BBA - Molecular Cell Research 1868 (2021) 119059

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BBA - Molecular Cell Research

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Review ☆ The role of GSK3 in metabolic pathway perturbations in cancer

David Papadopoli a,b,*, Michael Pollak a,b,c, Ivan Topisirovic a,b,c,d a Lady Davis Institute for Medical Research, 3755 Chemin de la Cote-Sainte-Catherine,ˆ Montr´eal, QC H3T 1E2, Canada b Gerald Bronfman Department of Oncology, McGill University, 5100 Maisonneuve Blvd West, Montr´eal, QC H4A 3T2, Canada c Department of Medicine, Division of Experimental Medicine, McGill University, 1001 D´ecarie Blvd, Montr´eal, QC H4A 3J1, Canada d Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montr´eal, QC H3G 1Y6, Canada

ARTICLE INFO ABSTRACT

Keywords: Malignant transformation and tumor progression are accompanied by significant perturbations in metabolic GSK3 programs. As such, cancer cells support high ATP turnover to construct the building blocks needed to fuel Cancer neoplastic growth. The coordination of metabolic networks in malignant cells is dependent on the collaboration mTOR with cellular signaling pathways. synthase 3 (GSK3) lies at the convergence of several signaling AMPK axes, including the PI3K/AKT/mTOR, AMPK, and Wnt pathways, which influence cancer initiation, progression and therapeutic responses. Accordingly, GSK3 modulates metabolic processes, including protein and lipid syn­ thesis, glucose and mitochondrial metabolism, as well as autophagy. In this review, we highlight current knowledge of the role of GSK3 in metabolic perturbations in cancer.

1. Introduction their structural similarities, the GSK3 isoforms are non-redundant [6]. Homozygous knockout mice of the more widely studied isoform, GSK3B, Cellular proliferation and survival require coordination of multifac­ are not viable due to, at least in part, induction of apoptosis in the liver eted signaling events. kinase 3 (GSK3) is a / [12]. However, GSK3A knockout mice are viable but exhibit impaired threonine kinase with a large number of established and putative sub­ spermatogenesis [13]. In addition, GSK3A knockouts demonstrate strates [1,2] that are implicated in various cellular functions [3]. elevated insulin sensitivity and increased hepatic glycogen storage [14]. Although originally discovered as a regulator of glycogen synthase, In mammals, GSK3α is inhibited by at Ser21 and acti­ GSK3 is involved in modulating numerous processes, including meta­ vated by Tyr279 phosphorylation, while phosphorylation at Ser9 and bolism, proliferation, apoptosis, autophagy, development, and differ­ Tyr216 signify suppression and activation of GSK3β, respectively [15]. entiation [4]. Phosphorylation of a target by GSK3 is often preceded by GSK3α and GSK3β are both inhibited by phosphorylation by protein priming , such as A (PKA), (PKC), kinase B (PKB/AKT) [16], 90-kDa (RSK) [17–19], protein kinases CK1 and CK2, and -dependent kinase-5 (CDK-5) 70-kDa ribosomal S6 kinase (S6K) [17,18], and PKA [15]. In contrast, [5,6]. GSK3-mediated phosphorylation frequently leads to inactivation PKC selectively inhibits GSK3β, while not appearing to effect GSK3α and proteasomal degradation of its targets [4]. Based on its broad [20]. The activity of GSK3 is also positively regulated through dephos­ function, GSK3 has been linked to several pathologies including cancer, phorylation of its inhibitory sites. The protein 1 (PP1) de­ diabetes, mood disorders, atherosclerosis, Alzheimer's disease, and phosphorylates Ser9 and activates GSK3β [21–23]. In turn, GSK3 Parkinson's disease, among others [4,7,8]. GSK3 exists in two isoforms collaborates with CK2 to inactivate inhibitor-2 GSK3α and GSK3β (encoded by GSK3A and GSK3B genes) [9]. Although (PPI2), a negative regulator of PP1 [23–26]. Thus, GSK3 attenuates GSK3 isoforms have unique N- and C-terminal regions, they share a the inhibition of PP1, thereby enhancing its own activity through a highly conserved catalytic domain (98%) [4,10]. Both isoforms are positive feedback loop [27]. In addition, CK2 and GSK3β also cooperate ubiquitously expressed, with highest expression in the brain and lowest to phosphorylate the phosphatase and tensin homologue (PTEN) in the pancreas, according to the Human Protein Atlas [11]. Despite [28,29], although the effect is unclear [30].

