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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