MOLECULAR & CELLULAR ONCOLOGY 2016, VOL. 3, NO. 3, e1045117 (3 pages) http://dx.doi.org/10.1080/23723556.2015.1045117
AUTHOR’S VIEW
Keeping autophagy in cheCK1 Jit Kong Cheong and David M. Virshup Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore
ABSTRACT
ARTICLE HISTORY
Mutant RAS-driven cancer cells cope with proliferative stress by increasing basal autophagy to maintain protein/organelle and energy homeostasis. We recently demonstrated that casein kinase 1 alpha (CK1a), a therapeutically tractable enzyme, is critical for fine-tuning the transcriptional regulation of mutant RASinduced autophagy and the development of mutant RAS-driven cancers.
Received 17 April 2015 Revised 20 April 2015 Accepted 21 April 2015 KEYWORDS
Autophagy; cancer; casein kinase 1; phosphorylation; RAS
Activating mutations in the RAS oncogene occur frequently in human cancers but are difficult to therapeutically target. In some cancers mutant RAS enhances autophagy,1 a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses, although the mechanism is not fully understood. We recently reported that casein kinase 1 alpha (CK1a), a ubiquitously expressed kinase, is a key negative feedback regulator of mutant RAS-induced autophagy gene transactivation.2 RNA interference (RNAi) or pharmacologic inhibition of CK1a upregulates basal autophagy and enhances autophagic flux in mutant RAS-driven human cancer cell lines. We found that the phosphoinositide 3-kinase–AKT–mammalian target of rapamycin (PI3K-AKT-mTOR) pathway of mutant RAS signaling increases the abundance of CK1a protein, which in turn phosphorylates and decreases the nuclear abundance of the forkhead box O3A (FOXO3A) transcription factor to suppress FOXO3A-mediated transactivation of autophagy-related genes. Importantly, CK1a and autophagy inhibitors [e.g., D4476:chloroquine (CQ)] combine in vitro and in vivo to block mutant RAS-driven cancer progression. These data offer important insights into CK1a-dependent transcriptional regulation of autophagy genes and new therapeutic combinations that are active in mutant RAS-driven cancers. The major finding of our study, that CK1a is a critical factor for the growth and/or survival of mutant RAS-driven cancer cells, is consistent with recent findings of Bowman and coworkers.3 Using genetically engineered mouse models, Bowman et al. elegantly showed that CK1a is required for mutant RASdriven lung tumorigenesis. They found that CK1a phosphorylates the Fas-associated death domain (FADD) protein to facilitate its nuclear entry for promotion of mitosis in mutant RASdriven lung cancer cells. Whereas we demonstrate that FOXO3A is the key CK1a-regulated substrate in mutant RASdriven colon and bladder cancer cells, they showed that CK1aphosphorylated FADD is absolutely required for mutant RASdriven lung tumorigenesis. Notably, although several studies
CONTACT Jit Kong Cheong © 2016 Taylor & Francis Group, LLC
have concluded that the tumor protein p53 (TP53, best known as p53) is negatively regulated by CK1a, no activation of p53 signaling was found in the current studies. The key substrate(s) of CK1a that promote mutant RAS-driven tumorigenesis differ among published studies. This may be due to specific differences in cell type and tissue of origin, and/or the different cooccurring oncogenic mutations present in different cancers, reflecting the complexity of mutant RAS signaling networks in cancers. It remains unclear how the combination of CK1a and autophagy inhibitors effectively induces cancer cell death. Could FADD play a vital role in mediating the death of mutant RAS-driven colon and bladder cancer cells triggered by build up of autophagic vesicles as a result of ineffective autophagy? Although Bowman et al. demonstrate that FADD plays a non-apoptotic role in mutant RAS-driven lung cancer cells, in several other reports FADD has been shown to be a key cytosolic autophagosome associatedmolecule that drives the activation of cell death pathways. FADD can be recruited to autophagosomes by the autophagy-related protein 5 (ATG5), where it interacts with and activates caspase 8 to trigger apoptosis.4 Alternatively, FADD can form a complex with ATG5, ATG12, and receptor-interacting protein kinases 1/3 (RIPK1/3) at autophagosomes in the absence of caspase 8 to trigger necroptosis.5 We show that depletion of FOXO3A transcription factor is sufficient to block D4476:CQ-induced growth arrest of mutant RASdriven colon and bladder cancer cells. Since FOXO3A is a master transcriptional regulator of a large number of core autophagy genes, including microtubule associated light chain 3 (LC3) and ATG12, its removal may lead to a reduction in the overall abundance of FADD-associated autophagosomes and activation of cell death in CK1a-depleted, autophagy-inhibited mutant RAS-driven cancer cells. We speculate that the knockdown of FADD and/or caspase 8 may also block cell death activation in these cancer cells.