☆ This article is part of a Special Issue entitled: GSK-3 and related kinases in cancer, neurological and other disorders edited by James McCubrey, Agnieszka Gizak and Dariusz Rakus. * Corresponding author at: Lady Davis Institute for Medical Research, 3755 Chemin de la Cote-Sainte-Catherine,ˆ Montr´eal, QC H3T 1E2, Canada. E-mail address: [email protected] (D. Papadopoli). https://doi.org/10.1016/j.bbamcr.2021.119059 Received 16 February 2021; Received in revised form 16 April 2021; Accepted 17 April 2021 Available online 12 May 2021 0167-4889/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/). D. Papadopoli et al. BBA - Molecular Cell Research 1868 (2021) 119059

Aside from phosphorylation, the activity of GSK3 is also dependent the translation of nuclear-encoded mRNAs encoding mitochondrial on its localization within cells. For instance, GSK3 targets transcription factors through inhibition of 4E-BPs [55]. The mTORC1/4E-BP axis also factors, such as c-MYC [31], SNAIL [32], and β-catenin [33], which regulates mitochondrial dynamics through MTFP1 [56]. In contrast, translocate from the cytosol to the nucleus. GSK3β was also suggested to mTORC2 is involved in the regulation of cytoskeleton and cell migration localize to mitochondria under stress, including ischemia/reperfusion in through PKCα [57]. mTORC2 also phosphorylates the hydrophobic the rat heart [34]. Conversely, GSK3α was reported to localize in nu­ motif (surrounding Ser473 in humans) of AKT, which controls both cleus, but not in mitochondria [35]. Collectively, these findings glucose and lipid metabolism [58–60], and directs signaling through the demonstrate distinct regulation and functional non-redundancy of GSK3 mitochondrial-associated ER membrane (MAM) to control mitochon­ isoforms. drial physiology [58–61]. In addition, mTORC2 controls ion transport In light of its involvement with signaling pathways implicated in through serum and glucocorticoid-regulated kinase 1 (SGK1) [62]. Both cancer, it is not surprising that GSK3 plays a prominent role in neoplasia. AKT and SGK1 negatively regulate apoptosis through the inhibition of To this end, GSK3 isoforms appear to play a dual role in cancer, whereby forkhead box protein O1/O3A (FOX01/3A) [57]. As a result, the PI3K/ both tumor promoting and tumor suppressive effects of GSK3 have been AKT/mTOR signaling modulates several metabolic nodes required by reported [36]. For instance, high GSK3 expression is associated with cancer cells to meet their high proliferative demands and promote their reduced relapse-free survival in breast cancer and GSK3 inhibitors survival and proliferation. suppress breast tumor growth in pre-clinical models [37]. In contrast, mTOR signaling is tightly regulated under periods of energetic stress AKT-dependent inhibition of GSK3 leads to a de-repression of GSK3 which at least in part occurs through the AMP-activated protein kinase substrates such as SNAIL, thus promoting breast tumorigenesis and (AMPK), a heterotrimeric serine/threonine kinase that is activated in disease progression [38]. These and similar studies therefore suggest response to metabolic stress and inhibited when ATP levels are high that the outcome of aberrant GSK3 activity in neoplasia is likely the [63]. Specifically,AMPK is activated by high AMP/ADP and through the result of effects on multiple signaling pathways, which are distinctively phosphorylation of liver kinase B1 (LKB1), -dependent pro­ affected through genetic and environmental alterations found in tein kinase kinase β (CAMKKβ), or TGF-β-activated kinase 1 (TAK-1) different cancer types. While Duda et al. provide an overarching review [63]. AMPK is also regulated independently of adenylate charge, of differential GSK3 involvement across a variety of malignancies [36], whereby glucose deprivation via the loss of fructose-1,6-bisphosphate herein we will focus on the potential roles of GSK3 in metabolic (FBP) binding to aldolase, allows for the activation of AMPK through reprogramming in cancer. the recruitment of Axin-LKB1 complex to the aldolase/V-ATPase/ Ragulator complex on [64]. Finally AMPK can be induced 2. GSK3: orcestrator of metabolic signaling pathways by ROS [65]. In general, AMPK promotes energy homeostasis by inhibiting ATP-consuming processes. It negatively regulates fatty acid 2.1. PI3K/AKT/mTOR and AMPK cross-talk biosynthesis through inhibition of acetyl-CoA carboxylase [66,67] and SREBP1 [68]. Due to high energy demand, protein synthesis is