Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers
Jit Kong Cheong, … , Andrew Thorburn, David M. Virshup
Jit Kong Cheong, … , Andrew Thorburn, David M. Virshup
Published March 23, 2015
Citation Information: J Clin Invest. 2015;125(4):1401-1418. https://doi.org/10.1172/JCI78018.
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Research Article Oncology

Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

Abstract

Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS–induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS–driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS–driven cancers.

Authors

Jit Kong Cheong, Fuquan Zhang, Pei Jou Chua, Boon Huat Bay, Andrew Thorburn, David M. Virshup

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Figure 1

CK1α suppresses RAS-induced basal autophagy.

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CK1α suppresses RAS-induced basal autophagy. (A) LC3B and CK1α are upreg...
(A) LC3B and CK1α are upregulated in BJ fibroblasts with oncogenic H-RASV12, regardless of p53 status. Cells were treated with 4-OHT for 48 hours. endo, endogenous. (B) CK1α depletion further upregulates LC3B-II. Eight hours after siRNA transfection, 4-OHT was added to cells for an additional 72 hours. (C) CK1α knockdown specifically increases LC3B-II levels. Cells were analyzed 48 hours after RNAi treatment. (D) D4476 potently increases LC3B-II levels in a mutant RAS–specific manner. Forty-eight hours after 4-OHT treatment, cell were incubated with DMSO or D4476 for an additional 6 hours. (E) D4476 increases LC3B-II in multiple RAS-mutant cell lines. Cells were treated with DMSO or D4476 (6 hours). (F) CK1α overexpression downregulates RAS-induced LC3B-II expression. Cells were transfected with pcDNA3.1 (EV, empty vector) or pcDNA3.1-CK1α-3HA. (G) CK1α inhibition increases LC3B-II–associated autophagosomes. Cells were exposed to the indicated siRNAs, DMSO, or D4476 (6 hours). Representative immunofluorescence images from three independent experiments are shown. DAPI stains the nuclei. Scale bars: 20 μm. (H) Quantification of LC3B foci–stained cells from G, using MetaMorph software (mean ± SD). One-way ANOVA with Dunnett’s test (for RNAi treatment) and Student’s t test (for drug treatment) were used to analyze statistical significance; ***P < 0.001; †P < 0.0001. Values are mean ± SD from 3 independent experiments. Fold expression change in the proteins of interest after normalization is indicated below protein blots. NS, nonspecific; hTERT, human telomerase catalytic subunit; st, SV40 small t antigen.