Current Understanding on EGFR and Wnt

Genes & Cancer4(11-12) 427 –446© The Author(s) 2013Reprints and permissions: /journalsPermissions.nav DOI: 10.1177/1947601913503341 IntroductionMalignancies of the central nervous system are generally classified as meningiomas, oligodendrogliomas, and astro-cytomas. Astrocytomas are further subdivided into pilo-cytic (grade I), diffuse (grade II), anaplastic (grade III), and glioblastoma multiforme (GBM; grade IV). GBM repre-sents the most common and aggressive primary brain tumor with a median survival of approximately 12 months after diagnosis. Despite aggressive therapeutic modalities includ-ing surgical resections followed by radiotherapy and the most up-to-date chemotherapeutic regimen, the prognostic statistics for GBM patients is discouraging. Treatment of GBM demands a multipronged attack, based on a compre-hensive understanding of the molecular principles govern-ing the growth and survival of malignant cells. The biggest challenges in GBM treatment are (a) the extremely narrow window for operative approaches, which is mostly pallia-tive and rarely curative; and (b) the blood–brain barrier, which poses a major challenge in successful drug delivery. Similarly, radiation therapy alone for GBM patients is unlikely to be curative. Therefore, most investigators and clinicians believe that development of better chemothera-peutic agents targeting multiple and specific aspects of gli-oma holds the best promise for patient outcome.1 More than 2 decades of research has identified important molecular events, such as overactivation of the EGFR and PI3K (phosphatidylinositide 3 kinase)/Akt pathways and inactivation of the p53/Rb pathways to be important con-tributors for glioma development. These alterations facili-tate cell survival, proliferation, and migration and thus are crucial for tumorigenesis.2 Another important factor that has recently emerged to be a significant determinant of gli-oma initiation and progression is the canonical Wnt/β-catenin signaling pathway. Apart from a direct survival advantage provided by Wnt signaling to the tumor cells,3 its role in stem cell biology is crucial.4 The widespread atten-tion the concept of cancer stem cells (CSCs) has gained in the last few years makes Wnt signaling an attractive target for glioma therapy.5However, it is surprising that very little work has been devoted to studying the crosstalk between these 2 signaling pathways in the context of glioma. Although published evi-dences are limited, in the last part of the review we focus on the collusion of EGFR and canonical Wnt pathways in gli-1Signal T ransduction in Cancer and Stem Cells Laboratory, Cancer Biology and Inflammatory Disorder Division, Indian Institute of Chemical Biology, Kolkata, India*Authors contributed equally.Corresponding Author:Mrinal K. Ghosh, Cancer Biology and Inflammatory Disorder Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road,Kolkata-700 032, India (Email: mrinal.res@).Current Understanding on EGFRand Wnt/β-Catenin Signaling inGlioma and Their Possible CrosstalkIndranil Paul1,*, Seemana Bhattacharya1,*, Anirban Chatterjee1,and Mrinal K. Ghosh1Submitted 9-Apr-2013; accepted 31-Jul-2013AbstractGlioblastoma multiformes (GBMs) are extensively heterogeneous at both cellular and molecular levels. Current therapeutic strategies include targeting of key signaling molecules using pharmacological inhibitors in combination with genotoxic agents such as temozolomide. In spite of all efforts, the prognosis of glioma patients remains dismal. Therefore, a proper understanding of individual molecular pathways responsible for the progression of GBM is necessary. The epidermal growth factor receptor (EGFR) pathway is probably the most significant signaling pathway clinically implicated in glioma. Not surprisingly, anti-EGFR