also GSK3 is a key effector of PI3K/AKT signaling [39]. Class I phos­ heavily restricted under periods of metabolic stress. AMPK inhibits phatidylinositol 3-kinases (PI3K) convert phosphoinositol 4,5-bisphos­ mRNA translation by suppressing mTORC1 signaling through the phate (PIP2) to phosphoinositol 3,4,5-bisphosphate (PIP3) and are phosphorylation of TSC2 [69] and the regulatory-associated protein of typically activated by receptor tyrosine kinases (RTK) such as insulin mTOR (RAPTOR) [70]. AMPK also suppresses translational elongation and insulin-like growth factor receptor [40]. PI3K signaling is frequently through the inhibition of the eukaryotic elongation factor 2 kinase dysregulated in cancer via e.g. activating mutations in PIK3CA and (eEF2K) [71]. In turn, AMPK promotes catabolic reactions such as lipid AKT1/2/3 or inactivating mutations in PTEN [41,42]. PI3K-produced oxidation, glycolysis, autophagy, and mitochondrial respiration [63]. PIP3, facilitates the phosphoinositide-dependent kinase 1 (PDK1)- Collectively, the activities of the PI3K/AKT/mTOR pathway and AMPK mediated phosphorylation of AKT [41]. Conversely, PTEN inhibits AKT are tightly orchestrated based on nutrient availability to maintain en­ by converting PIP3 to PIP2 [43]. AKT activates the mechanistic target of ergy homeostasis of the cell, and when dysregulated, are thought to play rapamycin complex 1 (mTORC1) through the inhibition of the TSC a major role in metabolic reprogramming in cancer. (tuberous sclerosis complex) [44], and subsequent activation of Ras homologue enriched in brain (RHEB) [45]. mTOR exists in two com­ 2.2. GSK3 orchestrates PI3K/AKT/mTOR and AMPK signaling plexes: mTORC1 and mTORC2. mTORC1 is a master regulator of anabolic processes needed for cell proliferation and growth [46]. For GSK3 activity is coordinated with the PI3K/AKT/mTOR pathway instance, mTORC1 induces protein synthesis by phosphorylating a through complex cross-talk mechanisms (Fig. 1). To this end, AKT is the number of factors involved in regulation of mRNA translation [47]. This most well studied regulator of GSK3, as AKT inhibits both GSK3α and includes the phosphorylation and inactivation of the eukaryotic trans­ GSK3β [16], thereby stimulating glycogen synthesis in response to in­ lation initiation factor 4E (eIF4E)-binding proteins (4E-BP1–3 in mam­ sulin [16,72]. Similarly, S6K1 acts to suppress GSK3, particularly in cells mals) that, in their unphosphorylated form, inhibit assembly of the with dysfunctional TSC1/TSC2, which exhibit high mTORC1 activity eukaryotic translation initiation factor 4F (eIF4F) complex. Moreover, [73]. In addition, mTORC1 has been reported to control GSK3β-medi­ mTORC1 phosphorylates and activates S6Ks (S6K1 and 2 in mammals) ated transcription of c-MYC and SNAI1 by preventing GSK3β localization [47]. S6Ks phosphorylate a number of proteins implicated in regulation to the nucleus [74]. Conversely, GSK3 controls mTOR activity through of translation including (rpS6), eIF4B, pro­ multiple mechanisms. For instance, S6K1 is a substrate of GSK3 (pri­ grammed cell death 4 (PDCD4) and eukaryotic translation elongation marily GSK3β), whereby it has been reported that GSK3β-mediated factor 2 kinase (eEF2K) [47]. Of note, eEF2K is also phosphorylated phosphorylation of S6K1 at Ser371 promotes its interaction with directly by mTORC1, whereby phosphorylation of eEF2K leads to its mTORC1 to enhance cell proliferation [75]. GSK3β was also suggested to inactivation and increase in translation elongation rates [48]. mTORC1 phosphorylate 4E-BP1 at Thr37/46 [76,77] and RAPTOR at Ser859 to also promotes lipogenesis through SREBP [49,50] and lipin1 [51], and support mTOR signaling [78]. Both GSK3 isoforms were shown to nucleotide biosynthesis through S6K, ATF4, and PRPS2 [52,53]. In phosphorylate the mTORC2 scaffolding protein, rapamycin-insensitive addition, mTORC1 inhibits autophagy, the process by which the cells companion of mTOR (RICTOR) at Thr1695 [79]. This is thought to clear out damaged proteins, organelles, and pathogens, and recycle induce FBXW7-directed degradation of RICTOR thereby suppressing amino acids via the Unc-51 like autophagy activating kinase 1 (ULK1) mTORC2 [79]. [54]. Emerging studies have shown that mTORC1 plays a major role in GSK3 is implicated in regulating cell fate in response to signals controlling mitochondrial functions. For instance, mTORC1 promotes emanating from nutrients and cellular energy status. For example, GSK3

