high 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 and interaction with stromal and inflammatory cells . these are orchestrated in a way that enables the tumor to invade the host organ 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 effect of anticancer 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 friedl9 ] . acquired resistance to chemotherapeutic and moleculartargeted agents involves distinct mechanisms , including genetic alterations to the drug targets , activation of surrogate prosurvival pathways , and interactions between tumor cells and components of the tumor environment [ reviewed in holohan et al.5 , ramos and bentiresalj6 and alexander and friedl9 ] . intratumor heterogeneity emerges in many tumors and often underlies their resistance to therapeutic agents.18 , 19 
  the processes of tumor invasion and therapy resistance and their 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 the ability to migrate and invade following the development of resistance to anticancer therapeutics ( suppl . the therapeutic modalities included conventional chemotherapeutic agents , different types of radiation therapy as well as bioactive compounds targeting epidermal growth factor ( egf ) receptor and vascular endothelial growth factor ( vegf ) . 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 all available 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  treatmentinduced metastasis these experimental and preclinical studies suggest that the invasive behavior of cancer cells and their acquired resistance to therapy may not be separate pathological properties and could , instead , represent interconnected processes [ reviewed in alexander and friedl9 ] . representative previous studies showing interrelationship between therapeutic stimuli / resistance and proinvasive phenotype in cancer s. shimozaki et al . aif , apoptosisinducing factor ; ar , androgen receptor ; bax , bcl2associated x protein ; bfgf , basic fibroblast growth factor ; bmi1 , b lymphoma momlv insertion region 1 homolog ; csc(s ) , cancer stemlike cell(s ) ; cxcl1 , chemokine ( cxc motif ) ligand 1 ; cxcr4 , cxc receptor type 4 ; dnmt3a , dna ( cytosine5)methyltransferase 3a ; egfr , epidermal growth factor receptor ; emt , epithelialmesenchymal transition ; erk , extracellular signalregulated kinase ; fak , focal adhesion kinase ; 5fu , 5fluorouracil ; hif , hypoxia inducible factor ; htert , human telomerase reverse transcriptase ; jnk , cjun nterminal kinase ; mgmt , omethylguanine dna methyltransferase ; mir , microrna ; mmp , matrix metalloproteinase ; mt1mmp , membrane type 1mmp ; nfb , nuclear factorb ; parp1 , poly [ adpribose ] polymerase 1;pgp , pglycoprotein ( multidrug resistance ) ; pi3k , phosphatidylinositol3kinase ; rb , retinoblastoma ; sparc , secreted protein , acidic , cysteine rich ; sr , supplementary reference no . ; timp , tissue inhibitor of mmp ; tnf , tumor necrosis factor ; tp53inp1 , tumor protein p53inducible nuclear protein 1 ; trail , tumor necrosis factorrelated apoptosisinducing ligand ; wave2 , was ( wiskottaldrich syndrome ) protein family member 2 . 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 molecules that mediate and interconnect these pathways include growth and transcription factors , chemokines , ras and rho family members ( e.g. ras , rac1 ) and cellmatrix 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 antiangiogenic agent . acquired resistance to bevacizumab leads to enhanced tumor cell invasion due to the metabolic shift to glycolysis and degradation and remodeling of tumor stromal tissues . a better understanding of the biological mechanisms underlying the tight association between tumor invasion and therapy resistance should provide a solid rationale for the development of innovative cancer treatments.9 gsk3 is a family of serine / threonine protein kinases comprising two highly conserved isoforms , gsk3 and gsk3 , that show approximately 85% overall homology and 98% homology in their kinase domains . gsk3 is constitutively active in normal cells and its activity is regulated by the differential phosphorylation at serine ( s ) residues 21 ( pgsk3 ) and 9 ( pgsk3 ) ( both inactive forms ) , and tyrosine ( y ) residues 279 ( pgsk3 ) and 216 ( pgsk3 ) ( both active forms ) ( fig . gsk3 regulates a diverse array of physiological cellular functions via the phosphorylation of and interaction with various proteins ( fig . although the two gsk3 isoforms share many substrates , they are not functionally identical and show some differences in their substrate specificity [ reviewed in beurel et al.10 ] . a growing number of studies have demonstrated that deregulated gsk3 activity contributes to the pathogenesis and progression of various diseases , including glucose intolerance , neurodegenerative disorders , and chronic inflammatory and immunological 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 have been developed in academic and pharmaceutical institutes over recent years . some are reported to inhibit both the  and  isoforms with different affinities , while others are specific for gsk3 ( reviewed in sr25,26 ) . among the latter compounds , it was reported that ara014418 does not inhibit the activity of 26 closely related kinases and is , therefore , considered to be highly specific for gsk3. 
