ISSN 1349-7006

Volume 107, Issue 10, October 2016

Issue Highlights
Phosphorylation-mediated regulation of
GSK3βInhibition of Hedgehog pathway inhibits
clonogenic growth of primary AML cells
Mechanisms of action of oncolytic virus therapy

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The official journal of the Japanese Cancer Association
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Cover of this issue. Subcellular localization of CtIP and γH2AX. See also Wu, et al. (pages 1406–1415 of this issue).

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Cancer Science (formerly Japanese Journal of Cancer Research) is the official journal of the Japanese Cancer Association, and is published monthly.
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Volume 107, Issue 10, October 2016

In This Issue
Glycogen synthase kinase-3b is apivotal mediator of cancer invasion
and resistance to therapy
Page 1363–72

Page 1363–72

Page 1422–29

Many types of human malignancies frustrate
both clinicians and patients with their resistance to multimodal therapy and high rate ofrecurrence in spite of aggressive treatment.
Research efforts have focused on identifying
genetic and epigenetic patterns that may leadto resistance, and identifying a common linkthat defines resilience across cancer subtypes
would be a major step forward in therapy. In
this issue of Cancer Science, Domoto andcolleagues provide a comprehensive review
on glycogen synthase kinase-3b (GSK-3b)
and its potential role as a driver of cancer
invasiveness and resistance to therapy. The
authors first provide the reader with a fundamental understanding of how cancers becomeinvasive and then detail how GSK-3b functions in cells under normal and malignant
conditions. The reader is left with a prospective on what next steps can be taken to better
understand GSK-3b and how this may lead tonovel anti-cancer agents.
doi: 10.1111/cas.13028

Small-molecule Hedgehog inhibitor
attenuates the leukemia-initiation
potential of acute myeloid
leukemia cells
Page 1422–29

Page 1373–79

Aberrant activation of the Hedgehog (Hh) pathway is thought to be linked to a variety of cancers, including acute myeloid leukemia (AML),
particularly in the role of maintaining the leukemic stem cell population; however, the particulars are not known. In this proof-of-concept
study, Fukushima et al. attempted to determine
how PF-04449913 (PF-913) works on the
G-protein inhibitor of Hh called Smoothened in

doi: 10.1111/cas.13067© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

both in vivo and in vitro models of AML. Theyshow that in vivo PF-913 works primarily on
the quiescent cell population, causing minimal
cell death, whereas in vitro reduced the tumor
burden in a xenotransplant system, likely byworking through inhibition self-renewal signatures and cell cycle progression. In addition,
PF-913 served to sensitize leukemic cells tocytosine arabinoside. This exciting work servesto deepen the understanding of this novel drug
and will hopefully guide treatment to improveoutcomes for AML patients.
doi: 10.1111/cas.13019

Oncolytic virus therapy: a new eraof cancer treatment at dawn
Page 1373–79
Cancer immunotherapy is an emerging fieldthat has already revolutionized the way clinicians treat patients and researchers focus theirefforts. In this timely review, Fukuhara et al.
provide a summary of oncolytic virus therapy,
a new cancer therapy that also utilizes hostimmunity, which seizes naturally occurring
viruses and targets them to destroy cancer
cells. The unique function of this therapy isthat these viruses do not function as transgene
carriers, but instead themselves are targetedanti-neoplastic agents. Local treatment withthe viruses not only causes a robust viral replication and killing of cancer cells but also
induces systemic antitumor immunity. The
authors outline the long history of oncolytic
virus therapy and describe how the field has
been reborn, which has led to several recent
approvals for novel cancer treatments. This
review provides excellent groundwork for
those interested in oncolytic virus therapy andsimultaneously updates those in the field of thelatest developments and emerging concepts.
doi: 10.1111/cas.13027

Cancer Sci | 2016

Review Article

Glycogen synthase kinase-3b is a pivotal mediator ofcancer invasion and resistance to therapy
Takahiro Domoto,1 Ilya V. Pyko,1,2 Takuya Furuta,2,3 Katsuyoshi Miyashita,1,2 Masahiro Uehara,1 Takeo Shimasaki,1,4
Mitsutoshi Nakada2 and Toshinari Minamoto1
1
Division of Translational and Clinical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa; 2Department of Neurosurgery, Graduate
School of Medical Science, Kanazawa University, Kanazawa; 3Department of Pathology, Kurume University School of Medicine, Kurume; 4Medical ResearchInstitute, Kanazawa Medical University, Uchinada, Ishikawa, Japan

Key words
Cancer, GSK3b, invasion, therapeutic target, therapy
resistanceCorrespondence
Toshinari Minamoto, Division of Translational and ClinicalOncology, Cancer Research Institute and Cancer Center,
Kanazawa University and Hospital, 13-1 Takara-machi,
Kanazawa 920-0934, Japan.
Tel: +81-76-265-2792; Fax: 81-76-234-4529;
E-mail: minamoto@staff.kanazawa-u.ac.jp
Funding Information
Japanese Ministry of Education, Science, Sports, Technology and Culture; Ministry of Health, Labour and Welfare;Japan Society for the Promotion of Science, ExtramuralCollaborative Research Grant of Cancer Research Institute,
Kanazawa University.
Received April 23, 2016; Revised July 26, 2016; Accepted
August 1, 2016
Cancer Sci 107 (2016) 1363–1372
doi: 10.1111/cas.13028

Tumor cell invasion and resistance to therapy are the most intractable biologicalcharacteristics of cancer and, therefore, the most challenging for current cancer
research and treatment paradigms. Refractory cancers, including pancreatic cancer
and glioblastoma, show an inextricable association between the highly invasive
behavior of tumor cells and their resistance to chemotherapy, radiotherapy and targeted therapies. These aggressive properties of cancer share distinct cellular pathways that are connected to each other by several molecular hubs. There isincreasing evidence to show that glycogen synthase kinase (GSK)-3b is aberrantly
activated in various cancer types and this has emerged as a potential therapeutictarget. In many but not all cancer types, aberrant GSK3b sustains the survival,
immortalization, proliferation and invasion of tumor cells, while also rendering
them insensitive or resistant to chemotherapeutic agents and radiation. Here we
review studies that describe associations between therapeutic stimuli/resistanceand the induction of pro-invasive phenotypes in various cancer types. Such cancers
are largely responsive to treatment that targets GSK3b. This review focuses on therole of GSK3b as a molecular hub that connects pathways responsible for tumor
invasion and resistance to therapy, thus highlighting its potential as a major cancer
therapeutic target. We also discuss the putative involvement of GSK3b in determining tumor cell stemness that underpins both tumor invasion and therapy resistance, leading to intractable and refractory cancer with dismal patient outcomes.