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Insulin, EGF Fig. 1. GSK3 acts as a central node of cellular signaling. Schematic depicting the cross-talk between GSK3 and AMPK, and mTOR signaling. GSK3 is negatively regu­ RTK lated by the PI3K/AKT axis, a pathway that is stimulated by insulin/EGF and is hyper­ activated through oncogenic perturbation of RTK that occur in the wide range of tumors. PI3K GSK3 is modulated by Wnt, which controls β-catenin. GSK3 activity is reduced following the phosphorylation by S6K1, and the cytoplasmic retention by mTORC1. In PDK1 turn, in concert with AMPK, GSK3 stimu­ lates TSC2, a suppressor of mTORC1. Conversely, GSK3 inhibits AMPK. mTORC1 TSC1 TBC1D7 is also directly influenced by GSK3 through DEPTOR PROTOR AKT TSC2 the phosphorylation of RAPTOR whereas mTOR GSK3 modulates mTORC2 via FBXW7- mLST8 RICTOR TSC Complex mediated degradation of RICTOR. GSK3 mSin1 has also been shown to directly modulate GSK3α major effectors of mTORC1 including 4E- mTORC2 BP1 and S6K1. Taken together, GSK3 plays Wnt GSK3β a multifaceted role in coordinating multiple signaling pathways including Wnt, mTOR GSK3 and AMPK. 4E-BP1: eukaryotic translation initiation factor 4E binding protein 1. AKT: AMPKγ AMPKβ . AMPK: AMP-activated RHEB protein kinase. DEPTOR: DEP domain- AMPKα containing mTOR-interacting protein. EGF: 4E-BP1 S6K1 Nuclear epidermal growth factor. FBXW7: F-box and Translocation AMPK WD repeat domain containing 7. GSK3α/β: glycogen synthase kinase 3 α/β. mLST8: mammalian lethal with SEC13 protein 8. DEPTOR RAPTOR mTOR: mechanistic target of rapamycin. mTOR mTORC1: mechanistic target of rapamycin mLST8 complex 1. mTORC2: mechanistic target of PRAS40 rapamycin complex 2. PDK1: phosphoinositide-dependent kinase 1. PI3K: mTORC1 phosphatidylinositol 3-kinase. PRAS40: proline-rich AKT substrate of 40kDa. PRO­ TOR: protein observed with RICTOR. RAPTOR: regulatory-associated protein of mTOR. RHEB: Ras homologue enriched in brain. RICTOR: rapamycin-insensitive com­ panion of mTOR. RTK: receptor tyrosine kinases. Sin1: stress-activated protein kinase-interacting protein 1. S6K1: 70-kDa ribosomal protein S6 kinase 1. TBC1D7: Tre2-Bub2-Cdc16 (TBC) 1 domain family, member 7. TSC1/2: tuberous sclerosis complex 1/2. coordinates with AMPK to repress mTORC1 and maintain energy ho­ promotes the expression of tumor-promoting genes such as VEGF and c- meostasis. An AMPK-priming phosphorylation promotes the GSK3- MYC, c-JUN, , MCL1, and MMPs, among others [83]. The activity mediated phosphorylation and activation of TSC2, which ultimately of β-catenin is regulated by a multiprotein “destruction” complex that impairs mTOR signaling and protein synthesis [80]. Thus, under con­ includes GSK3, adenomatous polyposis coli (APC), Axin, CK1, and ditions that induce AMPK activation, GSK3 restricts mTOR signaling by β-catenin itself [82]. GSK3 phosphorylates β-catenin, targeting it for targeting TSC2. In addition, GSK3 cooperates with AKT to inhibit AMPK proteasomal degradation [82]. Upon activation of Wnt signaling, GSK3 by phosphorylating it at T479 and S485 respectively [81]. Taken is re-organized within the destruction complex by Wnt co-receptor LDL together, these results indicate numerous feedback mechanisms and receptor related protein 5 (LRP5) and 6 (LRP6), which leads to relief of cross-talk between GSK3 and the PI3K/AKT/mTOR signaling [81] that β-catenin phosphorylation [84–87]. Notably, Wnt activates mTOR are likely to play central role in cellular energetics. independently on its effects on β-catenin, but Wnt-dependent stimula­ tion of mTOR requires GSK3, Dvl, Axin and APC [80]. Wnt appears to activate mTOR signaling by inhibiting GSK3-dependent phosphoryla­ 2.3. GSK3 is a central node of Wnt signaling tion of TSC2 and thus abolishing TSC1/2-mediated suppression of mTORC1 [80]. Considering that PI3K/AKT/mTOR and AMPK act as Wingless-related integration site (Wnt) proteins play a fundamental major regulators of cellular metabolism whereas dysregulation of PI3K/ role in GSK3 function and GSK3/mTOR cross-talk [80]. Wnt signaling is AKT/mTOR and Wnt signaling are frequently observed in neoplasia, we involved in the regulation of proliferation, differentiation, apoptosis, will elaborate on potential roles of GSK3 on metabolic reprogramming invasion and EMT and has a prominent roles in embryonic development (Fig. 2). and cancer [4]. Wnt comprises a family of secreted glycoproteins that bind to cell surface receptors such as Frizzled, thus initiating a signaling cascade that activates Dishevelled (Dvl) leading to accumulation of β-catenin in the nucleus [82]. β-catenin is a that