therapies mostly prevail for therapeutic purposes. The Wnt/β-catenin pathway is well implicated in multiple tumors; however, its role in glioma has only recently started to emerge. We give a concise account of the current understanding of the role of both these pathways in glioma. Last, taking evidences from a limited literature, we outline a number of points where these pathways intersect each other and put forward the possibility of combinatorially targeting them for treatment of glioma.Keywordsglioma, signaling, EGFR, β-catenin, crosstalkReview428 Genes & Cancer / vol 4 no 11-12 (2013)oma and try to point toward the possibility of combinatori-ally targeting them for the treatment of glioma.EGFR and Wnt Are Important Survival and Growth Signaling PathwaysEGFR Signaling. The epidermal growth factor receptor (EGFR; also ErbB1 or Her1) belongs to the ErbB family consisting of 4 receptors (ErbB or Her 1-4) and 13 polypep-tide extracellular ligands with a conserved EGF domain. Structurally, this 170 kDa protein consists of an extracellu-lar ligand-binding domain, a hydrophobic domain spanning the membrane, and a cytosolic tyrosine kinase domain. On binding of ligand to the extracellular domain, the receptor dimerizes (homo or heterodimers), leading to phosphoryla-tion of the cytosolic tail (autophosphorylation and/or cross-phosphorylation) with subsequent activation of several downstream kinases and finally transcription factors.6 On activation of the EGFR, various adaptor and effector mole-cules like Shc (Src homology 2 containing transforming protein); Grb2 and Grb7 (growth factor receptor-bound pro-tein); c-Crk-p38; PLC-γ (phospholipase C-γ); kinases—JAKs (Janus kinases), Src (c-Src tyrosine kinase), and PI3K; protein tyrosine phosphatases—SH P1 and 2; E3 ligase Cbl; and transcription factors like STAT (signal transducer and activator of transcription) 1, 3, and 5 are activated, triggering several signaling cascades—Ras/Raf (rat sarcoma/rapidly accelerated fibrosarcoma), MAPK (mitogen activated protein kinase), PKC (protein kinase C), PI3K/PKB (protein kinase B), JAK/STAT, and so on—which results in the activation of Erk (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), p38-MAPK, NF-κB (nuclear factor κ-light-chain-enhancer of activated B-cells), transcription factors like c-Myc, and so on, that ultimately regulate the expression of genes involved in proliferation, survival, differentiation, and migration7-9 (see Fig. 1).EGFR phosphorylated at residues Y1068 and Y1086 can recruit Grb2 directly or indirectly via tyrosine phosphory-lated Shc leading to translocation of Grb2/Sos complex to the membrane, where it activates Ras proteins, which in turn induce Raf kinases, MEKs and MAPKs, and Erk1/2 nuclear transport leading to cell proliferation by activating transcription factors like c-Myc and RSK (ribosomal S6 kinase).10 Recently, EGFR-mediated MAPK signaling has been shown to attenuate the Groucho-mediated gene repres-sion, establishing a node for crosstalk between the EGFR, Notch, Wnt, and TGF-β (transforming growth factor-β) sig-naling pathways.11-13 EGFR-mediated PI3K/ PDK1(phosphoinositide-dependent protein kinase 1)/Akt pathway plays a crucial role in sustained cell survival and proliferation. Phospho-EGFR-Y920 can dock PI3K, con-verting PIP2 to PIP3 (phosphatidyl inositol (4,5) bisphos-phate to (3,4,5) triphosphate), phosphorylating Akt, which inactivates the apoptotic cascade via Bad (Bcl2 associated death factor) and caspase-9.14 In PTEN (phosphatase and tensin homolog) null or mutated GBMs also, Akt is consti-tutively activated, thus providing survival signals.15 STATs are very important transcription factors downstream of EGFR responsible for cellular transformation and migra-tion.16,17 JAK dependent activation of STAT1 and STAT3 or JAK-independent