Glycogen synthase kinase 3 beta: can it be a target for oral cancer
© Mishra. 2010
Received: 11 November 2009
Accepted: 11 June 2010
Published: 11 June 2010
Despite progress in treatment approaches for oral cancer, there has been only modest improvement in patient outcomes in the past three decades. The frequent treatment failure is due to the failure to control tumor recurrence and metastasis. These failures suggest that new targets should be identified to reverse oral epithelial dysplastic lesions. Recent developments suggest an active role of glycogen synthase kinase 3 beta (GSK3 β) in various human cancers either as a tumor suppressor or as a tumor promoter. GSK3β is a Ser/Thr protein kinase, and there is emerging evidence that it is a tumor suppressor in oral cancer. The evidence suggests a link between key players in oral cancer that control transcription, accelerated cell cycle progression, activation of invasion/metastasis and anti-apoptosis, and regulation of these factors by GSK3β. Moreover, the major upstream kinases of GSK3β and their oncogenic activation by several etiological agents of oral cancer support this hypothesis. In spite of all this evidence, a detailed analysis of the role of GSK3β in oral cancer and of its therapeutic potential has yet to be conducted by the scientific community. The focus of this review is to discuss the multitude of roles of GSK3β, its possible role in controlling different oncogenic events and how it can be targeted in oral cancer.
Oral cancer is the sixth most common cancer in the world, and its incidence varies in different ecogeographic regions [1, 2]. Its occurrence is associated with exposure to smoking and alcohol consumption in the Western population. The majority of cases occur in Asia, where it is mainly associated with betel quid chewing . Poor oral hygiene and human papillomavirus (HPV) infection of oral epithelial cells are other etiological factors . In addition to genetic differences, other etiological factors promote the occurrence of this disease to different extents in different populations. Although there are several differences in disease occurrence and etiology between populations, there is one aspect of these tumors that is highly similar worldwide. Oral tumors are mainly asymptomatic initially, are aggressive, and frequently invade and migrate to distant organs, making them difficult to treat. This suggests that, although different predisposing factors activate various molecular pathways , eventually all of them may follow a common path thereafter to result in oral cancer.
Advances in recent decades in the surgical, radiotherapeutic and chemotherapeutic treatment of oral cancer have only modestly improved patient survival. Various approaches have been used for the clinical treatment of oral cancer patients in the last three decades, from non-targeted chemotherapy to highly targeted pharmacological inhibitors and specific monoclonal antibodies [3, 6]. Although targeted therapies yield better outcomes than non-targeted therapies, frequent treatment failure suggests the need for new treatments or targets for this disease. In oral cancer, active transcription of various genes leads to rapid cell division, faster invasion and reduction of cell death. Although it has been largely overlooked, there is a potential link between key players in oral cancer, including transcription factors, cell cycle regulators, invasion/metastasis-promoting factors, and cell survival regulators, and their regulation under the control of glycogen synthase kinase 3β (GSK3β).
GSK3β plays a major role in epithelial cell homeostasis . Its activity is regulated by site-specific phosphorylation of Tyr216/Ser9 residues . The regulated phosphorylation of Ser9GSK3β is the main cause of various pathological conditions, and it is upregulated in epithelial cancers. Many upstream kinases protein kinase A (PKA) , Akt/PKB , PKC , p90 ribosomal S6 kinase/MAPK-activating protein (p90RSK/MAPKAP)  and p70 ribosomal S6 kinase (p70S6K)  are known to phosphorylate Ser9 of GSK3β, depending on the cellular context and various upstream regulators. The oncogenic activation of these upstream signaling molecules is frequently reported in oral squamous cell carcinoma (OSCC) [14–16]. Many of these oncogenic pathways are activated by common etiological factors of this cancer. Overall, this evidence suggests the possible active involvement of GSK3β-mediated signaling in this neoplastic disease. This review attempts to correlate the established pathways of oral cancer with GSK3β signaling and discusses the potential of this kinase as a therapeutic target.