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Glutamine Metabolism

Protein Synthesis Autophagy GLS S6K1 4E-BP1 ULK1 eIF2Bε

GSK3α HK2 Lipid Glucose Biosynthesis GSK3β Metabolism SREBP1 GLUT1 GSK3 ACLY

PGC-1α GS Glycogen TFAM Synthesis DRP1 BiogenesisMitochondrial and Metabolism

Mitochondrial Fission

Fig. 2. Metabolic functions of GSK3. GSK3 plays complex and likely context-dependent roles in metabolism, including the biosynthesis of protein, lipids, and glycogen, nutrient transport/oxidation, and mitochondrial functions. GSK3 is associated with reduced protein synthesis through its regulation of eIF2Bε, a factor involved in ternary complex assembly. Conversely, it has been shown that GSK3 can promote translation through mTOR and directly phosphorylating S6K1 and/or 4E-BP1. GSK3 restricts fatty acid biosynthesis by repressing SREBP1, a master regulator of lipogenic genes, and inhibiting ACLY, a metabolic that catalyzes the breakdown of citrate to generate acetyl-CoA. Conversely, the isoform GSK3α promotes PPARα-dependent accumulation of fatty acids. GSK3 controls glucose metabolism through multiple mechanisms. It phosphorylates glycogen synthase (GS) to impede . GSK3 restricts glucose uptake through the inhibition of GLUT1 and impedes glycolysis through the regulation of HK2. Similarly, GSK3 perturbs glutamine metabolism by attenuating its uptake and oxidation through GLS. In addition, GSK3 controls mitochondrial bioenergetics by inhibiting PGC-1α and TFAM and regulates mitochondrial fission through DRP1. GSK3 also controls autophagy through modulation of ULK1. 4E-BP1: eukaryotic translation initiation factor 4E binding protein 1. ACLY: ATP citrate . DRP1: dynamin-related protein 1. eIF2Bε: eukaryotic translation initiation factor 2B epsilon. GLS: glutaminase. GS: glycogen synthase. GLUT1: glucose transporter 1. HK2: hexokinase 2. S6K1: 70-kDa ribosomal protein S6 kinase 1. PPARα: peroxisome proliferator activated receptor alpha. PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha. SREBP1: sterol regulatory element-binding protein 1. TFAM: mitochondrial transcription factor A. ULK1: Unc-51 like autophagy activating kinase 1.

3. GSK3 and cancer cell metabolism sequential phosphorylation of Ser644 and Ser640, which greatly impair the activity of glycogen synthase [5]. In addition, AMPK also inhibits Cell metabolism is highly rewired in neoplasia. Cancer cells can be glycogen synthase by phosphorylating it at Ser7 [96]. Insulin stimula­ highly glycolytic, even in the presence of ample oxygen [88,89]. These tion reverses the phosphorylation by GSK3 and AMPK on GS, hence observations have been exploited clinically through the use of 18F-deox­ promoting glycogen synthesis [97]. As a result, GSK3 plays a key role in yglucose positron emission tomography (FDG-PET) [90]. It is now well the perturbation of glucose storage. appreciated that cancer cells and tumors display metabolic plasticity, Furthermore, GSK3 influencesglucose import and catabolism. GSK3 known as the capability to adapt metabolic programs to support the inhibits glucose uptake and GLUT1 expression in a TSC2/mTOR- energetic/biosynthetic needs of the cell/tumor in different environ­ dependent manner [98]. Silencing of GSK3 increases glucose con­ ments or cancer states [91]. For instance, cancer cells exploit numerous sumption and the expression of hexokinase 2 (HK2) [99]. In addition, metabolic pathways to generate the ATP needed to supply anabolic activation of GSK3-deficient B cells using anti-CD4 and IL-4 promotes processes to stimulate neoplastic growth and survival of cancer cells glucose consumption and lactate production [100]. mTORC1 down­ [91,92]. Consequently, the coordination of metabolic processes is regulates GSK3α, which suppresses Foxk1/HIF-1α dependent expression crucial for cancer progression. of pro-glycolytic genes [101]. Glucose depletion also perturbs GSK3 activity [102]. When KRAS-transformed fibroblasts,which are known to 3.1. Glycogen/glucose metabolism be heavily dependent on glycolysis [103–105], are grown in low glucose conditions, they exhibit greater GSK3 inhibitory phosphorylation than GSK3 was originally discovered over 40 years ago as a kinase that cells grown in high glucose, and are more sensitive to GSK3 inhibitors inhibits glycogen synthase, the enzyme that catalyzes the transfer of [102]. Inhibition of GSK3 using lithium chloride (LiCl), coincides with glucose from UDP-glucose to glycogen [9,93,94]. In mammals, GSK3 AMPK activation and leads to a stronger anti-proliferative response in phosphorylates glycogen synthase at Ser652 after priming by CK2 at transformed cells compared to controls [102]. Taken together, these Ser656 [95]. The phosphorylation of Ser652 acts as the recognition site findings suggest that GSK3 acts to negatively regulate glucose for the phosphorylation of Ser648, which in turn promotes the metabolism.

