Trp53 and Rb1 regulate autophagy and ligand-dependent Hedgehog signaling
Catherine R. Cochrane, … , D. Neil Watkins, Jason E. Cain
Catherine R. Cochrane, … , D. Neil Watkins, Jason E. Cain
Published June 22, 2020
Citation Information: J Clin Invest. 2020;130(8):4006-4018. https://doi.org/10.1172/JCI132513.
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Research Article Cell biology Oncology

Trp53 and Rb1 regulate autophagy and ligand-dependent Hedgehog signaling

Abstract

Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.

Authors

Catherine R. Cochrane, Vijesh Vaghjiani, Anette Szczepny, W. Samantha N. Jayasekara, Alvaro Gonzalez-Rajal, Kazu Kikuchi, Geoffrey W. McCaughan, Andrew Burgess, Daniel J. Gough, D. Neil Watkins, Jason E. Cain

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Figure 1

Hh signaling and primary cilia formation in Trp53- and Rb1-mutant MEFs.

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Hh signaling and primary cilia formation in Trp53- and Rb1-mutant MEFs. ...
(A) Western blot analysis of SHH and actin expression in MEF cell lines and NIH 3T3 cells (n = 3 cell lines per genotype). (B) Gli1 expression in MEFs treated with PBS control, 1 μg/mL recombinant human SHH (rhSHH), or rhSHH and 400 nM LDE225 for 24 hours, normalized to the expression of β2-microglobulin (n = 4, mean ± SEM). ****P < 0.0001, 2-way ANOVA/Tukey’s test. (C) Immunofluorescence colocalization of acetylated tubulin (AcTUB) and ARL13B in MEFs in 10% serum or serum-free conditions. Primary cilia are highlighted by arrows. Scale bar: 5 μm. (D) Primary cilia frequency in MEFs in 10% serum or serum-free conditions (n = 4 cell lines, mean ± SEM). ***P < 0.001 and ****P < 0.0001, 2-way ANOVA/Tukey’s test. (E) Primary cilia frequency in p53Rb-KO MEFs in 10% serum or serum-free conditions following transfection with control (siCont) or Kif3a siRNA (n = 3, mean ± SEM). ****P < 0.0001, 2-way ANOVA/Tukey’s test. (F) Gli1 expression in p53Rb-KO MEFs in serum-free medium for 24 hours after transfection with control (siCont) or Kif3a siRNA, followed by treatment with PBS or 1 μg/mL rhSHH for 24 hours (n = 5, mean ± SEM, normalized to the expression of β2-microglobulin). ****P < 0.0001, 2-way ANOVA/Tukey’s test. (G) Immunofluorescence colocalization of PCNA and ARL13B in MEFs cultured in serum-free conditions. Primary cilia are highlighted by arrows. Scale bar: 2 μm. (H) Primary cilia in PCNA+ MEFs, quantified as percentage of total cell population, in 10% serum or serum-free conditions (n = 3 independent cell lines, mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 2-way ANOVA/Tukey’s test.