The GSK3 family and its regulation
GSK3 was discovered nearly three decades ago in rabbit skeletal muscle as a protein kinase that phosphorylates and inactivates glycogen synthase, the final enzyme of glycogen biosynthesis [17, 18]. GSK3 is a multifunctional Ser/Thr kinase with diverse roles in various human diseases, including diabetes, inflammation, neurological disorders and various neoplastic diseases [19, 20]. To date, two members of the mammalian GSK3 family (α and β) are known . They are ubiquitously expressed and highly conserved and are members of the CMGC family of protein kinases . Many of the substrates of GSK3 need a "priming phosphate" (which is a Ser/Thr residue) located four amino acids (aa) C-terminally from the site of phosphorylation . GSK3 is constitutively active in resting cells and undergoes a rapid and transient inhibition in response to a number of external signals. Physiological regulation of GSK3 activity by various upstream kinases [9–13] in different physiological and pathological condition is established .
GSK3β and its role in tumorigenesis
Paradoxical role of GSK3β in various human cancers
Explanation for Tumour Suppressor Role of GSK3β
Inactivation of GSK3β (higher pSer9GSK3β expression) 
Pharmacological inhibition of GSK3β in normal epithelial causes epithelial mesenchymal transition (EMT) and invasion 
Inactivation of GSK3β (higher pSer9GSK3β expression) 
Activation of GSK3β, can reverse EMT 
Inactivation of GSK3β (higher pSer9GSK3β expression) 
Inactivation of GSK3β (higher pSer9GSK3β expression) 
Pharmacological inhibition of GSK3 in breast epithelial causes EMT and invasion 
Salivary gland cancer
Inactivation of GSK3β (pSer9GSK3β) observed in this tumor 
Nasopharyngeal cancer (SCC)
Inactivation of GSK3β observed and positively correlated with its upstream inactivating kinase Akt 
Lung cancer (SCC)
Inactivation of GSK3β reported 
Adenocarcinoma of Lung
Higher level of inactivated of GSK3β (pSer9GSK3β) observed 
Inactivation of GSK3β reported 
Skin cancer (Basal cell carcinoma)
Inactivation of GSK3β reported 
Explanation for Tumour Promoter Role of GSK3β
Active GSK3β promotes growth 
Absence of inactive GSK3β (lower pSer9GSK3β expression) in tumors 
Absence of inactive GSK3β (lower pSer9GSK3β) in majority of tumors 
GSK3β promotes growth and use of pharmacological inhibitor promotes apoptosis 
Absence of inactive form of GSK3β (pSer9GSK3β) in these tumors 
Increase and active GSK3β expression 
Missplicing at the kinase domain causing active GSK3β 
Absence of inactive GSK3β (pSer9GSK3β) in these tumours 
Active GSK3β observed frequently and its pharmacological inhibition attenuates survival, proliferation and induce apoptosis 
GSK3β expression increases and it promotes cell division 
GSK3 activity favors replication of DNA and S-phase progression 
Inhibition of GSK3 activity leads to growth suppression 
Higher and active GSK3β expression observed 
Absence of inactive GSK3β (pSer9GSK3β) in these tumors 
Renal cell carcinoma
Activation of GSK3β observed in this tumor 
Nuclear accumulation of GSK3β and its pharmacological inhibition suppress growth 
Pharmacological inhibition of GSK3 induces cell death 
GSK3β and its control over transcription
Alteration of the transcriptional machinery is common in neoplastic diseases, including oral cancer [22, 23]. Oncogenic transcription factors (OTFs) alter the transcriptional machinery to regulate mRNA synthesis. GSK3β regulates the stability of various oncogenic TFs like the activator protein 1 (AP-1) , nuclear factor kappa B (NFκB) , c-Myc , β-catenin , Snail , Forkhead (FH) , CAAT-enhancer binding protein (C/EBPs) , and cAMP response element-binding (CREB)  by phosphorylation . Most of these TFs are physiological targets of GSK3β that undergo proteasomal degradation upon phosphorylation [8, 24–28]. AP-1 transcriptional activity is high in oral cancer tissue samples . Active GSK3β directly phosphorylates c-Jun at Thr239 which promotes its degradation . It is also known that in normal oral mucosa c-Jun is localized in the cytoplasm while it enters to the nucleus at the onset of oral carcinogenesis . Both Fos and Jun are phosphorylated and activated by mitogen activated protein kinase (MAPK) and c-Jun n-terminal kinase (JNK) kinase system [33, 34] may be due to inactive GSK3β. Moreover the expressions of