Abstract 4994: p53 and RB regulate Hedgehog responsiveness via autophagy-mediated ciliogenesis

Abstract

Abstract Hedgehog (Hh) signaling regulates patterning, cell-fate and self-renewal in development. Hh proteins signal via Smoothened (Smo), a G-protein coupled receptor whose activity is dependent on translocation to the primary cilium, a single immotile tubulin-based structure present on most mammalian cells. Activation of Smo results in the stabilization of GLI activator transcription factors, which in turn induce Hh pathway gene expression. Aberrant activation of the Hh pathway is implicated in initiation and progression of a wide range of cancers, yet very few contain genetic mutations of Hh pathway components that account for increased signaling. Instead, the majority of Hh-driven tumors exhibit ligand-dependent signaling but the mechanisms governing this are unknown. We show that genetic inactivation of Trp53 and or Rb1 in mouse embryonic fibroblasts (MEFs) promotes ciliogenesis and cell responsiveness to Hh ligand. Pampliega et al. (Nature 2013) have previously described a functional interaction between autophagy and ciliogenesis. Interestingly, Trp53KO, Rb1KO and Trp53;Rb1KO MEFs exhibit defective autophagic flux and reduced expression of genes associated with autophagy. siRNA knockdown of Atg5, Atg9b, Ctsd and Pik3cg in C57Bl/6 wild-type MEFs resulted in increased Hh ligand responsiveness. To explore this further in a disease setting we assessed an extensive panel of mouse osteosarcoma (mOS) cell lines. In contrast to radiation-induced mOS cell lines that demonstrated variable sensitivity, all genetically induced mOS cell lines (OsxCre;Trp53fl/fl;Rb1fl/fl) were highly sensitive to Hh ligand. In all cases Hh pathway activation could be inhibited by the small-molecule Smo-inhibitor, LDE225. Hh responsiveness correlated to primary cilia frequency with responsive cell lines demonstrating high cilia frequency while nonresponsive cell lines exhibited few if any cilia, under both normal or serum-deprived conditions. Similarly, autophagic flux was significantly reduced in Hh responsive compared to nonresponsive mOS cell lines. Consistent with these findings, in a panel of human osteosarcoma (hOS) cell lines, those with p53 and/or RB-deficient pathways are associated with reduced autophagy and increased primary cilia frequency. Pathway inhibition by LDE225 in in vivo allograft and xenograft models of highly ciliated mOS and hOS cell lines leads to reduced tumor growth, increased survival and intratumoral bone deposition, but has no effect on xenografts of a cell line lacking primary cilia. These data suggest that p53 and Rb control of genes required for autophagy regulates ciliogenesis and ultimately Hh pathway responsiveness to ligand, implicating p53 and Rb mutation status and primary cilia frequency as biomarkers for Hh-ligand sensitivity and potential responsiveness to Hh-inhibitor therapy. Citation Format: Jason E. Cain, Catherine R. Cochrane, Vijesh Vaghjiani, Anette Szczepny, Andrew McCaw, Kirstyn Carey, Luciano Martelotto, Maya Kansara, David Thomas, Carl Walkley, William H. Matsui, David N. Watkins. p53 and RB regulate Hedgehog responsiveness via autophagy-mediated ciliogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4994.