Email: [email protected]; David M. Virshup
Email: [email protected]
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J. K. CHEONG AND D. M. VIRSHUP
Figure 1. CK1 kinases are positive or negative regulators of autophagy initiation. The yeast homolog of CK1d/e, Hrr25, induces selective autophagy by phosphorylating the autophagy cargo receptors ATG19 (in nutrient-rich conditions), ATG34 (under nitrogen starvation), and ATG36 (for pexophagy). CK1a and CK1g2, on the other hand, were both identified as negative regulators of basal autophagy. CK1a, a downstream effector of mutant RAS, phosphorylates and destabilizes nuclear FOXO3A to limit transcriptional hyperactivation of basal autophagy. The mechanism that underlies suppression of autophagy by CK1g2 is currently unknown. ATG, autophagy-related; CK1, casein kinase 1; FOXO3A, forkhead box O3A; P, phosphorylation; PI3K-AKT-mTOR, phosphoinositide 3-kinase–AKT–mammalian target of rapamycin.
In a broader context, autophagy joins a long list of cellular processes that are regulated by the CK1 family of serine/threonine kinases.6 There are 6 human CK1 isoforms that are encoded by distinct genes (a, d, e, g1, g2, and g3). CK1a and CK1g2 were both identified in a kinome RNAi screen as negative regulators of basal autophagy.7 Although we have further characterized the role of CK1a as a suppressor of mutant RASinduced basal autophagy via transcriptional control,2 it remains unclear how the membrane-associated CK1g regulates basal autophagy in the cell. Conversely, the budding yeast CK1d/e homolog, Hrr25, activates selective autophagy pathways by phosphorylating the cargo receptors ATG19, ATG34, and ATG36.8,9 The Hrr25-dependent phosphorylation of ATG19, ATG34, and ATG36 in different cellular contexts promotes their interaction with the adaptor protein ATG11. ATG11 in turn directs cytosolic aminopeptidase-bound ATG19 (in nutrient-rich conditions) or ATG34 (under nitrogen starvation) receptors to the digestive vacuole of budding yeast. ATG11 may also recruit core ATG proteins to initiate the synthesis and extension of autophagosome membranes to mediate the clearance of superfluous peroxisomes (also known as pexophagy). In addition, Hrr25 promotes clathrin-mediated endocytosis in budding yeast by phosphorylating the early endocytic Eps15-like protein Ede1 to regulate the rate of endocytic site initiation.10 This may provide yet another avenue by which Hrr25 positively regulates autophagy initiation using clathrin-mediated endocytosis as a means to feed parts of the plasma membrane to the growing pool of autophagosomes. Although we did not observe a decrease in LC3B abundance in CK1d/e-depleted or CK1d/e-inhibited RASdriven cancer cells growing in optimal culture conditions,2 the involvement of CK1d/e in the activation of selective autophagy in mammalian cells under other pathophysiologic conditions cannot be ruled out.
Given that CK1 isoforms may differentially regulate autophagy in a context-dependent manner (Fig. 1), the optimal use of CK1 inhibitors alone or in combination with autophagy inhibitors for the treatment of human malignancies such as mutant RAS-driven cancers will require additional study. The development of more potent small molecules or biologics to specifically inhibit each CK1 isoform will aid in applying this new knowledge in the effective treatment of specific cancers.
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
Funding This work was supported by the Duke-NUS Signature Research Program funded by the Agency for Science, Technology and Research, Singapore, and the Ministry of Health, Singapore, and a Singapore Translational Research Investigator Award to DMV funded by the National Research Foundation and the National Medical Research Council (NMRC; NMRC/STaR/0017/2013). JKC was supported by an NMRC-CBRG New Investigator Grant (NMRC/BNIG/1078/2012).
References 1. Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, Kamphorst JJ, Chen G, Lemons JM, Karantza V, et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 2011; 25:460-70; PMID:21317241; http://dx.doi. org/10.1101/gad.2016311 2. Cheong JK, Zhang F, Chua PJ, Bay BH, Thorburn A, Virshup DM. Casein kinase 1alpha-dependent feedback loop controls autophagy in RAS-driven cancers. J Clin Invest 2015; 125:1401-18; PMID:25798617; http://dx.doi.org/10.1172/JCI78018 3. Bowman BM, Sebolt KA, Hoff BA, Boes JL, Daniels DL, Heist KA, Galban CJ, Patel RM, Zhang J, Beer DG, et al. Phosphorylation of
MOLECULAR & CELLULAR ONCOLOGY
4.