p65 (one of the NFκB family member) have been observed in oral cancer tissue samples [35, 36] and metastatic OSCC . GSK3β phosphorylates p65 at Ser468 and negatively regulate its activity by promoting its degradation . p65 might escape from its turnover because of inactivated GSK3β in OSCC. Recent report suggests active GSK3β physically interact with IκBα in normal epithelial cells . Moreover study in different system suggests that active GSK3β blocks NFκB dependent transcription, by preventing IκBα degradation . In normal epithelial cells NFκB activity is known to be inhibited by GSK3 . From all these evidences, it seems like NFκB activation in OSCC may be modulated, because of inactive GSK3β like that in other epithelial cancers . On the other hand, degradation of c-Myc and β-catenin is initiated by phosphorylation of GSK3β . The overexpression of c-Myc and β-catenin protein in OSCC is established [41–46]. The gene mutation on hot spots i.e. Thr58 of c-Myc and Ser33, Ser37, Thr41 and Ser45 of β-catenin abolishes phosphorylation by GSK3β results in preventing ubiquitination and proteasome mediated degradation of c-Myc [47–50]/β-catenin [46, 51–53] has been reported in various cancers but not so far in OSCC. In OSCC, c-Myc/β-catenin protein might get stability not because of missense mutation at these hot spot codons but because of inactivation of its phosphorylating kinase i.e. GSK3β it self. The activated Snail has been reported in OSCC . GSK3β is well known regulator of Snail which phosphorylates and that leads to Snail nuclear export and deregulation [28, 39, 55, 56]. Moreover, p53 is highly involved in OSCC . Though it is inactivated by mutation in nearly half of oral cancer population  the cause of its inactivation is still doubtful in the other half. p53 activity is regulated by active GSK3β, due to either physical association or phosphorylation and post-translational modification [58, 59]. It is possible that in OSCC cases without p53 mutations , p53 can be inactivated due to inactive GSK3β. These OTFs those are important in OSCC and are directly regulated possibly by GSK3β. Alteration of these TFs plays a vital role in various diseases, including OSCC.
GSK3β is a key player in OSCC
GSK3β can promote or suppress growth in different types of cancer (Table 1). The inactivation of GSK3β has been reported in most cancers of epithelial origin, such as skin, breast, and in cancers of the oral cavity, salivary glands, larynx, and esophagus . The basal level of inactivated GSK3β (pSer9GSK3β) in OSCC cell lines is very high [61–63] but can be decreased by inhibiting the GSK3β upstream inactivating pathway [61, 62]. A recent report suggests that activating GSK3β can reverse the epithelial-mesenchymal process in oral cancer . GSK3β-mediated signaling could explain numerous molecular disorders specific to oral cancer.
A) Cell cycle regulation
B) Nodal invasion by epithelial-mesenchymal transition
OSCC is a cancer of epithelial cells that invades surrounding tissues and frequently migrates to distant organs (metastasizes) . The extra cellular matrix (ECM) interaction is important for the survival of normal epithelial cells but this interaction is gradually lost in squamous cell carcinoma . The major ECM molecules implicated in OSCC development include collagen, fibronectin , tenascin  and laminin [54, 91, 93]. Many ECM molecules are indirect targets of GSK3β via Snail- or AP-1 [28, 94]. The degradation of basement membrane (collagen) by MMPs and its regulation by inactive GSK3β have been reported [95, 96]. Focal adhesion kinase (FAK) is overexpressed in preinvasive and invasive OSCC . Upregulation of FAK leads to migration, and its regulation by active NFκB is known in tongue squamous cell carcinoma cells (SCC25) [98, 99] possibly via inactive GSK3β. Another group of molecules, the integrins, are transmembrane, heterodimeric, cell-surface proteins (consisting of one α and one β subunit) that primarily function as cell adhesion molecules but also participate in signal transduction leading to cell migration, growth and oncogenesis. Human integrins are upregulated in OSCC [100, 101], and they are primarily controlled by those transcription factors regulated by GSK3β [102–104]. Recent evidence suggests a role for Snail in controlling multiple α/β-integrins and EMT in OSCC [54, 94, 105].
