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 10

CK1α inhibition synergizes with autophagy inhibition to hyperaccumulate autophagic vesicles and induce cell death in RAS-driven human cancers.

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CK1α inhibition synergizes with autophagy inhibition to hyperaccumulate ...
(A) D4476:CQ elevates LC3B expression in tumor xenografts. LC3B abundance in tumors was assessed by immunostaining and quantitation. A representative section is shown. The insets represent images taken under ×100 magnification in reduced size. Integrated density (IntDen) of LC3B immunostaining in multiple tumor sections (n = 4 per group) was quantified (ImageJ; Student’s unpaired t test). Scale bars: 200 μm. (B) D4476:CQ induces accumulation of autophagic vesicles (AV) in tumor xenografts. Representative transmission electron micrographs of multiple tumor xenografts are shown. Scale bars: 2 μm (blue), 0.2 μm (insets). Yellow arrows, autophagic vesicles; red N, nucleus; white arrow, mitochondria. (C) D4476:CQ upregulates FOXO3A, LC3B-II, and cleaved PARP in tumor xenografts. Representative immunoblots of xenograft lysates are shown. (D) D4476:CQ induces cell death in tumor xenografts. Drug-treated tumor xenografts were analyzed by TUNEL staining (see Methods). TUNEL-positive and TUNEL-negative immunostained xenograft sections were quantified (ImageJ), with data shown in the stacked bar chart. Scale bars: 50 μm. Data are mean ± SD of 4 xenografts per group. (E) D4476:CQ upregulates FOXO3A-responsive autophagy-related genes. qPCR profiling of xenografts after the indicated therapy is shown. Data are mean ± SD of triplicate experiments and presented as fold expression change relative to controls (1-way ANOVA with Dunnett’s test). Fold expression change in the proteins of interest after normalization is shown below protein blots. **P < 0.01; ***P < 0.001.