5.
6.
7.
FADD by the kinase CK1alpha promotes KRASG12D-induced lung cancer. Sci Signal 2015; 8:ra9; PMID:25628462; http://dx.doi.org/ 10.1126/scisignal.2005607 Pyo JO, Jang MH, Kwon YK, Lee HJ, Jun JI, Woo HN, Cho DH, Choi B, Lee H, Kim JH, et al. Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death. J Biol Chem 2005; 280:20722-9; PMID:15778222; http://dx.doi.org/10.1074/jbc.M413934200 Basit F, Cristofanon S, Fulda S. Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes. Cell Death Differ 2013; 20:1161-73; PMID:23744296; http://dx.doi.org/10.1038/cdd.2013.45 Cheong JK, Virshup DM. Casein kinase 1: complexity in the family. Int J Biochem Cell Biol 2011; 43:465-9; PMID:21145983; http://dx.doi. org/10.1016/j.biocel.2010.12.004 Szyniarowski P, Corcelle-Termeau E, Farkas T, Hoyer-Hansen M, Nylandsted J, Kallunki T, Jaattela M. A comprehensive siRNA screen
e1045117-3
for kinases that suppress macroautophagy in optimal growth conditions. Autophagy 2011; 7:892-903; PMID:21508686; http://dx.doi.org/ 10.4161/auto.7.8.15770 8. Pfaffenwimmer T, Reiter W, Brach T, Nogellova V, Papinski D, Schuschnig M, Abert C, Ammerer G, Martens S, Kraft C. Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19. EMBO Rep 2014; 15:862-70; PMID:24968893; http:// dx.doi.org/10.15252/embr.201438932 9. Tanaka C, Tan LJ, Mochida K, Kirisako H, Koizumi M, Asai E, SakohNakatogawa M, Ohsumi Y, Nakatogawa H. Hrr25 triggers selective autophagy-related pathways by phosphorylating receptor proteins. J Cell Biol 2014; 207:91-105; PMID:25287303; http://dx.doi.org/ 10.1083/jcb.201402128 10. Peng Y, Grassart A, Lu R, Wong CC, Yates J 3rd, Barnes G, Drubin DG. Casein kinase 1 promotes initiation of clathrin-mediated endocytosis. Dev Cell 2015; 32:231-40; PMID:25625208; http://dx.doi.org/ 10.1016/j.devcel.2014.11.014
AUTHOR’S VIEW
Keeping autophagy in cheCK1 Jit Kong Cheong and David M. Virshup Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore
ABSTRACT
ARTICLE HISTORY
Mutant RAS-driven cancer cells cope with proliferative stress by increasing basal autophagy to maintain protein/organelle and energy homeostasis. We recently demonstrated that casein kinase 1 alpha (CK1a), a therapeutically tractable enzyme, is critical for fine-tuning the transcriptional regulation of mutant RASinduced autophagy and the development of mutant RAS-driven cancers.
Received 17 April 2015 Revised 20 April 2015 Accepted 21 April 2015 KEYWORDS
Autophagy; cancer; casein kinase 1; phosphorylation; RAS
Activating mutations in the RAS oncogene occur frequently in human cancers but are difficult to therapeutically target. In some cancers mutant RAS enhances autophagy,1 a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses, although the mechanism is not fully understood. We recently reported that casein kinase 1 alpha (CK1a), a ubiquitously expressed kinase, is a key negative feedback regulator of mutant RAS-induced autophagy gene transactivation.2 RNA interference (RNAi) or pharmacologic inhibition of CK1a upregulates basal autophagy and enhances autophagic flux in mutant RAS-driven human cancer cell lines. We found that the phosphoinositide 3-kinase–AKT–mammalian target of rapamycin (PI3K-AKT-mTOR) pathway of mutant RAS signaling increases the abundance of CK1a protein, which in turn phosphorylates and decreases the nuclear abundance of the forkhead box O3A (FOXO3A) transcription factor to suppress FOXO3A-mediated transactivation of autophagy-related genes. Importantly, CK1a and autophagy inhibitors [e.g., D4476:chloroquine (CQ)] combine in vitro and in vivo to block mutant RAS-driven cancer progression. These data offer important insights into CK1a-dependent transcriptional regulation of autophagy genes and new therapeutic combinations that are active in mutant RAS-driven cancers. The major finding of our study, that CK1a is a critical factor for the growth and/or survival of mutant RAS-driven cancer cells, is consistent with recent findings of Bowman and coworkers.3 Using genetically engineered mouse models, Bowman et al. elegantly showed that CK1a is required for mutant RASdriven lung tumorigenesis. They found that CK1a phosphorylates the Fas-associated death domain (FADD) protein to facilitate its nuclear entry for promotion of mitosis in mutant RASdriven lung cancer cells. Whereas we demonstrate that FOXO3A is the key CK1a-regulated substrate in mutant RASdriven colon and bladder cancer cells, they showed that CK1aphosphorylated FADD is absolutely required for mutant RASdriven lung tumorigenesis. Notably, although several studies
CONTACT Jit Kong Cheong © 2016 Taylor & Francis Group, LLC
