Abstract B18: Myc governs a prostate epithelial differentiation program involving chromatin remodeling protein ING4 and Notch3: Disruption of which is necessary for human prostate cancer development

Myc is overexpressed in the majority of human prostate cancers and is a known determinant of cell fate, yet the cell of origin from which prostate cancers arise is controversial. Furthermore, the mechanisms by which oncogenes such as Myc disrupt prostate epithelial cell fate are poorly understood. Using a novel human in vitro differentiation model in which prostate basal epithelial cells are induced to differentiate into lumenal cells, we previously demonstrated that Myc-driven prostate cancer develops in an intermediate progenitor cell population whose full differentiation is derailed upon oncogenic transformation (Berger et al, Cancer Res 74:3357-68, 2014). In basal prostate epithelial cells, Myc is required for transient expression of the chromatin-binding protein ING4, which is required for lumenal cell differentiation. In human tissues, ING4 expression is lost in >60% of primary prostate tumors. Loss of ING4 prevented differentiation and was necessary for Myc-dependent tumorigenesis in vivo. ING4 loss generated Myc-dependent tumor cells co-expressing basal and lumenal markers, indicating Myc-dependent oncogenesis disrupted an intermediate step in the prostate epithelial differentiation program. Our objective for this study was to further elucidate the mechanisms by which Myc controls prostate epithelial cell fate. Myc is a known downstream target of Notch1, and several studies suggest Notch signaling is aberrant in prostate cancer; although the mechanistic details are vague. We found that Notch3 is required for lumenal cell differentiation and hypothesized that Notch3 expression is directly controlled by Myc. Inhibition of total Notch signaling with a γ-secretase inhibitor (RO4929097) prevented differentiation. Total Notch1 mRNA and protein levels change very little during differentiation; whereas both Notch3 mRNA and protein increase dramatically. Knock-down of Notch3 by shRNA blocked differentiation, while over expression of active Notch3 (NCID3) induced spontaneous differentiation. Less than 15% of the increase in Notch3 mRNA was attributable to increased mRNA stability, and required new protein synthesis. Temporally, Myc mRNA and protein levels increase prior to Notch3. Blocking Myc expression prevented Notch3 induction. The 2kb proximal promoter region of Notch3 lacked the elements that promote Notch3 induction. We identified a Notch3 enhancer element with Myc binding motifs that support differentiation-induced luciferase reporter activity. We further determined that p38α-MAPK is required for Myc and Notch3 induction. We are currently determining how Myc-dependent regulation of Notch3 influences Myc-dependent regulation of ING4. Thus, our studies demonstrate that at least 2 targets of Myc, ING4 and Notch3, control prostate epithelial cell fate, and that disruption of at least one of them is required for Myc-driven human prostate cancer development. In depth understanding of Myc-driven differentiation pathways will provide new insights into how oncogenic transformation by Myc in intermediate progenitor prostate epithelial cells gives rise to prostate cancer. Funding was provided by the Association for International Cancer Research, NIH/NCI CA154835, Department of Defense W81XWH-14-10479, and the Van Andel Research Institute. Citation Format: Sander B. Frank, Penny L. Berger, Cindy K. Miranti. Myc governs a prostate epithelial differentiation program involving chromatin remodeling protein ING4 and Notch3: Disruption of which is necessary for human prostate cancer development. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B18.