MMPs are a group of extracellular matrix/basement-degrading proteases. High levels of MMP-2, -3, and -9 have been associated with poor prognosis for patients with oral cancer, including the development of lymph node metastasis and poor survival [100, 106, 107]. The transcriptional activation of MMP-1,-3, and -9 is common in OSCC [108, 109], and they are all targets of AP-1, NFκB, C/EBPs or Snail, highlighting the importance of GSK3β-mediated signaling in the oral cancer invasion program [110–112].
The inhibition of apoptosis is a major cause of neoplastic disorders and an integral part of oral cancer pathogenesis. Abundant evidence suggests a possible role for active GSK3β in cell survival and apoptosis [123, 124]. Apoptosis is controlled by either the intrinsic (mitochondrial) or extrinsic pathway (activation of procaspase-8) [123, 125–128].
Higher levels of Bcl-2 and lower levels of Bax are frequently reported in oral cancer . A recent report suggests that, in an OSCC cell line, Bcl-2 expression is affected even by slight changes in the status of pSer9GSK3β . Active GSK3β blocks CREB-dependent expression of the anti-apoptotic protein Bcl-2 . Additionally, active GSK3β regulates p53 activity, which increases Bax protein levels to initiate apoptosis . Modulation of GSK3β can markedly increase p53-dependent activation of Bax, leading to cytochrome c release, loss of mitochondrial membrane potential and caspase-9 processing . Moreover, the physiological effect of p53 is governed by inactivation of GSK3β (pSer9 GSK3β)  (and not by pTyr216GSK3β). Inhibition of Akt (a well-known kinase upstream of GSK3β) can only induce tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -mediated apoptosis by regulating the levels of Bcl-2 and Bax in OSCC . All of this evidence suggests that the survival advantage of OSCC cells over the normal oral epithelium might be due to progressive inactivation of GSK3β, which could be responsible for an increased Bcl-2/Bax protein ratio [63, 125–127].
Oral cancer therapy and role of GSK3β signaling
Oral cancer etiology and intracellular signaling
The activation of established GSK3-inactivating upstream biological pathways by oral cancer-predisposing factors, such as tobacco, alcohol, and HPV, support the proposition of a causative role for GSK3β in OSCC. The role of carcinogens (from chewing and smoking tobacco) in oral cancer is firmly established [15, 140]. Smokers show elevated levels of adenyle cyclase (AC) and PKA activity in oral epithelial cells [141, 142]. Chewing areca nuts can lead to DNA damage and increased oxidative stress. The lime (calcium hydroxide) that coats the betel leaf promotes an alkaline oral environment, which activates Akt signaling . There is accumulating evidence that connects nicotine-induced tumorigenesis to the activation of MAPK signaling , activation of PI3K/Akt signaling  and blocking of cytochrome c-mediated apoptosis . Alcohol abuse increases the permeability of cells to carcinogens and activates PKA in cell culture . HPV activates Akt in epithelial keratinocytes [4, 147]. Moreover, a recent evaluation of epithelial tumors suggests that HPV infection can alter many biological pathways to maintain malignant processes by decreasing focal adhesion and up-regulating Wnt signaling and cell cycle genes . Therefore, it is logical to hypothesize that the inactivation of GSK3β contributes to oral cancer.
Evaluation of therapeutic potential and possible methods of targeting GSK3β in OSCC
Before selecting GSK3β as a therapeutic target in OSCC, its biological functions should be explored in detail. Though GSK3β has several isoforms, the isoform(s) specifically expressed in OSCC remain to be identified. If multiple isoforms are expressed, it will be important to understand their respective functions in oral cancer pathogenesis. The upstream cause of activation or inactivation of GSK3β as well as downstream target molecules and their status in OSCC should be thoroughly investigated at the patient level. Because it is an enzyme involved in regulating growth, cell cycle progression, apoptosis, and invasion, GSK3β may qualify as an ideal therapeutic target [123, 149] for OSCC. Because of its role in both extrinsic and intrinsic apoptotic pathways, and because active GSK3β is nontoxic to non-cancerous cells (e.g., in a knock-in mouse study replacing Ser9 of GSK3β with Ala) , targeting the GSK3β pathway might be helpful in reducing unwanted apoptosis (in normal cells) and increasing useful apoptosis (in cancer cells).
