with 20 μM MG132 to induce endogenous AR, or 40 μg pSVARo to express exogenous AR-fl, and treated with 10−8 M DHT with/without 10−5 M OHF. Vehicle control, 95% ethanol. VCaP and 22RV1 cells lines served as control PCa cell lines for AR-fl, AR-V1 and AR-V7 expression. Primer sets used to characterize AR-V expression are listed in Supplementary Table S2. Only data for AR-fl, AR-V1 and AR-V7 are shown since HPET and HuSLCs did not express any of the other AR-Vs tested (Supplementary Figure S3). (B-C) RT-qPCR analysis of endogenous AR-V1 and AR-V7 following treatment with DHT with/without OHF. HPET cells (upper panel) and HuSLCs (lower panel) were treated as in (A). In both HPET and HuSLCs, AR-V1 and AR-V7 expression was not observed under any conditions tested. Abbreviations: fl, full-length; V, Variant; DHT, dihydrotestosterone; OHF, hydroxyflutamide. Data are expressed as mean ± SEM; n = 4.
Figure 3.
Figure 3.. Induction of AR protein down-regulates sphere formation and cell proliferation.
(A) Top panel, sphere formation assay (2). HPET cells were treated with 2 μM MG132 to induce endogenous AR and 10−8 M DHT with/without 10−5 M OHF to increase or inhibit AR activity respectively. Vehicle control, 95% ethanol. Phase contrast images, 20x. Bottom left panel, quantification of the number of spheres/well. Bottom right panel, proliferation assay (19). HPET cells were treated as described in the top panel and total cell numbers/well were determined using the Trypan Blue Viability assay. AR expression inhibited sphere formation and cell proliferation which could be rescued by treatment with OHF. (B). Assays in HuSLCs were performed as described in (A) with HuSLCs being treated with 20 μM MG132 to induce endogenous AR. (C) Assays in HPET cells were performed as described in (A) with the modification that AR was exogenously expressed following transfection with 30 μg pSVARo plasmid. (D) Assay in HuSLCs cells were performed as described in (A) following transfection with 40 μg pSVARo plasmid. Data are expressed as mean ± SEM; n = 4. **, p<0.001; ***, p<0.0001.
Figure 4.
Figure 4.. MG132 treatment down-regulates expression of stem cell-associated markers and promotes luminal epithelial cell fate.
(A) HPET cells and HuSLCs were treated with 2 μM and 20 μM MG132 respectively and expression of the stem cell factors Oct4, Nanog, Sox2, Nestin and CD44 were determined by RT-qPCR. (B) Cells were treated with 2 μM and 20 μM MG132 respectively and expression of prostate luminal epithelial cells factors PSA, PAP, Nkx3.1, TMPRSS2 and FGF5 were determined by RT-qPCR. (C) Cells were treated with 2 μM and 20 μM MG132 respectively and expression of p63 and ChrA were determined by RT-qPCR. Primer sets to characterize expression of all of these factors are listed in Supplementary Table S3. Data are expressed as mean ± SEM; n = 4. *, p<0.01; **, p<0.001; ***, p<0.0001.
Figure 5.
Figure 5.. Induction of exogenous AR down-regulates expression of stem cell-associated markers and promotes luminal epithelial cell fate.
(A) HPET cells and HuSLCs were transfected with 30 μg and 40 μg of pSVARo respectively and treated with/without DHT with/without OHF as indicated. Expression of the stem cell factors Oct4, Nanog, Sox2, Nestin and CD44 were determined by RT-qPCR. (B) Cells were transfected and treated as in (A) and expression of prostate luminal epithelial cells factors PSA, PAP, Nkx3.1, TMPRSS2 and FGF5 were determined by RT-qPCR. (C) Cells were transfected and treated as in (A) and expression of p63 and ChrA were determined by RT-qPCR. Primer sets to characterize expression of all of these factors are listed in Supplementary Table S3. Data are expressed as mean ± SEM; n=4. *, p<0.01; **, p<0.001; ***, p<0.0001.
Figure 6.
Figure 6.. The E3 ligase MDM2 selectively degrades AR in prostate CSCs.
(A) HPET cells were transfected with either 30 μg pSVARo (i) or treated with 2 μM MG132 (ii) and transfected with increasing concentrations of the wild-type ubiquitin expression vector pRK5-HA-Ubiquitin-WT (HA-UbWt) to determine whether poly-ubiquitination regulated AR protein levels. In addition, HPET cells were transfected with increasing concentrations of a mutant ubiquitin plasmid pRK5-HA-Ubiquitin-KO (HA-UbKO, iii) which is incapable of adding ubiquitin molecules onto its target protein, to demonstrate that inhibiting AR poly-ubiquitination would prevent AR degradation. (B) HPET cells were transfected with increasing concentrations of shMDM2, shNEDD4, shMarchVII or shWWP2 plasmid and protein expression was analyzed by Western blot analysis. (C) Cells were co-transfected with 20 μg of shMDM2, 10 μg of shNEDD4, 2 μg of shMarchVII or 1ug of shWWP2 plasmid and the ARR2PB-luc and Renilla luciferase reporter gene constructs. Luciferase activity was determined. Positive controls, cells transfected with 30 μg pSVARo or treated with 2 μM MG132. NT, non-targeting shRNA control. (D) IP analysis was performed to determine whether MDM2 directly binds to AR. Cells were transfected with 20 μg non-targeting shNT control plasmid, 20 μg HA-UbWt, or 20 μg HA-UbKO and treated with 2 μM MG132 as indicated. Antibodies for Western blot analysis are listed in Supplementary Table S1. Data are expressed as mean ± SEM; n=4.
Figure 7.
Figure 7.. MDM2 maintains stemness in human prostate CSC-like cells.
(A) Sphere formation assay. HPET cells were transfected with 20 μg of shMDM2 plasmid and treated with 10−8 M DHT with/without 10−5 M OHF to increase or inhibit AR activity respectively. Control group, transfected with 20 μg of non-targeting shNT control plasmid and treated with vehicle control (95% ethanol). Phase contrast images, 20x. (B) Quantification of the number of spheres/well in (A). (C) Proliferation assay (19). HPET cells were transfected with 20 μg of shMDM2 plasmid and treated with 10−8 M DHT with/without 10−5 M OHF treated as indicated. Total cell numbers/well were determined using the Trypan Blue Viability assay. (D) HPET cells were transfected with 20 μg of shMDM2 and expression of the stem cell factors were determined by RT-qPCR. (E) HPET cells were transfected with 20 μg of shMDM2 and expression of prostate luminal epithelial cells factors were determined by RT-qPCR. Data are expressed as mean ± SEM; n = 4. (F) Schematic representation of MDM2-mediated regulation of AR expression in modulating self-renewal and epithelial cell specification. Details are provided in the main text.
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