activation of STAT 5b (docking at EGFR Y845)18 leads to their dimerization (homo and heterodi-mers) and nuclear translocation, where they act as tran-scription factors for several growth promoting genes like C-JuN, C-FOS, and so on.19,20 Interestingly, inhibiting the activity of PI3K and in turn Akt significantly increases the DNA-binding activity of STAT3 in u87MG and D54 cells. While considering this apparent contradiction, it should be noted that the major oncogenic role of STAT3 is because of the phosphorylation at Y705 while the negative regulation being discussed here concerns S727 and the other Ser/Thr residues at the C-terminal end of STAT3. Also, the phos-phorylation at S727 has opposing roles, both oncogenic and tumor suppressive, in a context-specific manner. The find-ing, however, indicates a crosstalk between the 2 survival pathways, which decides the cell fate via a common player, Mcl-1 in this case.21 Regulation of angiogenesis and metas-tasis are important functions of the EGFR. EGFR promotes angiogenesis by upregulating VEGF (vascular endothelial growth factor) and MMPs (matrix metalloproteinases).22 Also, phosphorylated EGFR (Y992) directly interacts with and activates PLC-γ, which regulates actin cytoskeletal reorganization and hence cell motility.23 Thus, in general, the multifaceted nature of the EGFR signaling in cancer can be clearly understood, which is being exploited as therapy, and some of the molecules in the EGFR pathway are impor-tant targets for drug development.The EGFR gene is a major target for alterations in glioma. EGFR is one of the major genetic factors affecting the pathogenesis and prognosis of GBM. Genetic amplifica-tion, elevated expression, and mutation of EGFR have been widely implicated in various cancers and its role in influ-encing multiple aspects of tumorigenesis is well docu-mented. Almost 50% to 60% of GBMs are genetically marked with overactivated EGFR and multiple functionally distinct forms of EGFR have been associated with glioma (see Table 1). The most common mutation is EGFRvIII (in-frame deletion spanning exons 2-7, imparting ligand- independent activation and high oncogenicity). In primary GBMs, EGFR aberrations are accompanied with mutations in tumor suppressors p16Ink4a, p19Arf, and PTEN, consti-tutively activating several oncogenes like STATs, Akt, Erk1/2, and so on, while secondary GBMs are mostly asso-ciated with mutation/deletions in p53, deregulating the cell cycle and also aberrant activation of PDGFR (platelet-derived growth factor receptor) pathway and IDH1EGFR and Wnt/β-catenin signaling crosstalk in glioma / Paul et al. 429(isocitrate dehydrogenase 1) mutations. In PTEN null/mutated GBMs, Akt is constitutively activated, providing survival signals.24,25Alternate mechanisms for activating EGFR in glioma . Apart from EGFR itself, a number of different mechanisms have been used by cancer cells to overactivate EGFR signaling. For example, the ligands of receptor tyrosine kinase (RTK) family such as PDGF, TGF-α, and EGF are often overex-pressed in glioma leading to positive feedback autocrine loops.35,36 Also, the need for EGFR overactivation can bebypassed or complemented by activating other RTKFigure 1. An overview of EGFR and Wnt/β-catenin signaling. EGFR signaling pathway . On binding of specific ligands, the EGF receptor dimerizes, activating an array of molecules including kinases, Grbs, and so on, initiating several downstream signaling cascades—MAPK, PI3K/Akt, JAK/STAT—which upregulate multiple transcription factors, anti-apoptotic and prosurvival proteins pushing the cells toward prolonged survival, proliferation, migration, and tumorigenesis. Wnt signaling pathway. In the absence of Wnt, β-catenin is captured by APC and Axin and forms a destruction complex in which its phosphorylation by the