Targeting GSK3 and Associated Signaling Pathways Involved in Cancer

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine (S/T) protein kinase. Although GSK-3 originally was identified to have functions in regulation of glycogen synthase, it was subsequently determined to have roles in multiple normal biochemical processes as well as various disease conditions. GSK-3 is sometimes referred to as a moonlighting protein due to the multiple substrates and processes which it controls. Frequently, when GSK-3 phosphorylates proteins, they are targeted for degradation. GSK-3 is often considered a component of the PI3K/PTEN/AKT/GSK-3/mTORC1 pathway as GSK-3 is frequently phosphorylated by AKT which regulates its inactivation. AKT is often active in human cancer and hence, GSK-3 is often inactivated. Moreover, GSK-3 also interacts with WNT/β-catenin signaling and β-catenin and other proteins in this pathway are targets of GSK-3. GSK-3 can modify NF-κB activity which is often expressed at high levels in cancer cells. Multiple pharmaceutical companies developed small molecule inhibitors to suppress GSK-3 activity. In addition, various natural products will modify GSK-3 activity. This review will focus on the effects of small molecule inhibitors and natural products on GSK-3 activity and provide examples where these compounds were effective in suppressing cancer growth.

Brain-derived neutrophilic factor/TrkB induced phosphorylation of GSK-3β which resulted in its inactivation and contributed to chemotherapeutic drug resistance. GSK-3β was acting as a tumor suppressor.
In vitro [2] Brain cancer GSK-3β Inhibition of AKT mediated phosphorylation of GSK-3β by an AKT inhibitor reduced cell growth. GSK-3β was acting as a tumor suppressor.
In vitro [3] Brain cancer GSK-3β GSK3β was linked with increased expression of TP53 and p21 Cip-1 in glioblastoma cells with wild-type p53 and with decreased Rb phosphorylation and expression of cyclindependent kinase 6, Treatment with GSK-3 inhibitor AR-A014418 sensitized GMB cells to temozolomide. GSK-3β was functioning as a tumor promoter.
Human tumor samples, in vitro studies. [4] Brain cancer GSK-3β Expression of high levels of GSK-3β was associated with poor prognosis. Treatment with a combination of temozolomide other drugs used to treat brain cancer improved prognosis. GSK-3β was acting as a tumor promoter.
In vitro, in vivo, clinical trial, 7 patients in clinical study [5] Brain cancer Suppression of GSK-3β by miR-101 restored sensitivity to temozomide in brain cancer.
In vitro, in vivo [6] GSK-3β was acting as a tumor promoter.
Breast cancer GSK-3β GSK-3β expression was associated with MCL1 expression and inactivation. GSK-3β was acting as a tumor suppressor. High MCL1 expression was associated with poor prognosis and high P-GSK-3β (inactive) expression.
In vitro, in vivo [11] Cervical cancer GSK-3β High expression of forkhead box M1 (FOXM1) transcription factor was associated with poor prognosis and it activated AKT and inactivated GSK-3β which resulted in higher SNAIL activity and poor prognosis. GSK-3β was acting as a tumor suppressor.
In vitro, human tumor samples [12] Colorectal cancer GSK-3β Nuclear accumulation of GSK-3β was observed in 39% (33/85) and associated with short overall survival, larger tumor size, distant metastasis and loss of membranous β-catenin. This loss was present in 37% and associated with poor survival. Nuclear expression of GSK-3β and loss of membrane β-catenin were present in CRC with worse Human tissue microarrays [13] prognosis. GSK-3β was functioning as a tumor promoter, Colorectal cancer GSK-3β GSK-3β increased NF-κB expression, inhibition of GSK-3 inhibited growth. GSK-3β was serving as a tumor promoter.
In vitro, in vivo. chemokine ELISA arrays from CRC patients [15] Gastric cancer GSK-3β P-GSK-3β (T216, active) was expressed in 46% of cases and associated with a good prognosis. GSK-3β was acting as a tumor suppressor.
Human tissue arrays containing 281 gastric cancer specimens and in vitro studies [16] Gastric cancer GSK-3β Higher GSK-3β levels were associated with a better prognosis. GSK-3β was acting as a tumor suppressor.
Gene expression profiling in 63 tumors [17] Hepatocellular carcinoma GSK-3β S9-P-GSK-3β was overexpressed in 50% of tumor tissues and was associated with a poor prognosis. GSK-3β was acting as a tumor suppressor.