  ( a ) comparison of the structural and functional domains of the two gsk3 isoforms , ( b ) the sites ( s9 and y216 ) of phosphorylation of gsk3 by different kinases regulating gsk3 activity , and ( c ) the substrates of gsk3 and proteins that interact with it . ( a ) gsk3 ( 51 kd ) and gsk3 ( 47 kd ) are products of their respective genes located in chromosomes 19q13 and 3q13 . the isoforms share high ( 98% ) homology of the catalytic domains , and gsk3 has a glycinerich extension at the nterminal side . blue and red narrow columns indicate the sites of serine ( s ) and tyrosine ( y ) phosphorylation , respectively . ( b ) the kinases indicated in blue phosphorylate gsk3s9 resulting in its inactivation , while those indicated in red phosphorylate gsk3y216 resulting in its activation . ( c ) gsk3 stabilizes / activates ( red arrows ) and destabilizes / inactivates ( blue lines ) various transcription factors as well as structural and functional proteins . ap1 , activator protein 1 ; apc , adenomatous polyposis coli ; bax , bcl2associated x protein ; bcl , bcell lymphoma ; c , cterminal of protein ; c / ebp , ccaat ( cytosinecytosineadenosineadenosinethymidine)enhancerbinding 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 ; fgd1/3 , fyve rhogef ( guanine nucleotide exchange factor ) and ph domaincontaining protein 1/3 ; fkhr , forkhead in rhabdomyosarcoma ; gly , glycine ; gr , glucocorticoid receptor ; gs , glycogen synthase ; gsk3 , glycogen synthase kinase 3 ; hif1 , hypoxia inducible factor1 ; hsf1 , heat shock transcription factor1 ; ilk , integrinlinked kinase ; ipf1/pdx1 , insulin promoter factor 1/pancreatic and duodenal homeobox 1 ; irs1 , insulin receptor substrate 1 ; lrp5/6 , lipoprotein receptorrelated 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 ( mitogenactivated protein kinase)/erk ( extracellular signalregulated kinase ) kinase ; mitf , microphthalmiaassociated transcription factor ; mlk3 , mixed lineage kinase 3 ; n , nterminal of protein ; nac , nascent polypeptideassociated complex subunit ; nfat , nuclear factor of activated tcells ; nfb , nuclear factorb ; nrf2 , nuclear factor erythroid 2related factor 2 ; p130rb , p130 retinoblastoma ; p21cip1 , p21 cdk ( cyclindependent 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 proliferatoractivated receptor ; pten , phosphatase and tensin homolog ; pyk2 , prolinerich tyrosine kinase 2 ; rar , retinoic acid receptor ; red1 , rnaediting deaminase 1 ; s , serine ; src3 , steroid receptor coactivator3 ; srebp , sterol regulatory elementbinding protein ; tsc2 , tuberous sclerosis complex 2 ; vdac , voltagedependent anion channel ; y , tyrosine of the two gsk3 isoforms , most studies in the field of oncology have focused on gsk3. this is partly because of the functional redundancy of the two isoforms in regulating the canonical wnt/catenin pathway , responsible for generating the most prevalent oncogenic signaling.23 , 24 based on its known effects on several protooncoproteins ( e.g. catenin , cyclin d1 and cmyc ) , cell cycle regulators ( e.g. p27 ) and mediators of emt ( e.g. snail ) ( fig . 1c ) , it has long been recognized that gsk3 suppresses tumor development [ reviewed in clevers and nusse,24 jope et al.,25 luo26 and mccubrey et al.27 ] , as discussed later ( table s1 ) . paradoxically , our earlier studies found increased expression and deregulated activity of gsk3 due to changes in the differential phosphorylation of s9 and y216 residues in gastrointestinal cancers , glioblastoma and osteosarcoma compared to respective normal cells and tissues.28 , 29 , 30 , 31 these observations suggested that gsk3 could participate in cancer development and progression , despite the general belief that it has tumorsuppressive functions.25 , 26 , 27 we and other research groups have shown that inhibition of gsk3 activity with specific pharmacological inhibitors , or inhibition of its expression by rna interference , can preferentially attenuate the survival and proliferation of tumor cells and induce them to undergo apoptosis . this effect has been observed not only in gastrointestinal and pancreatic cancer cells28 , 29 , 30 , 32 , 33 , 34 , 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 gynecological and urogenital cancers,39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 soft tissue sarcomas,48 , 49 hematological malignancies50 , 51 and lung cancers.52 , 53 this accumulating evidence firmly establishes gsk3 as a valuable target in cancer treatment.11 , 12 