kinases CK1α and GSK3β is facilitated, which, in turn, allows binding of β-T rCP , which subsequently mediates the proteasomal degradation of β-catenin. On binding of the Wnt ligand to its cognate receptors Fzd and LRP5/6 triggers the formation of Dvl–Fzd complexes and the phosphorylation of LRP by CK1γ, facilitating relocation of Axin to the membrane and inactivation of the destruction complex. This allows β-catenin to accumulate and enter the nucleus, where it interacts with members of the Tcf/Lef family and exerts its transcriptional activity. For a more comprehensive and detailed account of these pathways, the readers should consult excellent reviews available in the literature.Abbreviations: AR, amphiregulin; EPG, epigen; EREG, epiregulin; Cbl E3 ligase, casitas B-lineage lymphoma; mTOR, mammalian target of rapamycin; CK1γ, casein kinase 1 γ; uPAR, urokinase-type plasminogen activator receptor. EGF , EGFR, TGF α, LRP , Shc, PLC, Shp, MAPK, PI3K, PTEN, JAK, Stat, Dvl, Fzd, CK1α, GSK3β, APC, β-TRCP , Tcf, Lef, VEGF , IL, PPAR—see text.430 Genes & Cancer / vol 4 no 11-12 (2013)T able 1. Various Mutations in EGFR Leading to Its Constitutive Activity and Enhanced Oncogenicity in Glioma.Mutant Genetics Size (kDa)Functional Significance Occurrence References Overexpression WT Gene amplification170EGFR overexpression associated withtumoral proliferation37% to 58%26 WT No gene amplification12% to 38% In-frame deletion vI N-terminal deletion145T ranslation starts at 543. Structur-ally similar to v-erbB1 productand shows constitutive receptoractivation In a small number of human gliomas27-29 vIIΔ521-603 (exons14-15; cysteine-richregion)150Impairs disulphide bonds, binds ligand,enhanced TK activity; inducesgrowth advantage12% to 16%28-30 vIIIΔ6-273 (exons 2-7)140Highly oncogenic. Constitutive,ligand-independent, tyrosine kinaseactivity; imparts a selective growthadvantage Most common—55%to 62%28-30 vIVΔ959-1030 (exons25-27)167Associated with an overresponse inthe presence of exogenous ligands28-30 vV (958T or C-958)T runcated at the C-terminal at position958105Increased ligand-dependent kinaseactivity; shows an overresponse withexogenous ligands; imparts a selec-tive growth advantage11% to 17%28-30 vVIΔ6-273 and C-1030Oncogenic27, 31 vVIIΔ6-273 and C-958Oncogenic27, 31 Δ2-5Deletion of exons 2-5Oncogenic27, 31 vIII/Δ12-13Δ6-273 and Δ409-520(exons 2-7 and12-13)125Oncogenic27, 31 Δ14Deletion of 521-603Oncogenic27, 31 Δ14/vVΔ521-603 and trun-cated beyond 958Oncogenic27, 31 Δ25Deletion of exon 25Oncogenic27, 31DeletionLREA; Δ746-750Deletion of 4 con-served amino acids(LREA) in exon 19Oncogenic mutant with increased TKdomain dimerization and enhancedautophosphorylation32, 33T andem duplication TDM/2-7Exons 2-7 (codons6-273)180Constitutive ligand-independent acti-vation with high basal phosphoryla-tion TDM/18-25Exons 18-25 (codons664-1014)185Ligand-independent and constitutivelyautophosphorylated. Induces colonyformation in soft agar and rapidtumor growth in nude miceFound in A1235 andKE glioma cell lines27, 29, 31, 33 TDM/18-26Exons 18-26 (codons664-1030)190Poorly downregulated by ligand andconstitutively autophosphorylatedExpressed in A172glioma cells27, 29, 31, 33 TDM/I7-E8 (315T)Duplication in intron7 through exon 8(truncation at 315)Oncogenic27, 31, 34Point mutation L858R170Oncogenic mutant with increased TKdomain dimerization and enhancedautophosphorylation32, 33 572, 604Missense170Within the second cysteine-richregion of the extracellular domain.Probably have a role to play inreceptor activation28EGFR and Wnt/β-catenin signaling crosstalk in glioma / Paul et al.431signaling such as PDGFR or by functionally obliterating important negative regulators such as PTEN. As a result, several crucial downstream players of EGFR pathway such as Akt and Ras gain constitutive activity.37-39 