have concluded that the tumor protein p53 (TP53, best known as p53) is negatively regulated by CK1a, no activation of p53 signaling was found in the current studies. The key substrate(s) of CK1a that promote mutant RAS-driven tumorigenesis differ among published studies. This may be due to specific differences in cell type and tissue of origin, and/or the different cooccurring oncogenic mutations present in different cancers, reflecting the complexity of mutant RAS signaling networks in cancers. It remains unclear how the combination of CK1a and autophagy inhibitors effectively induces cancer cell death. Could FADD play a vital role in mediating the death of mutant RAS-driven colon and bladder cancer cells triggered by build up of autophagic vesicles as a result of ineffective autophagy? Although Bowman et al. demonstrate that FADD plays a non-apoptotic role in mutant RAS-driven lung cancer cells, in several other reports FADD has been shown to be a key cytosolic autophagosome associatedmolecule that drives the activation of cell death pathways. FADD can be recruited to autophagosomes by the autophagy-related protein 5 (ATG5), where it interacts with and activates caspase 8 to trigger apoptosis.4 Alternatively, FADD can form a complex with ATG5, ATG12, and receptor-interacting protein kinases 1/3 (RIPK1/3) at autophagosomes in the absence of caspase 8 to trigger necroptosis.5 We show that depletion of FOXO3A transcription factor is sufficient to block D4476:CQ-induced growth arrest of mutant RASdriven colon and bladder cancer cells. Since FOXO3A is a master transcriptional regulator of a large number of core autophagy genes, including microtubule associated light chain 3 (LC3) and ATG12, its removal may lead to a reduction in the overall abundance of FADD-associated autophagosomes and activation of cell death in CK1a-depleted, autophagy-inhibited mutant RAS-driven cancer cells. We speculate that the knockdown of FADD and/or caspase 8 may also block cell death activation in these cancer cells.
Email: [email protected]; David M. Virshup
Email: [email protected]
e1045117-2
J. K. CHEONG AND D. M. VIRSHUP
Figure 1. CK1 kinases are positive or negative regulators of autophagy initiation. The yeast homolog of CK1d/e, Hrr25, induces selective autophagy by phosphorylating the autophagy cargo receptors ATG19 (in nutrient-rich conditions), ATG34 (under nitrogen starvation), and ATG36 (for pexophagy). CK1a and CK1g2, on the other hand, were both identified as negative regulators of basal autophagy. CK1a, a downstream effector of mutant RAS, phosphorylates and destabilizes nuclear FOXO3A to limit transcriptional hyperactivation of basal autophagy. The mechanism that underlies suppression of autophagy by CK1g2 is currently unknown. ATG, autophagy-related; CK1, casein kinase 1; FOXO3A, forkhead box O3A; P, phosphorylation; PI3K-AKT-mTOR, phosphoinositide 3-kinase–AKT–mammalian target of rapamycin.
In a broader context, autophagy joins a long list of cellular processes that are regulated by the CK1 family of serine/threonine kinases.6 There are 6 human CK1 isoforms that are encoded by distinct genes (a, d, e, g1, g2, and g3). CK1a and CK1g2 were both identified in a kinome RNAi screen as negative regulators of basal autophagy.7 Although we have further characterized the role of CK1a as a suppressor of mutant RASinduced basal autophagy via transcriptional control,2 it remains unclear how the membrane-associated CK1g regulates basal autophagy in the cell. Conversely, the budding yeast CK1d/e homolog, Hrr25, activates selective autophagy pathways by phosphorylating the cargo receptors ATG19, ATG34, and ATG36.8,9 The Hrr25-dependent phosphorylation of ATG19, ATG34, and ATG36 in different cellular contexts promotes their interaction with the adaptor protein ATG11. ATG11 in turn directs cytosolic aminopeptidase-bound ATG19 (in nutrient-rich conditions) or ATG34 (under nitrogen starvation) receptors to the digestive vacuole of budding yeast. ATG11 may also recruit core ATG proteins to initiate the synthesis and extension of autophagosome membranes to mediate the clearance of superfluous peroxisomes (also known as pexophagy). In addition, Hrr25 promotes clathrin-mediated endocytosis in budding yeast by phosphorylating the early endocytic Eps15-like protein Ede1 to regulate the rate of endocytic site initiation.10 This may provide yet another avenue by which Hrr25 positively regulates autophagy initiation using clathrin-mediated endocytosis as a means to feed parts of the plasma membrane to the growing pool of autophagosomes. Although we did not observe a decrease in LC3B abundance in CK1d/e-depleted or CK1d/e-inhibited RASdriven cancer cells growing in optimal culture conditions,2 the involvement of CK1d/e in the activation of selective autophagy in mammalian cells under other pathophysiologic conditions cannot be ruled out.
