Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers
Jit Kong Cheong, Fuquan Zhang, Pei Jou Chua, Boon Huat Bay, Andrew Thorburn, David M. Virshup
Jit Kong Cheong, Fuquan Zhang, Pei Jou Chua, Boon Huat Bay, Andrew Thorburn, David M. Virshup
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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.