  there has been substantial interest in the molecular mechanisms by which gsk3 favors tumor progression and by which 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)bmediated prosurvival pathway,34 , 35 , 36 , 38 tumor cell resistance to apoptosis induced by tumor necrosis factorrelated apoptosisinducing ligand ( trail),41 and failure of the p53mediated tumor suppressor pathway or of the rbmediated cell cycle regulation.31 , 32 preservation of telomere length by maintaining activity of human telomerase reverse transcriptase ( htert ) and telomerase also immortalizes tumor cells in response to the aberrant activation of gsk3.30 cell proliferation pathways mediated by cmyc and cyclin d1 sustain tumor cell proliferation that is dependent on gsk3.36 , 39 in osteosarcoma and rhabdomyosarcoma , the induction of catenin signaling , a wellknown tumor suppressive mechanism in these cancers , has been linked to decreased cell proliferation following gsk3 inhibition ( s. shimozaki et al . , unpublished data).49 another recent study reported that mitotic catastrophe caused by disruption of centrosome biodynamics was associated with attenuated tumor cell survival and proliferation following gsk3 inhibition.54 importantly , gsk3 has little impact on the cell survival , immortalization and growth of normal cells , where the differential phosphorylation of s9 and y216 residues functions to fine tune gsk3 activity.28 , 29 , 30 , 31 , 34 the growing body of evidence on the role of aberrant gsk3 in sustaining and promoting cancer cell survival , immortalization and proliferation , together with the differential effects of gsk3 inhibition on cancer and normal cells , underpins the targeting of gsk3 as a novel cancer treatment.11 , 12 the dependency of cancer cells on gsk3 for their survival and proliferation has encouraged further studies on whether aberrant gsk3 participates in tumor cell invasion and therapy resistance , the two major determinants of patient outcome.3 , 4 , 5 , 6 as shown in table 1 , most tumors that acquire proinvasive phenotypes as they evade therapy are susceptible to experimental treatment involving inhibition of gsk3. here we review what is known about the involvement of gsk3 in tumor 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 
  while gsk3 plays pivotal roles in cytoskeletal organization , cell polarity and migration in the physiological 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 gsk3inhibiting agents , inhibited the migration and invasion of glioblastoma cells , but the mechanism underlying this effect was not clear.58 , 59 subsequently , we demonstrated that inhibition of gsk3 by a specific pharmacological inhibitor and by rna interference decreases the capacity for migration and invasion of pancreatic cancer and glioblastoma cells and of their tumors in animal models.60 , 61 morphologically , these effects were associated with reduced formation of lamellipodia and invadopodia , the horizontal and vertical cellular surface microarchitectures , respectively , that drive cell migration , stromal degradation and invasion.62 , 63 inhibition of gsk3 disrupted the functional subcellular colocalization of rac164 and cortactin63 with the cytoskeletal molecule factin in lamellipodia and invadopodia , respectively . these changes in the proinvasive phenotype of tumor cells following gsk3 inhibition coincide with the suppression of molecular pathways mediated by focal adhesion kinase ( fak),65 guanine nucleotide exchange factors ( gef)/rac164 , 66 and cjun nterminal kinase ( jnk ) . this results in decreased expression of matrix metalloproteinase ( mmp)2 and membrane type ( mt ) 1mmp . our observations therefore suggest that gsk3 sustains tumor invasion via the promotion of morphological and functional proinvasive cellular phenotypes and that the molecular axis involves fak , gefs / rac1 and jnk ( fig . 2).60 , 61 
  putative molecular pathway through which deregulated gsk3 promotes tumor cell migration and invasion . the exact molecular pathway by which gsk3 mediates the activation of fak ( open arrow ) remains to be determined . fak , focal adhesion kinase ; gdp , guanosine diphosphate ; gef , guanine nucleotide exchange factor ; gsk3 , glycogen synthase kinase 3 ; gtp , guanosine triphosphate ; jnk , cjun nterminal kinase ; mmp , matrix metalloproteinase ; mt1mmp , membrane type 1mmp ; circled p , phosphorylation subsequent to our studies,60 , 61 other workers have reported the proinvasive effect of gsk3 in pancreatic , colorectal and breast cancer cells in association with modulation of cytoskeletal organization and cytokinemediated matrix