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3.2. Mitochondrial metabolism, functions, and morphology neoplasms, whereby cancer cells must closely coordinate mRNA trans­ lation rates with energy metabolism. In support of this point, mTORC1 is Mitochondria play a crucial role in fuelling cancer cells with anabolic a critical regulator of both protein synthesis and cellular metabolism precursors and ATP, as well as controlling apoptosis, in order to sustain [47]. Indeed, emerging studies have stressed importance of mTOR proliferation and survival of cancer cells [106]. GSK3 plays several roles signaling to translational machinery in metabolic reprogramming and in regulating mitochondrial metabolism, which is also controlled adaptations of cancer cells to energetic stress [128–130]. Accordingly, through mTOR. mTORC1 regulates mitochondrial function and GSK3 is expected to exert a profound impact on global protein synthesis biogenesis through translation of nuclear-encoded mitochondria-related as well as the translation of mRNAs encoding metabolic factors via mRNAs (complex I and V, mitochondrial ribosomal proteins, and the modulation of mTORC1 activity. For instance, GSK3 is thought to mitochondrial transcription factor a (TFAM)) [55,107]. In addition, restrict protein synthesis through the activation of TSC2 and consequent mTORC1 regulates transcription of nuclear-encoded mitochondrial suppression of mTORC1 signaling [80]. Nonetheless, GSK3 may also genes through peroxisome-proliferator-activated receptor-γ coactivator- enhance protein synthesis through the phosphorylation of S6K1 [75]. It 1 alpha (PGC-1α) and transcription factor Yin-Yang 1 (YY1) [108]. was also reported that GSK3 may directly phosphorylate and inactivate Intriguingly, it has been reported that insulin promotes mitochondrial 4E-BP1, thus enhancing protein synthesis in renal cancer cells [76,77]. respiration through activation of PI3K/AKT and inactivation of GSK3, in In addition to its effects on mTORC1, GSK3 also regulates translation which GSK3 is considered as a major regulator of insulin-dependent initiation by modulating ternary complex (TC) assembly. The TC is mitochondrial respiration [109]. Insulin also promotes GSK3 inhibi­ comprised of eIF2, initiator tRNA (Met-tRNAi) and GTP and its function tion to suppress caspase activation and apoptosis [109]. In addition, is to deliver initiator tRNAMet (Met-tRNAi) to the 43S pre-initiation GSK3 stimulates the phosphorylation and degradation of PGC-1α, a complex [131]. Upon tRNA delivery and GTP hydrolysis, a multicom­ master regulator of mitochondrial biogenesis and bioenergetics ponent guanine nucleotide exchange factor eIF2B converts GDP associ­ [110–112]. The mitochondrial protease, Omi, alleviates the repression ated eIF2 to its