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 2

Autophagic flux in Trp53- and Rb1-mutant MEFs.

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Autophagic flux in Trp53- and Rb1-mutant MEFs. (A) Western blot analysis...
(A) Western blot analysis of LC3, P62, and actin expression in WT, p53-KO, Rb-KO, and p53Rb-KO MEF cell lines cultured in 10% serum or serum-free medium with or without 50 nM bafilomycin A1 (Baf-A) for 24 hours (n = 4 independent experiments). (B) Quantification of autophagic flux by Western blot in MEFs as shown in A (n = 4 independent experiments, mean ± SEM). Graph represents fold change in autophagic flux in serum-free medium (induced autophagy) compared with normal serum (basal autophagy). *P < 0.005; **P < 0.01 compared with WT control; 1-way ANOVA/Tukey’s test. (C) Immunofluorescence detection of LC3- and P62-positive puncta in MEF cell lines cultured in serum-free medium, with or without 50 nM Baf-A for 24 hours. Nuclei are counterstained with DAPI. Representative images are shown. Scale bar: 5 μm. (D) Quantitative analysis of the experiment depicted in C as puncta per cell (n = 4 independent cell lines, mean ± SEM). ****P < 0.0001, 1-way ANOVA/Bonferroni’s correction.