O

ne of the well-recognized but still poorly understoodcharacteristics of cancer is its ability to adapt and survive
in a harsh microenvironment. Tumor cells survive the hypoxic
and starved conditions by changing their morphological andbiological properties and by interacting with tumor-associatedstromal components.(1) Another striking characteristic of cancers is their ability to invade host organs and to resist therapeutic insults, thus limiting the success of curative tumor
resection and leading to tumor metastasis and therapy failure.
These pathological behaviors of cancer are associated withacquisition of the hallmark biological traits of cancer.(2–4)
Despite recent advances in cancer treatments, the existence ofunresectable and recurrent cancers that share the persistentcapacity for invasion, metastasis and therapy resistanceremains a challenge for current medical therapies.(5,6) Although
our knowledge of the molecular and biological mechanisms bywhich cancer evolves to the refractory stage is increasing,(2)few strategies have been established to attenuate the ability oftumors to invade or to prevent the failure of therapy.
Modern options for cancer treatment consist of surgery,
cytotoxic or cytostatic chemotherapeutics, radiation, molecular-

targeted and immunomodulating agents, and their multidisciplinary combination.(7,8) These treatments aim primarily to
reduce tumor cell survival and proliferation, but not to eliminate invasive ability or resistance to therapy. Therefore, understanding the pathways by which cancer cells acquire bothinvasive and therapy-resistant phenotypes is critical for thedevelopment of more efficient therapeutic strategies against
refractory cancers and, therefore, improvements in patient survival. Apart from the known mediators of invasion and therapy
resistance [reviewed in Alexander and Friedl(9)], one emerging
candidate is glycogen synthase kinase (GSK)-3b. This
molecule has been extensively implicated in critical cellbiology processes and has causal roles in common diseases
including glucose intolerance, neurodegenerative disorders andcancer.(10–12) Here we review previous studies that havereported on the association between therapeutic stimuli/resistance and induction of pro-invasive phenotypes in various cancer types. Many of these cancers have proven to be responsive
to experimental treatment which targets GSK3b. This review
focuses on the role of GSK3b as a molecular hub that connects
pathways responsible for tumor invasion and resistance to© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and distribution
in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made.

Cancer Sci | October 2016 | vol. 107 | no. 10 | 1363–1372

Review
GSK3b in cancer invasion and therapy resistancetherapy, thus highlighting this kinase as a promising multipurpose cancer therapeutic target. It also discusses a putative rolefor GSK3b in sustaining the tumor cell “stemness” that is central to both tumor invasion and therapy resistance, thereby
leading to intractable cancer and resulting in treatment
failure.(13–15)
Invasion and therapy resistance co-segregate in refractory
cancerHigh invasiveness and resistance to therapy are common biological and clinical characteristics of refractory cancer that represent major challenges for research and treatment. The
mechanisms involved in tumor invasion include cell motility
and migration, degradation of the extracellular matrix andinteraction with stromal and inflammatory cells. These areorchestrated in a way that enables the tumor to invade the hostorgan and often beyond.(3,4) An early morphological and functional change for tumor cells of epithelial origin to acquire a
proinvasive phenotype is epithelial to mesenchymal transition(EMT), altering cell behavior to resemble a mesenchymal
type.(16,17) The major modes by which cancers evade the effectof anti-cancer drugs include intrinsic (or constitutive) resistance and acquired resistance. Constitutive resistance to therapy may be due to mutational activation of signaling for cell
survival, cytoprotective alterations in the cell cycle and in
DNA repair ability, differences in the efficiency of drug uptake
and efflux by cancer cells, and insufficient drug delivery [reviewed in Alexander and Friedl(9)]. Acquired resistance tochemotherapeutic and molecular-targeted agents involves distinct mechanisms, including genetic alterations to the drug targets, activation of surrogate prosurvival pathways, andinteractions between tumor cells and components of the tumor
environment [reviewed in Holohan et al.(5), Ramos and Bentires-Alj(6) and Alexander and Friedl(9)]. Intra-tumor heterogeneity emerges in many tumors and often underlies theirresistance to therapeutic agents.(18,19)
The processes of tumor invasion and therapy resistance andtheir underlying mechanisms are often investigated as independent pathological events in cancer. However, recent studies(shown in Table 1) have demonstrated that tumor cells acquire
morphological and functional proinvasive phenotypes with theability to migrate and invade following the development ofresistance to anti-cancer therapeutics (Suppl. References [SR]1–24) (Data S1).(SR1–24) The therapeutic modalities included
conventional chemotherapeutic agents(SR1–15), different typesof radiation therapy(SR16–20) as well as bioactive compounds
targeting epidermal growth factor (EGF) receptor and vascularendothelial growth factor (VEGF).(SR21–24) These studies indicate the therapeutic insult and/or resistance elicits proinvasive
phenotypes and evokes the invasive capability of tumor cells
in a broad spectrum of cancer types, including breast, colorectal, pancreatic, ovarian and prostate cancers, as well as rare
intractable tumors (e.g. glioblastoma and osteosarcoma). A
large number of preclinical studies have demonstrated that allavailable cancer treatments, including surgery, facilitate metastatic tumor spread. Such treatment often results in therapeutic
benefit, but occasionally also results in resistance, leading to
the paradoxical concept of “treatment-induced metastasis” [reviewed in Ebos(20)]. These experimental and preclinical studies
suggest that the invasive behavior of cancer cells and theiracquired resistance to therapy may not be separate pathologicalproperties and could, instead, represent interconnected processes [reviewed in Alexander and Friedl(9)].
© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

