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 4

CK1α phosphorylates FOXO3A to regulate its protein abundance.

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CK1α phosphorylates FOXO3A to regulate its protein abundance. (A) A cons...
(A) A conserved CK1 phosphorylation motif in FOXO3A. (B) CK1α phosphorylates FOXO3A at S318 and S321 in vivo. Twenty-four hours after transfection with the indicated proteins, HEK293 cells were treated with the indicated siRNAs for an additional 48 hours prior analysis. (C) CK1α is the only cytoplasmic CK1 isoform that phosphorylates FOXO3A in vivo. CK1 isoforms were depleted with the two indicated independent siRNAs (48 hours) prior to analysis. (D) CK1α depletion or inhibition increases FOXO3A protein stability in a mutant K-RAS–dependent manner. Cells were incubated with siCtrl, siCK1α (no. 13 or 14, 48 hours), DMSO, or 5 μM D4476 (6 hours). (E) D4476 reduces FOXO3AS318/321 phosphorylation but increases total FOXO3A abundance only in mutant RAS-driven cancer cells. HT-29 and HCT-116 cells were treated with DMSO or D4476 (6 hours). (F) Knockdown of CK1α upregulates nuclear abundance of FOXO3A. HCT-116 and T24 cells were treated with the indicated siRNAs (48 hours) before analysis. (G) Small molecule regulators of CK1α control nuclear abundance of FOXO3A. HCT-116 cells were treated with the indicated compounds (6 hours) prior to analysis. (H) FOXO3A is required for CK1α regulation of autophagy genes. The indicated FOXO3A-dependent autophagy genes were assessed by qPCR after CK1α and FOXO3A depletion as indicated. Data are mean ± SD of triplicate experiments. Fold expression change in the proteins of interest after normalization is shown below protein blots. ***P < 0.001 by 1-way ANOVA with Dunnett’s test.