The file contains 6 supplementary figures. Figure S1 shows that AR protein expression is also induced following treatment with additional proteasomal inhibitors, MG115 and Epoxomicine. Figure S2 compares the levels of MDM2 expression, demonstrating that CSC-like HuSLC and HPET cells express higher levels of MDM2 as compared to standard PCa cell lines. Figure S3 demonstrates that HPET and HuSLCs only express AR-fl mRNA but not AR-V mRNA and this is confirmed in Figure S4 where HPET and HuSLCs only express AR-fl protein but not AR-V protein. Figure S5 provides information that induction of AR promotes expression of additional AR target genes, namely AR-regulated HES1 and HEY1 genes. Figure S6 compares p53 expression in CSC-like HPET and HuSLCs to that of LNCaP cells.
ARTICLE 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.