www.wileyonlinelibrary.com/journal/cas

Regardless of the tumor type and therapeutic agent, the proposed mechanisms for invasion and resistance are attributable
to molecular pathways that participate in tumor cell survival,transition to mesenchymal (EMT) and cancer stem cell (CSC)
phenotypes, migration and invasion with extracellular matrix
degradation, and drug efflux (Table 1). The reported moleculesthat mediate and interconnect these pathways include growth
and transcription factors, chemokines, RAS and Rho family
members (e.g. Ras, Rac1) and cell-matrix adhesion molecules(e.g. integrin family and focal adhesion kinase [FAK])(Table 1).(9) Bevacizumab is a humanized monoclonal antibody to VEGF used clinically as an anti-angiogenic agent.
Acquired resistance to bevacizumab leads to enhanced tumor
cell invasion due to the metabolic shift to glycolysis anddegradation and remodeling of tumor stromal tissues.(SR21–24)
A better understanding of the biological mechanisms underlying the tight association between tumor invasion and therapy
resistance should provide a solid rationale for the developmentof innovative cancer treatments.(9)Aberrant GSK3b in cancer

GSK3 is a family of serine/threonine protein kinases comprising two highly conserved isoforms, GSK3a and GSK3b, thatshow approximately 85% overall homology and 98% homology in their kinase domains. GSK3 is constitutively active innormal cells and its activity is regulated by the differential
phosphorylation at serine (S) residues 21 (pGSK3aS21) and 9(pGSK3bS9) (both inactive forms), and tyrosine (Y) residues
279 (pGSK3aY279) and 216 (pGSK3bY216) (both active forms)(Fig. 1a,b). GSK3 regulates a diverse array of physiologicalcellular functions via the phosphorylation of and interaction
with various proteins (Fig. 1c). Although the two GSK3 isoforms share many substrates, they are not functionally identicaland show some differences in their substrate specificity [reviewed in Beurel et al.(10)]. Negative regulation of GSK3
activity is desirable to maintain physiological cellular functions. A growing number of studies have demonstrated thatderegulated GSK3 activity contributes to the pathogenesis andprogression of various diseases, including glucose intolerance,
neurodegenerative disorders, and chronic inflammatory andimmunological diseases.(10,21) This points to GSK3 being an
attractive and druggable target for a broad spectrum of diseases.(22) Consequently, many GSK3 inhibitor compounds havebeen developed in academic and pharmaceutical institutes overrecent years. Some are reported to inhibit both the a and b isoforms with different affinities,(SR25–28) while others are specificfor GSK3b (reviewed in SR25,26). Among the latter compounds, it was reported that AR-A014418 does not inhibit theactivity of 26 closely related kinases and is, therefore, considered to be highly specific for GSK3b.(SR29)
Of the two GSK3 isoforms, most studies in the field of
oncology have focused on GSK3b. This is partly because of
the functional redundancy of the two isoforms in regulating
the canonical Wnt/b-catenin pathway, responsible for generating the most prevalent oncogenic signaling.(23,24) Based on its
known effects on several proto-oncoproteins (e.g. b-catenin,
cyclin D1 and c-Myc), cell cycle regulators (e.g. p27Kip1) andmediators of EMT (e.g. snail) (Fig. 1c), it has long been recognized that GSK3b suppresses tumor development [reviewed
in Clevers and Nusse,(24) Jope et al.,(25) Luo(26) and McCubrey
et al.(27)], as discussed later (Table S1).(SR30–58) Paradoxically,
our earlier studies found increased expression and deregulated
activity of GSK3b due to changes in the differential
Cancer Sci | October 2016 | vol. 107 | no. 10 | 1364

Review
Domoto et al.

www.wileyonlinelibrary.com/journal/cas

Table 1. Representative previous studies showing interrelationship between therapeutic stimuli/resistance and pro-invasive phenotype incancerTumor type

Therapeutic insults

Breast cancer

Tamoxifen
Tamoxifen

Adriamycin
Doxorubicin andcyclophosphamide
alone or in
combination
Colorectal cancer

Oxaliplatin

Erlotinib

Pancreatic cancer

Gemcitabine

Gemcitabine

Gemcitabine, 5-FU,
cisplatin
Gemcitabine
c-irradiation

Ovarian cancer

Erlotinib
Taxol, vincristine
Paclitaxel
Cisplatin, taxol

Prostate cancer

Taxol, vincristine
Docetaxel

Biological mechanism (Suppl.
Reference number)

Therapeutic effect of GSK3b inhibition andunderlying mechanism (Reference number)

EGFR pathway, enhanced tumor
cell motility and invasion(SR1)
EMT induction with EGFR
pathway-dependent b-catenin
activation(SR2)
Twist 1-meditaed EMT induction
and P-gp up-regulation(SR3)
TNF-a/NF-jB-mediatedamplification of CXCL1 paracrine
network between carcinoma,
myeloid, and endothelial
cells(SR4)
NF-jB-mediated EMT induction
with enhanced cell migrationand invasion(SR5)
EMT induction(SR21)

Suppression of invasion through dysregulationof actin-reorganization via down-regulationof WAVE2(68)

EMT induction with activation ofb-catenin and c-Met andacquisition of CSC
phenotype(SR6)
Acquisition of EMT and CSC
phenotypes with activation of
Notch pathway(SR7)
Gene expression profile
responsible for EMT
phenotype(SR8)
NF-jB-mediated acquisition of
EMT and CSC phenotypes(SR9)Tumor cell migration andinvasion with enhanced MMP-2
activity(SR16)
EMT induction(SR21)
Increased expression of twist(SR10)
Acquisition of EMT andmetastatic potential(SR11)
Acquisition of EMT via
endothelin A receptor-mediatedpathway(SR12)
Increased expression of twist(SR10)
Gene expression profile
responsible for EMT
phenotype(SR13)