178 patients with HCC after curative partial hepatectomy [18] Hepatocellular carcinoma GSK-3β Protein arginine methyltransferase 9 (PRMT9) activation of PI3K/AKT resulted in decreased GSK-3β activity and increased SNAIL signaling. GSK-3β was acting as a tumor suppressor In vitro, in vivo, human tumor samples [19] Laryngeal Cancer GSK-3β Suppression of miR-27a interaction with GSK-3β altered laryngeal differentiation in response to retinoic acid treatment. GSK-3β. GSK-3β was acting as a tumor suppressor.
In vitro, human tumor samples [20] Laryngeal Cancer GSK-3β Alterations in the Tat Genetic deletion of GSK-3β in mice led to myelodysplastic disease syndrome (MDS), subsequent deletion of GSK-3α led to AML. Different roles of GSK-3α and GSK-3β in MDS progression into AML. GSK-3α and GSK-3β were acting as tumor suppressors.
In vitro, in vivo, in human AML patients [23] Leukemia GSK-3α GSK-3α was a target in AML. GSK-3α was serving as a tumor promoter.
Chemical small molecule screening, in vitro, in vivo [24] Leukemia (AML) GSK-3α and GSK-3β GSK-3α and GSK-3β phosphorylation leading to their inhibition correlated with poor prognosis. S21-P-GSK3α and S9-P-GSK-3β positively correlated with phosphorylation of AKT, BAD, and P70S6K, and negatively correlated with βcatenin and FOXO3A. GSK-3α and GSK-3β were serving as tumor suppressors In vitro, human patient samples, reverse phase protein analysis (RPPA) in a cohort of 511 AML patients [25] Leukemia (Natural Killer Cells cytotoxic to AML) GSK-3β and GSK-3α (GSK-3β) expression was elevated in AML-NK cells and decreased their activity as NK cells. Inhibition of GSK-3 restored NK cytotoxicity by increasing TNF-α production. GSK-3 was serving as a tumor suppressor.
In vitro, in vivo [26] Lung cancer GSK-3β High levels of TGFβ induced integrin β3/AKT, inhibited GSK-3β activity, and induced SNAIL activity and promoted metastatic potential. GSK-3β was acting as a tumor suppressor.
In vitro, in vivo, clinical data base [27] Lung cancer GSK-3α CREB induced GSK-3α which promoted lung cancer cell growth. GSK-3α was acting as a tumor promoter.
In vitro, in vivo, human tumors [28] Lung cancer GSK-3α and GSK-3β Tivantinib was initially thought to be a c-MET inhibitor. Subsequently, GSK-3α and GSK-3β were determined to be targets of tivantinib in lung cancer cells. GSK-3α and GSK-3β were acting as tumor promoter In vitro [29] Lung cancer (non-small cell) GSK-3α and GSK-3β GSK-3β levels were elevated in 41% of human NSCLC samples and led to increased proliferation in comparison to normal tissues. GSK-3β was acting as a tumor promoter.
In vitro, in vivo, human tumor samples [32] Myeloma GSK-3α and GSK-3β Treatment with Thiadiazolidinone (TDZD; a GSK-3 non-competitive inhibitor) resulted in Forkhead transcription factors (FOXO3a) activation. TDZD induced apoptosis in primary myeloma cells but not in normal CD34 cells. GSK-3 was acting as a tumor promoter.
In vitro, in vivo [34] Oral Cancer GSK-3β AKT and GSK-3β expression was associated with a poor prognosis. Phosphorylated Human tumor specimens (118 patient samples [35] GSK-3β (inactive) was associated with cervical lymph node (CLN) metastasis. GSK-3β was acting as a tumor suppressor. and normal controls).
Oral squamous cell cancer GSK-3α and GSK-3β Links between GSK-3α and GSK-3β and cyclin D1 and TP53. Inactive GSK-3β was expressed at higher levels than inactive GSK-3α. Inactive GSK-3β was detected at increased percentages in older patients (40->70 years old) than younger patients (<40 years old). GSK-3β was acting as a tumor suppressor.
In vitro, in vivo, human tumor samples [37] Ovarian cancer GSK-3β GSK-3 expression was associated with increased tumor growth, poor prognosis and chemoresistance. GSK-3 was functioning as a tumor promoter.
In vitro, in vivo, 71 human tumor samples. [38] Ovarian cancer GSK-3β Constitutively active GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. GSK-3 inhibition prevented the tumor formation of the tumor in nude mice. GSK-3 was acting as a tumor promoter.
In vitro, in vivo [39] Pancreatic cancer GSK-3α and GSK-3β GSK-3 promoted NF-κB activity. GSK-3β may have been the more important isozyme in regulating in NF-κB. GSK-3β was acting as a tumor promoter.

Human tumors
and in vitro studies. [40] Pancreatic cancer GSK-3β Inhibition of GSK-3 activity caused stabilization of β-catenin activity. GSK-3β expression was a strong prognosticator in PDAC. High expression of GSK-3β was associated with better survival. PDAC Patients with GSK-3β expression > than the third quartile (Q3) had a 46% reduced risk of dying of Immunofluorescence on human tumor microarray from 163 patients. [41] pancreatic cancer. GSK-3β was acting as a tumor suppressor.

