Numb^-/low Enriches a Castration-Resistant Prostate Cancer Cell Subpopulation Associated with Enhanced Notch and Hedgehog Signaling.

Abstract

<b>Purpose:</b> To elucidate the role and molecular mechanism of Numb in prostate cancer and the functional contribution of Numb^-/low prostate cancer cells in castration resistance.<b>Experimental Design:</b> The expression of Numb was assessed using multiple <i>Oncomine</i> datasets and prostate cancer tissues from both humans and mice. The biological effects of the overexpression and knockdown of Numb in human prostate cancer cell lines were investigated <i>in vitro</i> and <i>in vivo</i> In addition, we developed a reliable approach to distinguish between prostate cancer cell populations with a high or low endogenous expression of Numb protein using a Numb promoter-based lentiviral reporter system. The difference between Numb^-/low and Numb^high prostate cancer cells in the response to androgen-deprivation therapy (ADT) was then tested. The likely downstream factors of Numb were analyzed using luciferase reporter assays, immunoblotting, and quantitative real-time PCR.<b>Results:</b> We show here that Numb was downregulated and negatively correlated with prostate cancer advancement. Functionally, Numb played an inhibitory role in xenograft prostate tumor growth and castration-resistant prostate cancer development by suppressing Notch and Hedgehog signaling. Using a <i>Numb</i> promoter-based lentiviral reporter system, we were able to distinguish Numb^-/low prostate cancer cells from Numb^high cells. Numb^-/low prostate cancer cells were smaller and quiescent, preferentially expressed Notch and Hedgehog downstream and stem-cell-associated genes, and associated with a greater resistance to ADT. The inhibition of the Notch and Hedgehog signaling pathways significantly increased apoptosis in Numb^-/low cells in response to ADT.<b>Conclusions:</b> Numb^-/low enriches a castration-resistant prostate cancer cell subpopulation that is associated with unregulated Notch and Hedgehog signaling. <i>Clin Cancer Res; 23(21); 6744-56. ©2017 AACR</i>.