Other epithelial cancer
Endometrial

Ionizing radiation

EMT induction with enhanced
cell migration(SR17)

Bladder

Taxol, vincristine

Increased expression of twist(SR10)

TongueCisplatin

Nasopharyngeal

Taxol, vincristine

EMT induction and enhanced cell
migration and invasion inassociation with increased BMI1
by down-regulation of miR-200b
and miR-15b(SR14)
Increased expression of twist(SR10)

Cancer Sci | October 2016 | vol. 107 | no. 10 | 1365

Suppression of tumor cell survival andproliferation by inhibition of hTERT/
telomerase and promoting p53-dependent
apoptosis(28–30,32,33) and invasion by downregulation of WAVE2,(68) and enhancing 5-FU
effect via PARP1-dependent and AIF-mediatednecroptosis(73)Suppression of tumor cell survival andproliferation(30) by inhibition of NF-jB
transcriptional activity,(34,35) synergistic withgemcitabine by restoration of Rb function(60)and inhibition of TP53INP1-mediated DNA
repair,(70) increase in radiosensitivity andsuppression of invasion via FAK/Rac1/MMP-2
and CXCR4/MMP-2 pathways(60,67)

Suppression of tumor cell proliferation by
decrease in cyclin D1 expression(39)Suppression of tumor cell survival andproliferation by eliminating TRAIL resistance,
repressing AR activity,(41–44) and attenuationof metastasis by depleting CSC population(78)Suppression of tumor cell survival byinterrupting ERK-mediated prosurvival
pathway(40)Suppression of tumor cell survival andproliferation by inhibition of NF-jB
transcriptional activity(45)Not shown

Not shown© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Review
GSK3b in cancer invasion and therapy resistancewww.wileyonlinelibrary.com/journal/cas

Table 1 (Continued)

Tumor type

Therapeutic insults

Glioblastoma

Doxorubicin

Sublethal
irradiation

Ionizing radiation

Bevacizumab

Bevacizumab

Bevacizumab

Osteosarcoma

Low-dose photon
irradiation

Biological mechanism (Suppl.
Reference number)

Therapeutic effect of GSK3b inhibition andunderlying mechanism (Reference number)

Therapeutic effect andenhancement of doxorubicin
effect by a new anti-invasive
small molecule (IB)(SR15)
Enhanced tumor cell migrationand invasion involving avb3
integrin, MMP-2, MMP-9, MT1MMP, TIMP-2 and BCL-2/BAX
rheostat(SR18)
Enhanced tumor cell migration
with increased expression of b3
and b1 integrins(SR19)
Resistance to the therapy is
associated with up-regulation of
MMP-2, MMP-9, MMP-12,
TIMP1, SPARC and HIF-2a, andwith activation of bFGFmediated alternate angiogenesis
pathway(SR22)
Treatment of tumor xenograft isassociated with decrease of
mitochondria, induction of
glycolytic metabolites (lactate
and alanine) and HIF-1a, andactivation of PI3K pathway(SR23)
Resistance to the therapy
induced genes associated with amesenchymal origin, cellularmigration/invasion, andinflammation.(SR24)
Enhanced cell migration andinvasion concomitant with upregulation of avb3 integrin(SR20)

Suppression of tumor cell survival andproliferation by restoring p53/p21 pathway
and inhibition of c-Myc, NF-jB and abnormal
glycolysis,(31,36) synergistic with temozolomide
and increase in radiosensitivity via
Rb-mediated and c-Myc/DMNT3A/MGMT
pathways,(31,69) suppression of invasion(58,59,61)
by inhibition of FAK/Rac1/JNK pathway(61) and
induction of CSC differentiation(37,77)

Suppression of tumor cell survival andproliferation by inhibition of NF-jB
transcriptional activity(38) and restoringb-catenin osteosarcoma suppressor(S. Shimozaki et al.)††S. Shimozaki et al., unpublished observation. AIF, apoptosis-inducing factor; AR, androgen receptor; BAX, Bcl-2-associated X protein; bFGF, basic
fibroblast growth factor; BMI1, B lymphoma Mo-MLV insertion region 1 homolog; CSC(s), cancer stem-like cell(s); CXCL1, chemokine (C-X-C motif)
ligand 1; CXCR4, CXC receptor type 4; DNMT3A, DNA (cytosine-5)-methyltransferase 3A; EGFR, epidermal growth factor receptor; EMT, epithelialmesenchymal transition; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; 5-FU, 5-fluorouracil; HIF, hypoxia inducible factor;
hTERT, human telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; MGMT, O6-methylguanine DNA methyltransferase; miR, micro-RNA;
MMP, matrix metalloproteinase; MT1-MMP, membrane type 1-MMP; NF-jB, nuclear factor-jB; PARP1, poly [ADP-ribose] polymerase 1;P-gp, Pglycoprotein (multidrug resistance); PI3K, phosphatidyl-inositol-3-kinase; Rb, retinoblastoma; SPARC, secreted protein, acidic, cysteine rich; SR,
supplementary reference No.; TIMP, tissue inhibitor of MMP; TNF-a, tumor necrosis factor-a; TP53INP1, tumor protein p53-inducible nuclear protein 1;
TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WAVE2, WAS (Wiskott-Aldrich syndrome) protein family member 2.

phosphorylation of S9 and Y216 residues in gastrointestinal
cancers, glioblastoma and osteosarcoma compared to respective normal cells and tissues.(28–31) These observations suggested that GSK3b could participate in cancer development
and progression, despite the general belief that it has tumorsuppressive functions.(25–27) We and other research groups
have shown that inhibition of GSK3b activity with specificpharmacological inhibitors, or inhibition of its expression by
RNA interference, can preferentially attenuate the survival andproliferation of tumor cells and induce them to undergo apoptosis. This effect has been observed not only in gastrointestinal
and pancreatic cancer cells(28–30,32–35) but also in glioblastoma,(31,36,37) osteosarcoma (S. Shimozaki, N. Yamamoto, T.
Domoto, H. Nishida, K. Hayashi, H. Kimura, A. Takeuchi, S.
Miwa, K. Igarashi, T. Kato, Y. Aoki, T. Higuchi, M. Hirose,