Similarly, in a subset of GBM, monoallelic loss of NF-κB has been shown to cooperate with EGFR to negatively affect patient prog-nosis.40 Furthermore, observation that the bispecific EGFR/ HER2 inhibitor AEE788 displays radiosensitizing effect on EGFR overexpressing cells suggests that at least in certain treatment modalities the responsiveness of therapy depends on the relative ratio between EGFR and HER2 and not on any individual receptor.EGFR is involved in sustenance of glioma stem cells (GSCs). EGFR signaling regulates proliferation and migration of neural stem cells (NSCs). The balance between EGFR sig-naling and a classical stem-cell signaling like Notch decides the proportion of cells in the stem cell niche.41 Hence, the tumor microenvironment is a crucial factor in the mainte-nance of tumor initiating and self-renewing capacity of the CSCs. Like normal NSCs, the GSCs probably maintain their undifferentiated state and multipotency due to regulation of signaling in their niche.42 Constitutively activated EGFR signaling (EGFRvIII) in GBM leads to activation of Akt, which can directly phosphorylate Smad5, which in turn leads to overexpression of ID3 (inducer of differentiation 3) and the cytokines like GRO1 (growth regulated alpha pro-tein), interleukins (IL-6 and IL-8) supporting proliferation, angiogenesis, and acquisition of GSC-like characteristics. This signaling cascade imparts tumorosphere-forming abil-ity to the GSCs and can be inhibited by EGFR inhibitors.43 The growing tumor mass around the GSCs are often exposed to several stresses like hypoxia, low pH, nutrient deprivation at later stages,44 and over 2 decades of research has established hypoxia to promote tumor cell proliferation, survival, angiogenesis, and motility, by triggering the adap-tive transcriptional mechanisms. During hypoxic stress, the stem cell properties of neural progenitors (NPCs) are enhanced,45 and also CD133+ (cluster of differentiation 133 positive) GSCs proliferate.46 One of the key factors induced by hypoxia is H IF-1α (hypoxia-inducible factor 1α),47 which is also regulated by EGF and other growth promoting signals like bFGF (basic fibroblast growth factor), PDGF, IGF (insulin-like growth factor), although to a lesser extent. Studies have revealed the crucial role of EGFR signaling cascade in the maintenance of GSCs, wherein EGF is capa-ble of promoting sphere formation and self-renewal of the CD133+ subpopulation. This can be inhibited by specific pharmacological inhibitors like Gefitinib and AG1478 (tyrosine kinases), PD98059 (Erk1/2, MAPK), LY294002 (PI3K), and Rapamycin (mTOR).48 Hypoxia inhibits differ-entiation of these CD133+ GSCs and supports their prolif-eration mainly by upregulating HIF-1α and VEGF (acting as a paracrine signal) and also by activating the EGFR downstream signaling molecules PI3K/Akt and Erk1/2, which in turn regulate HIF-1α.46,49Targeting EGFR signaling for glioma therapy. EGFR signal-ing has been classically the central target for GBM therapy. The past 3 to 4 decades has seen the development of several strategies to curb EGFR activities ranging from small mol-ecule inhibitors to engineered antibodies and viruses. Blocking the tyrosine kinase activity of EGFR has been the most attractive strategy in antitumor therapy. One approach has been to use several competitive and noncompetitive kinase inhibitors like erlotinib, gefitinib, and other second-generation inhibitors like afatinib, dacomitinib, often in combination with radiation and temozolomide. Another approach in targeting EGFR is using monoclonal antibodies (mAbs), like cetuximab, nimotuzumab, and so on, which have also shown preliminary antitumor effects in GBM. Apart from EGFR itself, various downstream effectors of the cascade have also been targeted with varying degrees of success (see Table 