Prostate Cancer Both
GSK-3α and GSK-3β were detected at higher levels in 25/79 and 24/79 tumor samples respectively, in comparison to normal prostatic tissue. GSK-3α was elevated in low Gleason sum score tumors while GSK-3β was expressed in high Gleason tumors, and both isoforms correlated with high expression of the androgen receptor (AR).
Treatment with a GSK-3 inhibitor suppressed proliferation. GSK-3 was functioning as a tumor promoter.
In vitro, in vivo and in 79 human tumor samples [42] Renal Cell Carcinoma GSK-3β miR-199a downregulated GSK-3β and suppressed growth of RCC. GSK-3β was acting as a tumor promoter.
Human tumor samples and in vitro. [43] Renal Cell Carcinoma GSK-3β miR-203a targeting GSK-3β was detected at high levels in RCC and associated with a poor prognosis. miR-203a was overexpressed in 27 of 40 (68%) RCC patient samples. GSK-3β was acting as a tumor suppressor.
Human tissue arrays [45] Tongue (oral) cancer GSK-3β GSK-3β was detected at lower levels in 39% of patient samples in comparison to normal epithelial cells and was associated with reduced survival. In contrast, cyclinD, a target of GSK-3β was detected at higher levels in 65.9% of samples and was associated with a poor prognosis. GSK-3β was acting as a tumor suppressor 41 Human tissue samples, immunohistochemistry.

Lithium chloride
Lithium chloride inhibited GSK-3 which suppressed proliferation in Eca-109 human esophageal cancer cells. GSK-3 was functioning as a tumor promoter. [1]

SB21673
SB21673 inhibits GSK-3α and GSK-3β. c-JUN degradation was enhanced by SB21673 and breast cancer tumorigenesis was inhibited. [6] SB216763, GSK inhibitor XIII, and AR-A014418 SB216763 and the GSK inhibitor III suppressed ARtranscriptional activity as well as AR expression in prostate cancer cells. In contrast, AR-A014418 stimulated proliferation. [7] Lithium chloride, SB216763, and GSK-3 IX (BIO) Treatment of MLL LSC with GSK-3 inhibitors resulted in reversion of MLL LSCs to a pre-LSC stage and reduced their growth. [8]

GSK-3 inhibitor treatment of CD8+ T cells inhibited TBX21 (Tbet) expression and decreased PD-1 expression and increased cytolytic T cell responses.
[12] LY2090314, tideglusib, SB415286 GSK-3 inhibitors and NK cells Treatment of NK cells with GSK-3 inhibitors LY2090314, tideglusib or SB415286, increased TNF-α levels and cytotoxicity towards AML cells. [13] SB216763 and GMB-specific CAR-T cells Treatment with GSK-3 inhibitor of antigen specific CAR-T cells lowered PD-1 expression and promoted long term survival, memory and tumor elimination. [14] Enzastaurin Enzastaurin was initially developed as a PKC-β inhibitor. One of its targets is GSK-3. It has been examined in clinical [15] studies with various cancer types, often in combination with bevacizumab. SB415286 or LiCl and TRAIL Inhibition of GSK-3 enhanced the induction of apoptosis mediated by TRAIL in gastric cancer cells. [16]

CHIR99021 and paclitaxel
Effects of combination of the GSK-3 inhibitor CHIR99021 and paclitaxel on lung cancer. [17] SB415286, RO 318220, lithium chloride and paclitaxel SB415286 inhibits both GSK-3α and GSK-3β. RO 318220 inhibits PKC and GSK-3. More mitotic arrest was observed when GSK-3 inhibitors were combined with paclitaxel than in the absence of the GSK-3 inhibitors. [18] LY2090314 and nab-paclitaxel LY2090314 suppressed TAK1 levels. LY2090314 plus nabpaclitaxel combined treatment increased the survival of mice in orthotopic pancreatic tumor models. AR-A014418 and gemcitabine GSK-3 inhibitor suppressed some of the genes induced by gemcitabine that are involved in drug resistance of PDAC cells. [22]