R.M. Hoffman, T. Minamoto, H. Tsuchiya, unpublished data,
2016)(38) and other malignant neoplasms such as gynecologicaland urogenital cancers,(39–47) soft tissue sarcomas,(48,49) hematological malignancies(50,51) and lung cancers.(52,53) This accumulating evidence firmly establishes GSK3b as a valuabletarget in cancer treatment.(11,12)
There has been substantial interest in the molecular mechanisms by which GSK3b favors tumor progression and bywhich inhibition of its activity or expression attenuates tumor
cell survival, immortalization and proliferation. The reported
mechanisms for tumor cell survival include the nuclear factor(NF)-jB-mediated prosurvival pathway,(34–36,38) tumor cell
resistance to apoptosis induced by tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL),(41) and failure of the p53mediated tumor suppressor pathway or of the Rb-mediated cell

© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Cancer Sci | October 2016 | vol. 107 | no. 10 | 1366

www.wileyonlinelibrary.com/journal/cas

Review
Domoto et al.

Fig. 1. (a) Comparison of the structural and functional domains of the two GSK3 isoforms, (b) the sites (S9 and Y216) of phosphorylation of
GSK3b by different kinases regulating GSK3b activity, and (c) the substrates of GSK3b and proteins that interact with it. (a) GSK3a (51 kD) andGSK3b (47 kD) are products of their respective genes located in chromosomes 19q13 and 3q13. The isoforms share high (98%) homology of thecatalytic domains, and GSK3a has a glycine-rich extension at the N-terminal side. Blue and red narrow columns indicate the sites of serine (S)and tyrosine (Y) phosphorylation, respectively. (b) The kinases indicated in blue phosphorylate GSK3b-S9 resulting in its inactivation, while thoseindicated in red phosphorylate GSK3b-Y216 resulting in its activation. (c) GSK3b stabilizes/activates (red arrows) and destabilizes/inactivates (bluelines) various transcription factors as well as structural and functional proteins. AP-1, activator protein 1; APC, adenomatous polyposis coli; BAX,BCL2-associated X protein; BCL, B-cell lymphoma; C, C-terminal of protein; C/EBP, CCAAT (cytosine-cytosine-adenosine-adenosine-thymidine)enhancer-binding protein; cdc25A, cell division cycle 25 homolog A; CREB, cAMP (cyclic adenosine monophosphate) response element binding
protein; CRMP2, collapsin response mediator protein 2; eIF2B, eukaryotic initiation factor 2B; FAK, focal adhesion kinase; FGD-1/3, FYVE RhoGEF(guanine nucleotide exchange factor) and PH domain-containing protein 1/3; FKHR, forkhead in rhabdomyosarcoma; Gly, glycine; GR, glucocorticoid receptor; GS, glycogen synthase; GSK3b, glycogen synthase kinase 3b; HIF-1a, hypoxia inducible factor-1a; HSF-1, heat shock transcription
factor-1; ILK, integrin-linked kinase; IPF1/PDX1, insulin promoter factor 1/pancreatic and duodenal homeobox 1; IRS1, insulin receptor substrate
1; LRP5/6, lipoprotein receptor-related protein 5/6; MafA, musculoaponeurotic fibrosarcoma oncogene homolog A; MAP1B/2C, microtubule associated protein 1B/2C; Mcl1, myeloid cell leukemia 1; mCRY2, mouse cryptochrom 2; MDM2, mouse double minute 2 homolog; MEK, MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) kinase; MITF, microphthalmia-associated transcription factor; MLK3,
mixed lineage kinase 3; N, N-terminal of protein; NAC-a, nascent polypeptide-associated complex subunit-a; NF-AT, nuclear factor of activated Tcells; NF-jB, nuclear factor-jB; Nrf2, nuclear factor erythroid 2-related factor 2; p130RB, p130 retinoblastoma; p21CIP1, p21 CDK (cyclin-dependent kinase)-interacting protein 1; p90RSK, p90 ribosomal protein S6 kinase; PDH, pyruvate dehydrogenase; PKA, protein kinase A; PKC, protein
kinase C; PP2A, protein phosphatase 2A; PPAR, peroxisome proliferator-activated receptor; PTEN, phosphatase and tensin homolog; Pyk-2, proline-rich tyrosine kinase 2; RAR, retinoic acid receptor; Red1, RNA-editing deaminase 1; S, serine; SRC-3, steroid receptor coactivator-3; SREBP,
sterol regulatory element-binding protein; TSC2, tuberous sclerosis complex 2; VDAC, voltage-dependent anion channel; Y, tyrosine

cycle regulation.(31,32) Preservation of telomere length bymaintaining activity of human telomerase reverse transcriptase(hTERT) and telomerase also immortalizes tumor cells in
response to the aberrant activation of GSK3b.(30) Cell proliferation pathways mediated by c-Myc and cyclin D1 sustain
tumor cell proliferation that is dependent on GSK3b.(36,39) In
osteosarcoma and rhabdomyosarcoma, the induction of b-catenin signaling, a well-known tumor suppressive mechanism inthese cancers, has been linked to decreased cell proliferation
following GSK3b inhibition (S. Shimozaki et al., unpublisheddata).(49) Another recent study reported that mitotic catastrophe
caused by disruption of centrosome biodynamics was associated with attenuated tumor cell survival and proliferation following GSK3b inhibition.(54) Importantly, GSK3b has little
impact on the cell survival, immortalization and growth of normal cells, where the differential phosphorylation of S9 andY216 residues functions to fine tune GSK3b activity.(28–31,34)
The growing body of evidence on the role of aberrant GSK3b
in sustaining and promoting cancer cell survival, immortalization and proliferation, together with the differential effects of
GSK3b inhibition on cancer and normal cells, underpins thetargeting of GSK3b as a novel cancer treatment.(11,12)
Cancer Sci | October 2016 | vol. 107 | no. 10 | 1367

