Human prostate luminal cell differentiation requires NOTCH3 induction by p38-MAPK and MYC

Abstract

Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that p38-MAPK regulation of NOTCH3, via MYC, is required for luminal differentiation. Inhibition (SB202190, BIRB796) or knockdown of p38α and/or p38δ prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation by MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA/protein levels and was diminished upon MYC inhibition (10058-F4, JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements by a combination of eRNA detection (BruUV-seq) and luciferase reporter assays. Lastly, we found that NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC, and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation, and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.