2 and references therein).Wnt Signaling. Canonical Wnt signaling (β-catenin depen-dent) regulates a wide set of genes that in turn orchestrate diverse cellular functions such as morphogenesis, differen-tiation, and proliferation. Consequently, aberrant activation of the canonical Wnt pathway has been found to be the driver of several human cancers including glioma. In the absence of Wnt ligands, the cell gets rid of excess β-catenin by assembling a multiprotein destruction complex com-posed mainly of Axin, APC (adenomatous polyposis coli), and GSK3β (glycogen synthase kinase 3-β), on which β-catenin is first phosphorylated by CK1α (casein kinase 1-α), followed by GSK3β and then ubiquitinated by the E3 ligase β-TrCP (β-transducin repeat containing protein) to be subsequently degraded through the 26S proteasome system. In the absence of nuclear β-catenin, the Tcf/Lef (T-cell spe-cific transcription factor/lymphoid enhancer-binding fac-tor) family of transcription factors (Tcf1, Tcf3, Tcf4, and Lef1) recruit the transcriptional co-repressors Groucho/ TLE and efficiently repress expression of their target genes. The interaction of Wnt with Fzd/LRP (Frizzled/lipoprotein-receptor related protein) changes the state of things in ways for which a number of biochemical and cellular mecha-nisms have been proposed: first, it may trigger the forma-tion of Dishevelled (Dvl)–Fzd complex, which in turn, sequesters Axin (and possibly GSK3β) to the plasma mem-brane thus blocking the formation of destruction com-plex85-90; second, Wnt induced phosphorylation of LRP6 at PPPSPXS motifs by GSK3β and CK1γ may create docking sites for Axin, facilitating its sequestration on the mem-brane and inactivation of the destruction complex91,92; third, Wnt induced phosphorylation of PPPSPXS motifs of LRP6 may inactivate the catalytic pocket of GSK3β, thus directly blocking its activity against β-catenin93-96; fourth,432T a b l e 2. P h a r m a c o l o g i c a l C o m p o u n d s T a r g e t i n g E G F R a n d W n t /β-C a t e n i n P a t h w a y s i n G l i o m a .P h a r m a c o l o g i c a l I n h i b i t o r s /D r u g s [C o m p a n y ]M o d e o f A c t i o nC l i n i c a l O u t c o m e R e f e r e n c e sT a r g e t i n g E G F R p a t h w a y M R 1 i m m u n o t o x i nE n g i n e e r e d c h i m e r a o f s cF v a n d P s e u d o m o n a s e x o t o x i n A . R e c e p t o r b l o c k e r ; c y t o t o x i c t o EG F R v I I I e x p r e s s i n g c e l l s A g o o d t a r g e t f o r p r e c l i n i c a l t r i a l s 50T P -38R e c o m b i n a n t c h i m e r a o f T G F -α f u s e d w i t h a m u t a t e d f o r m o f P s e u d o m o n a s e x o t o x i n P E -38. T a r g e t s E G F R s i g n a l i n g 20% p a t i e n t s h a v e s h o w n r e s p o n s e i n p h a s e I t r i a l s i n r e c u r r e n t m a l i g n a n t b r a i n t u m o r s 51I M C -C 225/C e t u x i m a b /E r b i t u x [I m C l o n e ]M o n o c l o n a l a n t i b o d y a g a i n s t t h e e x t r a c e l l u l a r d o m a i n o f E G F R , b l o c k i n g l i g a n d a c t i v i t y I n d u c e s a p o p t o s i s a n d i n h i b i t s a n g i o g e n e s i s . I t h a s n o t b e e n i m p l e m e n t e d i n c l i n i c a l t r i a l s a s t h e m o d e o f d e l i v e r y i s s t i l l u n d e r s t u d y 52, 53E M D 55900/m A b 425 [M e r c k ]A m u r i n e m o n o c l o n a l a n t i b o d y a g a i n s t E GF R D i d n o t s u c c e s s f u l l y g e t t h r o u g h p h a s e I /I I t r i a l s i n r e c u r r e n tG B M p a t i e n t s 54G e f i t i n i b /Z D 1839/I R E S S A [A s t r a Z e n e c a ]R e v e r s i b l e t y r o s i n e k i n a s e i n h i b i t o r ; c o m p e t e s w i t h A T P f o r b i n d i n g t o t h e r e c e p t o r I n p h a s e I I c l i n i c a l t r i a l s f o r r e c u r r e n t