GSK3b mediates both the invasive phenotype and therapy
resistance in refractory cancer

The dependency of cancer cells on GSK3b for their survival
and proliferation has encouraged further studies on whether
aberrant GSK3b participates in tumor cell invasion and therapy
resistance, the two major determinants of patient outcome.(3–6)
As shown in Table 1, most tumors that acquire pro-invasive
phenotypes as they evade therapy are susceptible to experimental treatment involving inhibition of GSK3b. Here we
review what is known about the involvement of GSK3b intumor invasion and resistance to therapy based on our previous
studies in pancreatic cancer and glioblastoma. These are representative of lethal tumors and are characterized by high invasive capacity and resistance to available therapies.(55,56)
GSK3b and cancer invasion. While GSK3b plays pivotal roles
in cytoskeletal organization, cell polarity and migration in thephysiological processes of organogenesis and wound healing,(57) little is known about its role in cancer cell motility,
migration and invasion. Earlier studies showed that lithium
chloride and some indirubins, both classical GSK3-inhibiting
agents, inhibited the migration and invasion of glioblastoma© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Review
GSK3b in cancer invasion and therapy resistancecells, but the mechanism underlying this effect was notclear.(58,59) Subsequently, we demonstrated that inhibition of
GSK3b by a specific pharmacological inhibitor and by RNA
interference decreases the capacity for migration and invasionof pancreatic cancer and glioblastoma cells and of their tumorsin animal models.(60,61) Morphologically, these effects wereassociated with reduced formation of lamellipodia andinvadopodia, the horizontal and vertical cellular surface
microarchitectures, respectively, that drive cell migration, stromal degradation and invasion.(62,63) Inhibition of GSK3b disrupted the functional subcellular colocalization of Rac1(64) andcortactin(63) with the cytoskeletal molecule F-actin in lamellipodia and invadopodia, respectively. These changes in the
pro-invasive phenotype of tumor cells following GSK3b inhibition coincide with the suppression of molecular pathways
mediated by focal adhesion kinase (FAK),(65) guanine nucleotide exchange factors (GEF)/Rac1(64,66) and c-Jun N-terminal
kinase (JNK). This results in decreased expression of matrix
metalloproteinase (MMP)-2 and membrane type (MT) 1-MMP.
Our observations therefore suggest that GSK3b sustains tumor
invasion via the promotion of morphological and functional
pro-invasive cellular phenotypes and that the molecular axis
involves FAK, GEFs/Rac1 and JNK (Fig. 2).(60,61)
Subsequent to our studies,(60,61) other workers have reported
the proinvasive effect of GSK3b in pancreatic, colorectal andbreast cancer cells in association with modulation of cytoskeletal organization and cytokine-mediated matrix degradation.(67,68). These studies highlight the pivotal role of GSK3b
in tumor invasion and probably also in metastatic spread.
GSK3b and cancer therapy resistance. The combination ofmultiple agents having different targets and mechanisms ofactions is important in the treatment of cancer in order to optimize the therapeutic effects and minimize the adverse effects.
This is particularly relevant for the treatment of refractory cancer, and, hence, molecular-targeted therapy is often combinedwith conventional chemotherapeutics and/or radiation therapy,
as well as with other targeted agents.(7,8) As discussed above,
accumulating evidence on the effects of GSK3b inhibition
against cancer cell survival and proliferation has led us toFig. 2. Putative molecular pathway through which deregulated
GSK3b promotes tumor cell migration and invasion. The exact molecular pathway by which GSK3b mediates the activation of FAK (openarrow) remains to be determined. FAK, focal adhesion kinase; GDP,
guanosine diphosphate; GEF, guanine nucleotide exchange factor;
GSK3b, glycogen synthase kinase 3b; GTP, guanosine triphosphate;
JNK, c-Jun N-terminal kinase; MMP, matrix metalloproteinase;
MT1-MMP, membrane type 1-MMP; circled P, phosphorylation.
© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

www.wileyonlinelibrary.com/journal/cas

address whether this could be used in combination withchemotherapy and irradiation.
The standard treatment modalities prescribed are often ineffective in patients with refractory tumors such as pancreaticcancer and glioblastoma.(55,56) We have shown that administration of a GSK3b-specific pharmacological inhibitor (e.g.
AR-A014418)(SR29) at a dose below that which results in atherapeutic effect when given on its own can significantly
enhance the cytotoxic effects of temozolomide and gemcitabine.(31,60,69,70) These are the standard chemotherapeutic
agents prescribed for patients with glioblastoma and pancreaticcancer, respectively.(55,56) Low-dose GSK3b inhibitor given in
combination with chemotherapeutic agents had additive andsynergistic effects. Investigation of the underlying molecularmechanism is crucial to justify treatment combinations of
GSK3b inhibitor with other anti-cancer therapeutics (Fig. 3).
In glioblastoma, the combined effect was attributed tomodulation of the mouse double minute (Mdm)2/p53 andc-Myc-mediated pathways resulting in epigenetic silencing of
O6-methylguanine DNA methyltransferase (MGMT), a clinically proven biomarker of temozolomide efficacy(56) in tumor
cells (Fig. 3b).(31,69) In pancreatic cancer, inhibition of tumor
GSK3b activity decreased cyclin D1/cyclin-dependent kinase(CDK)4/6 complex-dependent phosphorylation of Rb tumor
suppressor protein.(60) One of the important functions of Rb is
to bind the oncogenic transcription factor E2F1 and inhibit its
transcriptional activity.(71,72) GSK3b may, therefore, sensitize
pancreatic cancer to gemcitabine by downregulating theexpression of ribonucleotide reductase, a well-known pharmacological biomarker of gemcitabine(55) that is regulated by theRb/E2F pathway (Fig. 3a).(71,72) In addition to modulation ofthis pathway, we found that downregulation of the TP53INP1
gene (encoding tumor p53-induced-nuclear-protein 1) involved
in DNA repair is associated with the chemosensitizing effectof GSK3b inhibitor.(70) The GSK3b inhibitor used in thesestudies also sensitized both pancreatic cancer and glioblastoma
to ionizing radiation.(31,60) This radio-sensitizing effect may
depend on the restoration of Rb function following GSK3b
inhibition, resulting in the inability of E2F1 to induce the transcription of thymidylate synthase and thymidine kinase(Fig. 3a).
Other recent studies have also reported that GSK3b inhibition allows therapy-resistant colon and pancreatic cancer cells
to become susceptible to 5-fluorouracil (5-FU),(73) and renal
cell carcinoma cells to become susceptible to a synthetic multikinase inhibitor (sorafenib) that targets growth signaling andangiogenesis.(74) Together, these studies suggest that GSK3b
participates in multiple molecular pathways used by various
cancer types to evade chemotherapy, radiotherapy and targeted
therapies.
Collectively, a growing body of experimental evidence supports the notion that GSK3b is a molecular “hub” that mediates and connects various pathways responsible for tumor
invasion and resistance to therapy. Together with the role of
GSK3b in promoting tumor cell survival and proliferation andits differential functions between tumor and normal cells [reviewed in Miyashita et al.(11) and McCubrey et al.(12)], thisevidence reinforces the promise of novel cancer therapeuticstrategies that target GSK3b.
Future perspectives