degradation.67 , 68 . these studies highlight the pivotal role of gsk3 in tumor invasion and probably also in metastatic spread . the combination of multiple agents having different targets and mechanisms of actions 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 , moleculartargeted therapy is often combined with conventional chemotherapeutics and/or radiation therapy , as well as with other targeted agents.7 , 8 as discussed above , accumulating evidence on the effects of gsk3 inhibition against cancer cell survival and proliferation has led us to address whether this could be used in combination with chemotherapy and irradiation . the standard treatment modalities prescribed are often ineffective in patients with refractory tumors such as pancreatic cancer and glioblastoma.55 , 56 we have shown that administration of a gsk3specific pharmacological inhibitor ( e.g. ara014418 ) at a dose below that which results in a therapeutic 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 pancreatic cancer , respectively.55 , 56 lowdose gsk3 inhibitor given in combination with chemotherapeutic agents had additive and synergistic effects . investigation of the underlying molecular mechanism is crucial to justify treatment combinations of gsk3 inhibitor with other anticancer therapeutics ( fig . , the combined effect was attributed to modulation of the mouse double minute ( mdm)2/p53 and cmycmediated pathways resulting in epigenetic silencing of o6methylguanine dna methyltransferase ( mgmt ) , a clinically proven biomarker of temozolomide efficacy56 in tumor cells ( fig . 3b).31 , 69 in pancreatic cancer , inhibition of tumor gsk3 activity decreased cyclin d1/cyclindependent kinase ( cdk)4/6 complexdependent 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 gsk3 may , therefore , sensitize pancreatic cancer to gemcitabine by downregulating the expression of ribonucleotide reductase , a wellknown pharmacological biomarker of gemcitabine55 that is regulated by the rb / e2f pathway ( fig . 3a).71 , 72 in addition to modulation of this pathway , we found that downregulation of the tp53inp1 gene ( encoding tumor p53inducednuclearprotein 1 ) involved in dna repair is associated with the chemosensitizing effect of gsk3 inhibitor.70 the gsk3 inhibitor used in these studies also sensitized both pancreatic cancer and glioblastoma to ionizing radiation.31 , 60 this radiosensitizing effect may depend on the restoration of rb function following gsk3 inhibition , resulting in the inability of e2f1 to induce the transcription of thymidylate synthase and thymidine kinase ( fig . ( a ) putative molecular pathway that links gsk3 activity with the resistance of pancreatic cancer cells to dna damage induced by gemcitabine and ionizing radiation . the effects of gsk3 on e2fdependent gene transcription and on the expression of rr , ts and tk remain to be determined . cdk , cyclindependent kinase ; e2f , e2 factor ; circled p , phoshorylation ; rb , retinoblastoma ( tumor suupressor protein ) ; rr , ribonucleotide reductase ; tk , thymidine kinase ; ts , thymidylate synthase . gsk3 inhibition results in cmyc activation directly and via activation of cateninmediated signaling , which consequently increases recruitment of dnmt3a by cmyc to the mgmt promoter , thus increasing de novo 
 dna methylation in the mgmt promoter . other recent studies have also reported that gsk3 inhibition allows therapyresistant colon and pancreatic cancer cells to become susceptible to 5fluorouracil ( 5fu),73 and renal cell carcinoma cells to become susceptible to a synthetic multikinase inhibitor ( sorafenib ) that targets growth signaling and angiogenesis.74 together , these studies suggest that gsk3 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 gsk3 is a molecular  hub  that mediates and connects various pathways responsible for tumor invasion and resistance to therapy . together with the role of gsk3 in promoting tumor cell survival and proliferation and its differential functions between tumor and normal cells [ reviewed in miyashita et al.11 and mccubrey et al.12 ] , this evidence reinforces the promise of novel cancer therapeutic strategies that target gsk3. while gsk3 plays pivotal roles in cytoskeletal organization , cell polarity and migration in the physiological 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 gsk3inhibiting agents , inhibited the migration and invasion of glioblastoma cells , but the mechanism underlying this effect was not clear.58 , 59 subsequently , we demonstrated that inhibition of gsk3 by a specific pharmacological