G B M 55E r l o t i n i b /O S I -774/T a r c e v a [O S I P h a r m a c e u t i c a l s ]E GF R t y r o s i n e k i n a s e i n h i b i t o r I n p h a s e I I t r i a l s ; c o a d m i n i s t e r e d w i t h t e m o z o l o m i d e d u r i n g a n d f o l l o w i n g r a d i a t i o n t h e r a p y 56-58V a n d e t a n i b /Z D 6474/Z A C T I M A [A s t r a Z e n e c a ]D u a l i n h i b i t o r o f V E G F R 2 a n d E G F R t y r o s i n e k i n a s e a c t i v i t y ; m o r e e f f e c t i v e i n E G F R v I I I e x p r e s s i n g G B M c e l l s Q u a l i f i e d i n p h a s e I I t r i a l s . A l s o t e s t e d f o r a d m i n i s t r a t i o n w i t h c a r b o p l a t i n 59, 60A E E 788 [N o v a r t i s ]R e v e r s i b l e t y r o s i n e k i n a s e i n h i b i t o r ; t a r g e t s E G F R , E r bB 2 a n d V E G F R 2P r e s e n t l y i n p h a s e I /I I t r i a l s i n G B M /r e c u r r e n t c a s e s 61 G W -572016/L a p a t i n i b [G l a x o S m i t h K l i n e ]A d u a l i n h i b i t o r o f E G F R a n d E r b B 2U n d e r e a r l y c l i n i c a l t r i a l s i n p h a s e I I 56, 62 C I -1033 [P f i z e r /W a r n e r -L a m b e r t ]I r r e v e r s i b l e p a n -E r b B t y r o s i n e k i n a s e i n h i b i t o r S u p p r e s s e s g r o w t h i n x e n o g r a f t m o d e l s o f G B M a n d h a s s h o w n p r o m o s i n g r e s u l t s i n p h a s e I /I I c l i n i c a l t r i a l s 63 A f a t i n i b I r r e v e r s i b l e i n h i b i t o r o f E G F R U n d e r c l i n i c a l t r i a l s 64 D a c o m i t i n i b I r r e v e r s i b l e i n h i b i t o r o f E G F R U n d e r c l i n i c a l t r i a l s 64 N i m o t u z u m a b H u m a n i z e d m o n o c l o n a l a n t i b o d y a g a i n s t E G F R U n d e r c l i n i c a l t r i a l s 64 C D X -110A n E G F R v I I I p e p t i d e v a c c i n e I n p h a s e I I t r i a l s 64 W o r t m a n n i n [W a k o ]I r r e v e r s i b l e i n h i b i t o r o f P I 3K R a d i o s e n s i t i z e s G B M c e l l s i n v i t r o ; t o b e t e s t e d c l i n i c a l l y 65 L Y 294002 [E l i L i l l y ]I n h i b i t o r o f P I 3K ; r e v e r s i b l e c o m p e t i t i v e i n h i b i t o r o f A T P A c t i v e a g a i n s t g l i o m a c e l l l i n e s i n v i t r o b u t h a s n o t m o v e d i n t o c l i n i c a l t r i a l s 66 R a p a m y c i n /S i r o l i m u s [W y e t h ]I n h i b i t s A k t p a t h w a y b y t a r g e t i n g m T O R . R a p a m y c i n s h o w s c y t o s t a t i c e f f e c t o n x e n o g r a f t s o f g l i o b l a t o m a s a n d m e d u l l o b l a s t o m a s i n a P T E N -d e p e n d e n t m a n n e r . C C I -779 i n h i b i t s G B M p r o l i f e r a t i o n i n v i t r o A l t h o u g h e f f e c t i v e i n p r e c l i n i c a l s t u d i e s , d u e t o i n s t a b i l i t y i n s o l u t i o n , r a p a m y c i n h a s n o t b e e n u s e d i n c l i n i c a l t r i a l s . C C I -779 h a s e n t e r e d p h a s e I I t r i a l s i n r e c u r r e n t G B M s . R A D 001 h a s a l s o s h o w n p r o m i s i n g r e s u l t s i n p r e c l i n i c a l t r i a l s 67-69C C I -779 (e s t e r a n a l o g o f R a p a m y c i n )/T e m s i r o l i m u s [W y e t h ] R A D 001 (R a p a m y c i n a n a l o g d r u g )/E v i r o l i m u s [N o v a r t i s ] L Y 317615/E n z a s t a u r i n [E l i L i l l y ]S e r i n e /t h r e o n i n e k i n a s e i n h i b i t o r ; t a r g e t s P K C -β, A k t , a n d G S K 3βD i d n o t s h o w m u c h p r o m i s e i n p h a s e I I I t r i a l s i n r e c u r r e n t G B M s , a l t h o u g h s h o w e d g o o d r e s p o n s e i n p h a s e I /I I t r i a l s , w h e n c o a d m i n i s t e r e d w i t h t e m o z o l o m i d e d u r i n g a n d /o r f o l l o w i n g r a d i a t i o n t h e r a p y 70, 