Given that CK1 isoforms may differentially regulate autophagy in a context-dependent manner (Fig. 1), the optimal use of CK1 inhibitors alone or in combination with autophagy inhibitors for the treatment of human malignancies such as mutant RAS-driven cancers will require additional study. The development of more potent small molecules or biologics to specifically inhibit each CK1 isoform will aid in applying this new knowledge in the effective treatment of specific cancers.
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
Funding This work was supported by the Duke-NUS Signature Research Program funded by the Agency for Science, Technology and Research, Singapore, and the Ministry of Health, Singapore, and a Singapore Translational Research Investigator Award to DMV funded by the National Research Foundation and the National Medical Research Council (NMRC; NMRC/STaR/0017/2013). JKC was supported by an NMRC-CBRG New Investigator Grant (NMRC/BNIG/1078/2012).
References 1. Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, Kamphorst JJ, Chen G, Lemons JM, Karantza V, et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 2011; 25:460-70; PMID:21317241; http://dx.doi. org/10.1101/gad.2016311 2. Cheong JK, Zhang F, Chua PJ, Bay BH, Thorburn A, Virshup DM. Casein kinase 1alpha-dependent feedback loop controls autophagy in RAS-driven cancers. J Clin Invest 2015; 125:1401-18; PMID:25798617; http://dx.doi.org/10.1172/JCI78018 3. Bowman BM, Sebolt KA, Hoff BA, Boes JL, Daniels DL, Heist KA, Galban CJ, Patel RM, Zhang J, Beer DG, et al. Phosphorylation of
MOLECULAR & CELLULAR ONCOLOGY
4.
5.
6.
7.
FADD by the kinase CK1alpha promotes KRASG12D-induced lung cancer. Sci Signal 2015; 8:ra9; PMID:25628462; http://dx.doi.org/ 10.1126/scisignal.2005607 Pyo JO, Jang MH, Kwon YK, Lee HJ, Jun JI, Woo HN, Cho DH, Choi B, Lee H, Kim JH, et al. Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death. J Biol Chem 2005; 280:20722-9; PMID:15778222; http://dx.doi.org/10.1074/jbc.M413934200 Basit F, Cristofanon S, Fulda S. Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes. Cell Death Differ 2013; 20:1161-73; PMID:23744296; http://dx.doi.org/10.1038/cdd.2013.45 Cheong JK, Virshup DM. Casein kinase 1: complexity in the family. Int J Biochem Cell Biol 2011; 43:465-9; PMID:21145983; http://dx.doi. org/10.1016/j.biocel.2010.12.004 Szyniarowski P, Corcelle-Termeau E, Farkas T, Hoyer-Hansen M, Nylandsted J, Kallunki T, Jaattela M. A comprehensive siRNA screen
e1045117-3
for kinases that suppress macroautophagy in optimal growth conditions. Autophagy 2011; 7:892-903; PMID:21508686; http://dx.doi.org/ 10.4161/auto.7.8.15770 8. Pfaffenwimmer T, Reiter W, Brach T, Nogellova V, Papinski D, Schuschnig M, Abert C, Ammerer G, Martens S, Kraft C. Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19. EMBO Rep 2014; 15:862-70; PMID:24968893; http:// dx.doi.org/10.15252/embr.201438932 9. Tanaka C, Tan LJ, Mochida K, Kirisako H, Koizumi M, Asai E, SakohNakatogawa M, Ohsumi Y, Nakatogawa H. Hrr25 triggers selective autophagy-related pathways by phosphorylating receptor proteins. J Cell Biol 2014; 207:91-105; PMID:25287303; http://dx.doi.org/ 10.1083/jcb.201402128 10. Peng Y, Grassart A, Lu R, Wong CC, Yates J 3rd, Barnes G, Drubin DG. Casein kinase 1 promotes initiation of clathrin-mediated endocytosis. Dev Cell 2015; 32:231-40; PMID:25625208; http://dx.doi.org/ 10.1016/j.devcel.2014.11.014