inhibitor and by rna interference decreases the capacity for migration and invasion of pancreatic cancer and glioblastoma cells and of their tumors in animal models.60 , 61 morphologically , these effects were associated with reduced formation of lamellipodia and invadopodia , the horizontal and vertical cellular surface microarchitectures , respectively , that drive cell migration , stromal degradation and invasion.62 , 63 inhibition of gsk3 disrupted the functional subcellular colocalization of rac164 and cortactin63 with the cytoskeletal molecule factin in lamellipodia and invadopodia , respectively . these changes in the proinvasive phenotype of tumor cells following gsk3 inhibition coincide with the suppression of molecular pathways mediated by focal adhesion kinase ( fak),65 guanine nucleotide exchange factors ( gef)/rac164 , 66 and cjun nterminal kinase ( jnk ) . this results in decreased expression of matrix metalloproteinase ( mmp)2 and membrane type ( mt ) 1mmp . our observations therefore suggest that gsk3 sustains tumor invasion via the promotion of morphological and functional proinvasive cellular phenotypes and that the molecular axis involves fak , gefs / rac1 and jnk ( fig . 2).60 , 61 
  putative molecular pathway through which deregulated gsk3 promotes tumor cell migration and invasion . the exact molecular pathway by which gsk3 mediates the activation of fak ( open arrow ) remains to be determined . fak , focal adhesion kinase ; gdp , guanosine diphosphate ; gef , guanine nucleotide exchange factor ; gsk3 , glycogen synthase kinase 3 ; gtp , guanosine triphosphate ; jnk , cjun nterminal kinase ; mmp , matrix metalloproteinase ; mt1mmp , membrane type 1mmp ; circled p , phosphorylation . subsequent to our studies,60 , 61 other workers have reported the proinvasive effect of gsk3 in pancreatic , colorectal and breast cancer cells in association with modulation of cytoskeletal organization and cytokinemediated matrix degradation.67 , 68 . these studies highlight the pivotal role of gsk3 in tumor invasion and probably also in metastatic spread . the combination of multiple agents having different targets and mechanisms of actions 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 , moleculartargeted therapy is often combined with conventional chemotherapeutics and/or radiation therapy , as well as with other targeted agents.7 , 8 as discussed above , accumulating evidence on the effects of gsk3 inhibition against cancer cell survival and proliferation has led us to address whether this could be used in combination with chemotherapy and irradiation . the standard treatment modalities prescribed are often ineffective in patients with refractory tumors such as pancreatic cancer and glioblastoma.55 , 56 we have shown that administration of a gsk3specific pharmacological inhibitor ( e.g. ara014418 ) at a dose below that which results in a therapeutic 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 pancreatic cancer , respectively.55 , 56 lowdose gsk3 inhibitor given in combination with chemotherapeutic agents had additive and synergistic effects . investigation of the underlying molecular mechanism is crucial to justify treatment combinations of gsk3 inhibitor with other anticancer therapeutics ( fig . , the combined effect was attributed to modulation of the mouse double minute ( mdm)2/p53 and cmycmediated pathways resulting in epigenetic silencing of o6methylguanine dna methyltransferase ( mgmt ) , a clinically proven biomarker of temozolomide efficacy56 in tumor cells ( fig . 3b).31 , 69 in pancreatic cancer , inhibition of tumor gsk3 activity decreased cyclin d1/cyclindependent kinase ( cdk)4/6 complexdependent 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 gsk3 may , therefore , sensitize pancreatic cancer to gemcitabine by downregulating the expression of ribonucleotide reductase , a wellknown pharmacological biomarker of gemcitabine55 that is regulated by the rb / e2f pathway ( fig . 3a).71 , 72 in addition to modulation of this pathway , we found that downregulation of the tp53inp1 gene ( encoding tumor p53inducednuclearprotein 1 ) involved in dna repair is associated with the chemosensitizing effect of gsk3 inhibitor.70 the gsk3 inhibitor used in these studies also sensitized both pancreatic cancer and glioblastoma to ionizing radiation.31 , 60 this radiosensitizing effect may depend on the restoration of rb function following gsk3 inhibition , resulting in the inability of e2f1 to induce the transcription of thymidylate synthase and thymidine kinase ( fig . ( a ) putative molecular pathway that links gsk3 activity with the resistance of pancreatic cancer cells to dna damage induced by gemcitabine and ionizing radiation . the effects of gsk3 on e2fdependent gene transcription and on the expression of rr , ts and tk remain to be determined . cdk , cyclindependent 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 gsk3 signaling in glioblastoma . gsk3 inhibition results in cmyc activation directly and via activation of cateninmediated signaling , which consequently increases recruitment of dnmt3a by cmyc to the mgmt promoter , thus increasing de novo 