The activation status of upstream molecules and the inactivation of GSK3β should be tested in different patients because each patient has a different lifestyle, etiological factors and genetic abnormalities. GSK3β can be inactivated by different upstream molecules in different oral tumors, even in the same patient. Inhibiting the upstream molecules pharmacologically by using peptide competitors and blocking phosphorylation at Ser9 certainly will keep GSK3β in an active state. The crystal structure of GSK3β peptide with an activated Akt ternary complex has been reported [151–154]. This may enable the design of small molecules that will disrupt the interaction of upstream kinases and GSK3β [Therapeutic strategy-I, Fig. 4] and thus prevent inhibitory kinases from associating with GSK3β. After checking the status of those patients who have inactivated GSK3β, Adenoviral vector carrying Ala9GSK3β may be tested along with other (chemo/radio) therapy, or with Ad-p53 (WT), which is known to block the progression of oral cancer to a certain extent . However, although the chances are remote, some OSCC tumors will contain active GSK3β. It will be easy to test the inhibitors of GSK3 in these cases. The use of LiCl and SB-216763 in ovarian cancer ; LiCl and TDZD-8 in prostate cancer ; TDZD-8, SB-216763 and AR-A014418 in colorectal cancer [158, 159]; LiCl, SB-216763, and TDZD-8 in myeloma ; TDZD-8 in AML and AML progenitor and stem cell cancer ; and LiCl and AR-A014418 in pancreatic cancer [161–163] has been evaluated, with positive outcomes. Almost all GSK3 inhibitors are able to inhibit two isoforms of GSK3 (α & β) with similar potency. The production and clinical evaluation of small-molecule inhibitors of particular isoforms will improve the chances of successful treatment in the future. Recent advancements in molecular biology have proven the effectiveness of small RNA interference (RNAi) in reducing the level of one protein by promoting mRNA degradation. This has been tried in an animal model of OSCC and as an alternative therapeutic strategy in patients who have developed drug resistance [164, 165]. Similarly, RNAi has been used to counteract the overexpression of GSK3β in pancreatic , gastrointestinal , and prostate cancer , and it may be tried for OSCC.
The goal of cancer drug discovery is to design non-toxic therapeutics that will be free of side effects. Thanks to a deepening understanding of cell biology and technological advancements, the concept of cancer therapy is being fine-tuned every day. Beginning with metabolic enzyme targeting using folate and methotrexate, to targeting of DNA polymerase and topoisomerase (tamoxifen), to selective hormonal targeting of estrogens/androgens via their nuclear hormone receptors, to the more recent advancement of targeting human growth factor receptor kinases and their effectors, the gradual improvements in our understanding of cancer biology have led to new and numerous therapeutics. Recent developments in molecular research have led to the hypothesis of "oncogene addiction," which suggest the continuous dependence of tumor cells on these oncogenes . The inactivation of GSK3β in OSCC may behave like an oncogene, and its gradual/sustained inactivation may promote oral cancer. Though most of the upstream and downstream targets and their expression status correlate with the understanding of GSK3β inactivation, real, direct assessment should be attempted. If the activated form of GSK3β is non-toxic to normal oral epithelial cells, as was found in animal models , then the manipulation of the activated GSK3β provides hope for treating oral cancer. Unlike other molecules, GSK3β is one of the most attractive targets and is well understood because of extensive prior research on it. Therefore, it should be evaluated thoroughly as a potential target for the treatment of oral cancer.
The author apologizes to those workers whose works have not been included. RM acknowledges his mentor, Prof. A. Rana, Prof. B.R. Das, and Prof. D.P. Sarkar for what he learns from them in his scientific career and personal life.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.