GTP-bound form, thereby recycling TC [132]. GSK3 of GSK3 on PGC-1α, to restore mitochondrial number and expression of phosphorylates Ser535 on eIF2Bε [133,134], which is primed by the mitochondrial components [112]. Inactivation of GSK3 by LiCl upre­ phosphorylation on Ser539 by the dual-specificity, tyrosine-phosphor­ gulates PGC-1α and TFAM [113], as well as the PGC-1α related coac­ ylated and regulated kinase (DYRK) [135]. This leads to decrease in TC tivator PRC [114], which are all involved in mitochondrial biogenesis. recycling and suppression of translation initiation [136]. Inactivation of GSK3 also increases oxygen consumption and mito­ Amino acids have also been demonstrated to indirectly alter GSK3 chondrial number [100]. Overall, these findingsshow that GSK3, and in activity, and vice versa, GSK3 has been shown to regulate amino acid particular GSK3β acts as a negative regulator of mitochondrial meta­ levels and utilization. Increasing the concentration of amino acids in bolism. In addition, GSK3 impacts mitochondrial morphology, which culture media enhances the inhibitory phosphorylation of both GSK3 has been implicated in alterations in mitochondrial functions in cancer isoforms [137]. Branched chain amino acids (BCAA), such as leucine, [115]. GSK3β mediates the phosphorylation and increases activity of stimulate mTOR while inactivating GSK3β [138]. In addition, cancer dynamin-related protein 1 (DRP1) in neuronal cells, whereby DRP1 acts cells can impinge on glutamine metabolism as a source of nitrogen for as a key factor in mitochondrial fission [116,117]. Conversely, GSK3 producing amino acids, and as a source of carbon to fuel bioenergetics inhibition by LiCl has been reported to reduce mitochondrial fission through the TCA cycle [139]. In lung squamous cell carcinoma, inacti­ [118]. Taken together, emerging data show that GSK3 may act a major vation of GSK3β increases the stability of c-MYC and c-JUN, which regulator of mitochondrial functions and dynamics, both of which have promote the expression of glutaminase (GLS), an enzyme that converts been reported to be dysregulated in a variety of cancers [115]. However, glutamine to glutamate [99]. GLS1 overexpression also increases there are some caveats to these studies, as lithium has many effects on inhibitory phosphorylation of GSK3β in hepatocellular carcinoma cells cells aside from GSK3, and the effects on mitochondrial biogenesis may [140]. These data support the idea that GSK3 restricts amino acid be primarily mediated via PGC-1α and its many functions [119]. metabolism in cancer cells. It therefore appears that the availability of amino acids and mTOR signaling are key components that dictate the 3.3. Lipid synthesis impact of GSK3 on protein synthesis and amino acid metabolism.