Combination of GSK-3 inhibitors with other inhibitors or agonists
9-ING-41 and either chloroquine and bafilomycin 9-ING-41 have been examined either by itself or in combination with autophagy inhibitors chloroquine and bafilomycin on RCC lines [23] lithium chloride, SB216763, inhibitor IX (BIO) and NF-κB inhibitors PDTC parthenolide, or BAY 11-7082 and chemotherapeutic drugs.
Combining GSK-3, NF-κB inhibitors and certain chemotherapeutic drugs resulted in increased osteosarcoma death both in vitro and in animal xenograft studies. [24] AR-A014418 and Troglitazone Treatment of prostate cancer cells with GSK-3 inhibitor and PPAR agonist suppressed NF-κB activity increased cell death. [25] 6BIO and AR-ASO 6BIO improved the targeting of antisense oligonucleotide (ASO) inhibitor and resulted in increased inhibition of AR signaling. [26] AR-A014418, 5-chloro-2,4dihydroxpyridine (CDHP) and 5FU GSK-3β inhibitor AR-A014418 induced head and neck cancer stem cells [CD44 (high)/ESA (low)] to undergo mesenchymal-to-epithelial transition (MET) back to CD44 (high)/ESA (high) cells. Furthermore, this combined treatment induced the cells to differentiate. [27] Inhibitors originally developed to target other signaling molecules which also target/inhibit GSK-3 activity

Tivantinib
Tivantinib was initially developed as a c-MET inhibitor but it was subsequently determined to target GSK-3α and GSK-3β in lung cancer cells. [28] GDC-0941 GDC-0941 is a PI3K inhibitor. It increased the sensitivity of GBM cells to radiotherapy and reduced chemoresistance to temzolomide. [29] AktX. Lithium chloride AktX is an AKT inhibitor. The effects AktX and lithium chloride on brain cancer cells were determined. AktX [30] suppressed AKT and increased GSK-3β expression and inhibited glioma cell proliferation.

Curcumin
Curcumin suppressed Syk activity which inhibited AKT and induced GSK-3 activity and inhibited B lymphoma growth. [35]

Curcumin and Tetrahydrocurcumin
Curcumin induced GSK-3 activity and inhibited WNT/βcatenin signaling and suppressed azoxymethaneinduced colon carcinogenesis. [36] Berberine Berberine inhibited AKT which resulted in GSK-3 activity in melanoma cells treated with alpha melanocyte stimulating hormone (α-MSH). Berberine suppressed induction of microphthalmia-associated transcription factor (MITF) and tyrosinase activity. [37] Berberine and lapatinib Combining berberine with the dual EGFR and HER receptor inhibitor lapatinib decreased lapatinib-resistance of breast cancer cells. Treatment with berberine and lapatinib induced higher levels of ROS and increased GSK-3 activity and decreased c-MYC levels. [38] Resveratrol Resveratrol increased GSK-3 activity which suppressed WNT/β-catenin signaling and decreased invasion and migration in breast cancer cells. [39]

Apocynin
The effects of apocynin and resveratrol on pancreatic cancer cells were mediated by decreased levels of phosphorylated GSK-3β and ERK1/2 present in the nucleus. [40] Microsclerodermin A Microsclerodermin A inhibited NF-κB activity in PDAC. Potential involvement of GSK-3. [41] Caffeine Caffeine inhibited JB6 mouse epidermal cells proliferation by suppression of AKT and activation of GSK-3. [42] Indirubin Indirubin inhibited GSK-3 and cyclin dependent kinase activity in leukemia cells. Indirubin may have competed for the ATP binding sites in the kinase domains of the proteins. [43] Tetrandrine Tetrandrine inhibited AKT which resulted in GSK-3 activation in colon cancer cells. [44] Differentiation-inducing factor-1 Differentiation-inducing factor-1 inhibited AKT and induced GSK-3 activity in colon cancer cells which resulted in apoptosis. [45]

Dioscin
The effects of dioscin on proliferation were examined with osteosarcoma cells. Dioscin inhibited AKT activity which resulted in GSK-3 activation. [46] Nimbolide Nimbolide inhibited PI3K activity in oral cancer cells which resulted in increased GSK-3 activity and inhibition of cytoprotective autophagy. [47] Oridonin Oridonin increased GSK-3 expression which resulted in c-MYC degradation and growth inhibition and apoptosis in leukemia cells. [48] Apicidin Apicidin resistance in HCC may result from decreased GSK-3 activity and increased WNT/β-catenin activity. [49] Wogonin Wogonin inhibits cell growth and induces apoptosis by inhibiting the expression of GSK-3β in lung cancer cells. [50] Sulforaphane Sulforaphane treatment resulted in induction of miR-19 and suppression of GSK-3β and increased WNT/β-catenin expression. [51] Butyrate Butyrate induced ROS and miR-22/SIRT-1 pathway in hepatic cancer cells which resulted in suppression of AKT, increased PTEN and GSK-3 and apoptosis. [52] Ursolic acid Treatment of ovarian carcinoma cells with ursolic acid resulted in inhibition of GSK-3 and induction of apoptosis [53] Gambogenic acid Gambogenic acid stimulated GSK-3 activity and inhibited growth in GBM cells. [54]