Targeting of the biological pathways that link tumor invasion
and therapy resistance is clearly an attractive prospect for theCancer Sci | October 2016 | vol. 107 | no. 10 | 1368

www.wileyonlinelibrary.com/journal/cas

Review
Domoto et al.

Fig. 3. (a) Putative molecular pathway that links
GSK3b activity with the resistance of pancreaticcancer cells to DNA damage induced by
gemcitabine and ionizing radiation. The effects of
GSK3b on E2F-dependent gene transcription and on
the expression of RR, TS and TK remain to be
determined. CDK, cyclin-dependent kinase; E2F, E2
factor; circled P, phoshorylation; Rb, retinoblastoma(tumor suupressor protein); RR, ribonucleotide
reductase; TK, thymidine kinase; TS, thymidylate
synthase. (b) Regulation of MGMT expression by
GSK3b signaling in glioblastoma. GSK3b inhibition
results in c-Myc activation directly and via
activation of b-catenin-mediated signaling, whichconsequently increases recruitment of DNMT3A by
c-Myc to the MGMT promoter, thus increasing denovo DNA methylation in the MGMT promoter. The
methylated status of the MGMT promoter increases
the sensitivity of glioblastoma to temozolomide.
DNMT3A, DNA (cytosine-5)-methyltransferase 3A;
MGMT, O6-methylguanine DNA methyltransferase.

development of innovative cancer treatments. Another important challenge is to understand the biology of cancer initiatingor stem-like cells (CSC) so that “stemness” phenotypes canalso be targeted. Based on the clonal evolution model oftumorigenesis and on the normal stem cell concept, CSC areconceptually defined as a population of tumor cells with selfrenewing and pluripotent capacity.(13) CSC have been identified in a wide range of hematopoietic malignancies and solid
tumors and are assumed to be progenitors of the entire neoplastic cell population. One of the most striking characteristics
of CSC is their influence on tumor cell plasticity, intra-tumor
heterogeneity, invasion and metastasis, therapy resistance andtumor relapse after surgery and adjuvant treatment.(14,75) Eradication of CSC is, therefore, of paramount clinical importancefor combatting refractory cancer.(15,76)
GSK3b has pivotal functions in normal cell biology(10,21,22)
and is also central to the processes of cancer cell survival, proliferation, invasion and therapy resistance, as discussed above.
A number of studies have reported that CSC phenotypes shareunique molecular pathways and tumor–environment interactions that are also associated with tumor invasion and therapy
resistance.(14,75) This leads us to propose a working hypothesis
whereby GSK3b underlies the basal mechanism for sustaining
the CSC phenotype (Fig. 4). Recent studies have shown that
GSK3b negatively controls the differentiation of malignant
glioma cells,(77) and that GSK3b inhibition results in depletionof cancer stem-like or progenitor-like cells and attenuation ofmetastatic spread in prostate cancer.(78) Consistent with thephysiological roles of GSK3b in negatively regulating canonical Wnt/b-catenin and hedgehog signaling,(10,23,24) inhibitionof GSK3b is a prerequisite for the maintenance of “stemness”
phenotypes in embryonic and hematopoietic stem cells.(79,80)
Therefore, future work should aim to understand the influenceof GSK3b on both tumor and normal stem cell phenotypes.
This knowledge can be applied for the development of novel
cancer treatments that target GSK3b.
One of the concerns about targeting GSK3b for cancer treatment is that its inhibition may promote the progression ofexisting tumors [reviewed in Takahashi-Yanaga(22), Jopeet al.,(25) Luo(26) and McCubrey et al.(27)]. A number ofCancer Sci | October 2016 | vol. 107 | no. 10 | 1369

Fig. 4. Involvement of GSK3b in the representative pathological hallmarks of cancer. GSK3b positively regulates the distinct molecularpathways and participates in survival, proliferation, migration andinvasion of tumor cells and their insensitivity and resistance to cancer
therapy. The cancer stemness phenotypes might underlie the processof these pathological hallmarks. Abbreviations are defined in
Figures 1, 2 and 3.