 dna methylation in the mgmt promoter . other recent studies have also reported that gsk3 inhibition allows therapyresistant colon and pancreatic cancer cells to become susceptible to 5fluorouracil ( 5fu),73 and renal cell carcinoma cells to become susceptible to a synthetic multikinase inhibitor ( sorafenib ) that targets growth signaling and angiogenesis.74 together , these studies suggest that gsk3 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 gsk3 is a molecular  hub  that mediates and connects various pathways responsible for tumor invasion and resistance to therapy . together with the role of gsk3 in promoting tumor cell survival and proliferation and its differential functions between tumor and normal cells [ reviewed in miyashita et al.11 and mccubrey et al.12 ] , this evidence reinforces the promise of novel cancer therapeutic strategies that target gsk3. targeting of the biological pathways that link tumor invasion and therapy resistance is clearly an attractive prospect for the development of innovative cancer treatments . another important challenge is to understand the biology of cancer initiating or stemlike cells ( csc ) so that  stemness  phenotypes can also be targeted . based on the clonal evolution model of tumorigenesis and on the normal stem cell concept , csc are conceptually 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 , intratumor heterogeneity , invasion and metastasis , therapy resistance and tumor relapse after surgery and adjuvant treatment.14 , 75 eradication of csc is , therefore , of paramount clinical importance for combatting refractory cancer.15 , 76 
  gsk3 has pivotal functions in normal cell biology10 , 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 share unique 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 gsk3 underlies the basal mechanism for sustaining the csc phenotype ( fig . recent studies have shown that gsk3 negatively controls the differentiation of malignant glioma cells,77 and that gsk3 inhibition results in depletion of cancer stemlike or progenitorlike cells and attenuation of metastatic spread in prostate cancer.78 consistent with the physiological roles of gsk3 in negatively regulating canonical wnt/catenin and hedgehog signaling,10 , 23 , 24 inhibition of gsk3 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 influence of gsk3 on both tumor and normal stem cell phenotypes . this knowledge can be applied for the development of novel cancer treatments that target gsk3. involvement of gsk3 in the representative pathological hallmarks of cancer . gsk3 positively regulates the distinct molecular pathways and participates in survival , proliferation , migration and invasion of tumor cells and their insensitivity and resistance to cancer therapy . one of the concerns about targeting gsk3 for cancer treatment is that its inhibition may promote the progression of existing tumors [ reviewed in takahashiyanaga22 , jope et al.,25 luo26 and mccubrey et al.27 ] . a number of studies have focused on the putative tumor suppressive role of gsk3 ( table s1 ) . many of these report inactivation or activation of gsk3 as a mediating event in pathways leading to tumor progression or suppression , respectively . an inverse association between tumor expression of gsk3 ( either total or active form pgsk3 ) and the survival of cancer patients has been reported . other studies found causal links between pharmacological gsk3 inhibition and/or depletion of gsk3 expression ( e.g. gene knockout and rna interference ) or activity ( e.g. recombinant kinasedead or constitutively active mutant form ) and tumor cell survival , proliferation and susceptibility to cancer therapies . gsk3 inhibition strategies and the development of gsk3targeted agents therefore require careful evaluation to determine whether the tumor promoting function of gsk3 is counteracted by its putative tumor suppressor function in different cancer types . the development and administration of gsk3 inhibitors