In addition to glucose, malignant cells require high levels of fatty 3.5. Autophagy acids for processes such as membrane biogenesis [91]. mTORC1 pro­ motes lipid synthesis through the activation of SREBP1, a master regu­ The recycling of cellular components via autophagy has been sug­ lator of the expression of lipogenic genes [49,50] and by repressing the gested to play a complex role in cancer maintenance and progression. inhibitory action of lipin1 [51]. Conversely, GSK3 downregulates the Many autophagy-related genes, including BIF-1 and UVRAG are transcriptional activity of SREBP1c [120] and in concert with the F-box frequently deleted or have reduced expression in tumors [141]. and WD repeat domain-containing 7 (FBW7) E3 ubiquitin stim­ Conversely, the breakdown of cellular material provides the cell with ulates the degradation of SREBP1a protein [121]. In addition, GSK3 metabolic building blocks for anabolic reactions, as well as substrates to impedes lipid synthesis through phosphorylation of ATP-citrate lyase be catabolized for ATP generation [141]. However, autophagy appears (ACLY) at Thr446 and Ser450 [122,123]. Accordingly, inactivation of to play a dual role in cancer progression. Autophagy can suppress tumor GSK3 by LiCl promotes fatty acid accumulation [124]. Interestingly, formation through accumulation of reactive oxidative species, which GSK3α but not GSK3β, has been reported to promote PPARα-mediated induces inflammation,genomic instability, and cell death [141]. On the increase in fatty acid accumulation and uptake in cardiac and liver cells other hand, autophagy provides metabolic resources that enhances [125]. These results suggest that although GSK3 appears to block fatty tumor cell survival at later stages [141]. Upon nutrient deprivation, acid biosynthesis, GSK3α may play a pro-lipogenic role in some contexts. AMPK phosphorylates ULK1 to enhance autophagy to promote cell To this end, the role of GSK3 in perturbations of lipid metabolism in survival [142,143]. In nutrient-replete conditions, mTORC1 phosphor­ cancer remains largely unclear. ylates and inhibits ULK1 and also ATG13, which is another subunit of the ULK complex [143–146]. mTORC1 also inhibits autophagy by 3.4. Protein synthesis and amino acid metabolism regulating the expression of autophagy-related genes through inhibition of TFEB [147], and activation of the acetyltransferase p300 [148]. In Protein synthesis is a cellular process that is tightly regulated due to return, ULK1 can phosphorylate and inhibit RAPTOR, thereby reducing its pivotal role in regulation of gene expression along with its high en­ mTORC1 signaling [149]. It is worth noting that mTORC2 can also ergy demands [126,127]. Protein synthesis is frequently dysregulated in perturb autophagy by regulating the expression of autophagy-related

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