studies have focused on the putative tumor suppressive role of
GSK3b (Table S1).(SR30–58) Many of these report inactivationor activation of GSK3b as a mediating event in pathways leading to tumor progression or suppression, respectively. An
inverse association between tumor expression of GSK3b (either total or active form pGSK3bY216) and the survival of cancer patients has been reported.(SR33,36,37) Other studies foundcausal links between pharmacological GSK3b inhibition and/or
depletion of GSK3b expression (e.g. gene knockout and RNAinterference) or activity (e.g. recombinant kinase-dead or constitutively active mutant form) and tumor cell survival, proliferation(SR40,52) and susceptibility to cancer therapies.(SR32,34,58)
GSK3b inhibition strategies and the development of GSK3btargeted agents therefore require careful evaluation to determine whether the tumor promoting function of GSK3b iscounteracted by its putative tumor suppressor function in different cancer types.
The development and administration of GSK3b inhibitors
for the treatment of chronic diseases, including diabetes© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Review
GSK3b in cancer invasion and therapy resistancewww.wileyonlinelibrary.com/journal/cas

mellitus, neurodegenerative disorders and cancer, requires serious awareness of the safety issues. A major concern is whether
and how GSK3b can be targeted to treat disease, because its
systemic inhibition may lead to unwanted effects by disrupting
multiple biological pathways (Fig. 1c). Most notably, longterm inhibition of GSK3b may initiate tumorigenesis due to itsrole in suppressing proto-oncogenic pathways, in particularthat are mediated by b-catenin [reviewed in TakahashiYanaga(22), Jope et al.,(25) Luo(26) and McCubrey et al. (27)]. A
previous study showed that genetic deletion of GSK3b inmammary epithelial cells resulted in b-catenin activation andinduced intraepithelial neoplasia that progressed to the development of adenosquamous carcinoma.(SR51) Moreover, overexpression of wild-type and constitutively active mutant GSK3b
in
12-O-tetradecanoylpholbor-13-acetate(TPA)-mediated,transformation-resistant mouse epidermal cells suppressed
EGF-mediated and TPA-mediated anchorage-independent
growth in soft agar and tumorigenicity in rodents(Table S1).(SR56) However, there is currently no direct evidence to support tumor development in vivo following treatment with a GSK3b inhibitor [reviewed in Miyashita et al.(11)and SR27]. As discussed in previous studies that report cancer
therapeutic effects of GSK3b inhibition (Table 1), none of theavailable experimental GSK3b inhibitors induces neoplastictransformation of non-neoplastic (normal) cells or tumor development in experimental animal models [reviewed in Miyashita
et al.(11) and SR27]. Long-term prescription of lithium, theonly GSK3b inhibitor approved for the treatment of bipolar
disorder since the 1950s, has not been associated with anincreased risk of cancer or death from cancer.(SR59) Post-translational regulation of GSK3b activity via the phosphorylationof S9 and Y216 (pGSK3bS9 versus pGSK3bY216) (Fig. 1b) in
response to various stimuli could partly underlie a mechanismthat protects normal cells from the detrimental effects of
GSK3b inhibition.
Despite the concerns outlined above, clinical trials for neurodegenerative diseases and cancer have tested some seedpharmacological GSK3(b) inhibitor compounds and also
approved medicines with the ability to inhibit GSK3b activity(Table S2). The former trials include AZD-1080 (AstraZeneca)for the treatment of Alzheimer’s disease (phase I), and
NP031112 (tideglusive; Noscira SA) for the treatment of progressive supranuclear palsy (NCT01049399: phase IIb)(SR60,61)
and of Alzheimer’s disease (NCT01350362: phase II).(SR62,63)
Clinical trials for cancer treatment have used LY2090314 (Eli
Lilly) alone for acute leukemia (NCT01214603: phase II), andthe same compound in combination: (i) with gemcitabine, thecombined folate, 5-FU and oxaliplatin (FOLFOX) regimen or
the combined gemcitabine and nab-paclitaxel regimen formetastatic pancreatic cancer (NCT01632306: phase I/II); and(ii) with pemetrexed and carboplatin for advanced or metastatic solid cancer (NCT01287520: phase I).(SR64,65) Our groupis undertaking the phase I/II clinical trials by repurposing
approved GSK3b-inhibiting medicines (combined cimetidine,lithium, olanzapine and valproate regimen) in combination
withgemcitabineforadvancedpancreaticcancer(UMIN000005095) and with temozolomide for recurrent
glioblastoma (UMIN000005111) (T. Furuta, H. Sabit, D. Yu,
K. Miyashita, M. Kinoshita, M. Kinoshita, Y. Hayashi, Y.
Hayashi, T. Minamoto, M. Nakada, unpublished data, 2016).
Currently, information regarding the side effects of GSK3b
inhibitors is limited because the clinical trials have evaluatedonly a small number of seed compounds and also because
lithium chloride is the only currently approved inhibitor forclinical use. It is, therefore, difficult to predict what serious
adverse events, if any, will occur in patients treated with
GSK3b inhibitors. The extent of GSK3b inhibition and the differential effects between normal and diseased cells/tissues(therapeutic window) are critical determining factors for therapeutic efficacy and undesirable side effects. It is clear that
GSK3b activity is deregulated in various pathologicalconditions, including many, but not all, cancer types(Table 1).(10–12,22,25–27) Identifying the range of GSK3b inhibition that is efficient for treatment of disease but which results inminimal detrimental effects to normal tissues and organs will be
important for the clinical application of GSK3b inhibitors.
Numerous studies have shown that, in addition to differential
phosphorylation at S9 and Y216 residues (Fig. 1a,b), the subcellular localization of GSK3b together with various upstream
pathways can regulate the expression and activity of this kinase[reviewed in Beurel et al.(10) and Takahashi-Yanaga(22)]. New
classes of GSK3b inhibitors that spatially and temporally control the expression and activity of GSK3b may therefore be
required to reduce the risk of adverse events such as carcinogenesis that may be associated with long-term GSK3b inhibition.

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© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Acknowledgments
We thank Dr Barry Iacopetta (University of Western Australia) for
critical review of the manuscript. This study was supported by Grantin-Aids for Scientific Research (TD, IVP, TS, MN and TM) from theJapanese Ministry of Education, Science, Sports, Technology andCulture; from the Ministry of Health, Labour and Welfare, from theJapan Society for the Promotion of Science, and the ExtramuralCollaborative Research Grant of Cancer Research Institute, Kanazawa
University.

Disclosure Statement
The authors have no conflict of interest to declare.

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Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this article:
Table S1. Previous studies reporting the putative tumor suppressor roles of GSK3b.
Table S2. Clinical trials of GSK3b inhibitors for treatment of diseases.
Data S1. Supplementary references.

© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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