for the treatment of chronic diseases , including diabetes mellitus , neurodegenerative disorders and cancer , requires serious awareness of the safety issues . a major concern is whether and how gsk3 can be targeted to treat disease , because its systemic inhibition may lead to unwanted effects by disrupting multiple biological pathways ( fig . most notably , longterm inhibition of gsk3 may initiate tumorigenesis due to its role in suppressing protooncogenic pathways , in particular that are mediated by catenin [ reviewed in takahashiyanaga22 , jope et al.,25 luo26 and mccubrey et al . a previous study showed that genetic deletion of gsk3 in mammary epithelial cells resulted in catenin activation and induced intraepithelial neoplasia that progressed to the development of adenosquamous carcinoma . moreover , overexpression of wildtype and constitutively active mutant gsk3 in 12otetradecanoylpholbor13acetate ( tpa)mediated , transformationresistant mouse epidermal cells suppressed egfmediated and tpamediated anchorageindependent growth in soft agar and tumorigenicity in rodents ( table s1 ) . however , there is currently no direct evidence to support tumor development in vivo following treatment with a gsk3 inhibitor [ reviewed in miyashita et al.11 and sr27 ] . as discussed in previous studies that report cancer therapeutic effects of gsk3 inhibition ( table 1 ) , none of the available experimental gsk3 inhibitors induces neoplastic transformation of nonneoplastic ( normal ) cells or tumor development in experimental animal models [ reviewed in miyashita et al.11 and sr27 ] . longterm prescription of lithium , the only gsk3 inhibitor approved for the treatment of bipolar disorder since the 1950s , has not been associated with an increased risk of cancer or death from cancer . posttranslational regulation of gsk3 activity via the phosphorylation of s9 and y216 ( pgsk3 versus pgsk3 ) ( fig . 1b ) in response to various stimuli could partly underlie a mechanism that protects normal cells from the detrimental effects of gsk3 inhibition . despite the concerns outlined above , clinical trials for neurodegenerative diseases and cancer have tested some seed pharmacological gsk3( ) inhibitor compounds and also approved medicines with the ability to inhibit gsk3 activity ( table s2 ) . the former trials include azd1080 ( 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 ) and of alzheimer 's disease ( nct01350362 : phase ii ) . clinical trials for cancer treatment have used ly2090314 ( eli lilly ) alone for acute leukemia ( nct01214603 : phase ii ) , and the same compound in combination : ( i ) with gemcitabine , the combined folate , 5fu and oxaliplatin ( folfox ) regimen or the combined gemcitabine and nabpaclitaxel regimen for metastatic pancreatic cancer ( nct01632306 : phase i / ii ) ; and ( ii ) with pemetrexed and carboplatin for advanced or metastatic solid cancer ( nct01287520 : phase i ) . our group is undertaking the phase i / ii clinical trials by repurposing approved gsk3inhibiting medicines ( combined cimetidine , lithium , olanzapine and valproate regimen ) in combination with gemcitabine for advanced pancreatic cancer ( 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 gsk3 inhibitors is limited because the clinical trials have evaluated only a small number of seed compounds and also because lithium chloride is the only currently approved inhibitor for clinical use . it is , therefore , difficult to predict what serious adverse events , if any , will occur in patients treated with gsk3 inhibitors . the extent of gsk3 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 gsk3 activity is deregulated in various pathological conditions , including many , but not all , cancer types ( table 1).10 , 11 , 12 , 22 , 25 , 26 , 27 identifying the range of gsk3 inhibition that is efficient for treatment of disease but which results in minimal detrimental effects to normal tissues and organs will be important for the clinical application of gsk3 inhibitors . numerous studies have shown that , in addition to differential phosphorylation at s9 and y216 residues ( fig . 1a , b ) , the subcellular localization of gsk3 together with various upstream pathways can regulate the expression and activity of this kinase [ reviewed in beurel et al.10 and takahashiyanaga22 ] . new classes of gsk3 inhibitors that spatially and temporally control the expression and activity of gsk3 may therefore be required to reduce the risk of adverse events such as carcinogenesis that may be associated with longterm gsk3 inhibition .