71P K I 166 [N o v a r t i s ]R e v e r s i b l e k i n a s e i n h i b i t o r ; d u a l a c t i v i t y a g a i n s t E G F R a n d E r b B 2P o t e n t i a l l y c o n t r o l s p r o g r e s s i o n o f G B M . I n d u c e s a p o p t o s i s i n g l i o m a c e l l l i n e s e i t h e r a l o n e o r i n c o m b i n a t i o n w i t h o t h e r d r u g s i s u n d e r s t u d y . H a s b e e n d i s c o n t i n u e d i n c l i n i c a l t r i a l s72(c o n t i n u e d )433P h a r m a c o l o g i c a l I n h i b i t o r s /D r u g s [C o m p a n y ]M o d e o f A c t i o nC l i n i c a l O u t c o m e R e f e r e n c e s G e n i s t e i n (a p l a n t d e r i v e d i s o f l a v o n e )T y r o s i n e k i n a s e i n h i b i t o r , t a r g e t s E G F RI n v i v o s t u d i e s p r o v e i t c a n c r o s s b l o o d –b r a i n b a r r i e r a n d a f f e c t s G B M c e l l i n v a s i v e n e s s i n v i t r o . S h o w s a s y n e r g i s t i c e f f e c t i n g c o m b i n a t i o n w i t h B C N U 73 R e o v i r u s e sO n c o l y t i c v i r u s ; t a r g e t s R a s /M A P K p a t h w a y a s i t s e l e c t i v e l y r e p l i c a t e s i n R a s a c t i v a t e d c e l l sE f f e c t i v e i n x e n o g r a f t m o d e l s o f g l i o m a a n d h a s b e e n i n p h a s e I /I I t r i a l s i n C a n a d a a n d h a s b e e n a p p r o v e d b y t h e U SF D A 74, 75 o H S V -M G 18LO n c o l y t i c r e c o m b i n a n t h e r p e s s i m p l e x v i r u s t a r g e t s G S C s a n d i s e f f e c t i v e i n c o m b i n a t i o n w i t h s m a l l m o l e c u l e i n h i b i t o r s o f P I 3K /A k t l i k e L Y 294002T h e s a f e t y o f o H S V t h e r a p y h a s b e e n s h o w n i n c l i n i c a l t r i a l s f o r G B M ; h o w e v e r , e f f i c a c y r e m a i n s a n e c d o t a l a n d n e e d s i m p r o v e m e n t76T a r g e t i n g W n t /β-c a t e n i n p a t h w a y A s p i r i n [B a y e r ]C O X -2 i n h i b i t o r ; c o n v e r t s β-c a t e n i n t o a p h o s p h o r y l a t e d f o r m i n c a p a b l e o f a c t i v a t i n g d o w n s t r e a m t a r g e t g e n e s P h a s e I I T r i a l f o r p r i m a r y p r o p h y l a x i s o f v e n o u s t h r o m b o e m b o l i s m i n G l i o b l a s t o m a p a t i e n t s77 V a l d e c o x i b /B e x t r a [G .D . S e a r l e ]A b r o g a t e s W n t s i g n a l i n g b y C O X -2 i n h i b i t i o nH a s b e e n i n u s e d i n c o m b i n a t o r i a l c h e m o t h e r a p y w i t h o t h e r d r u g s 78 R o f e c o x i b /V i o x x [M e r c k ] C e l e c o x i b /C e l e b r e x [P f i z e r ]I n h i b i t s W n t s i g n a l i n g b y C O X -2 i n h i b i t i o n a n d a l s o t a r g e t s C D 133+ c e l l s I s u s e d a s a r a d i o s e n s i t i z i n g d r u g i n c l i n i c a l a p p l i c a t i o n s78 I s o q u e r c e t i n (a p l a n t f l a v o n o i d )I n h i b i t s W n t s i g n a l i n g b y c h a n g i n g β-c a t e n i n n u c l e a r l o c a l i z a t i o n I n h i b i t s G B M c e l l p r o l i f e r a t i o n b u t d o e s n o i n d u c e a p o p t o s i s 79 X A V 939T a n k y r a s e i n h i b i t o r —i n c r e a s e s A x i n 1 a n d 2 l e v e l s l e a d i n g t o d e c r e a s e d β-c a t e n i n s t a b i l i z a t i o n , a c c u m u l a t i o n , a n d t r a n s c r i p t i o n a l a c t i v i t y , t u r n i n g o f f W n t s i g n a l i n gI m p a r t s r a d i o s e n s i t i z a t i o n t o G B M c e l l s 5, 80T a r g e t i n g b o t h p a t h w a y s Q u e r c e t i n (a p l a n t d e r i v e d f l a v o n o i d )D o w n r e g u l a t e sE r k a n d A k t s i g n a l i n g a n d s u r v i v i n . A l s o r e g u l a t e s W n t /β-c a t e n i n s i g n a l i n g v i a G S K 3βI n d u c e s a p o p t o s i s i n h u m a n g l i o m a c e l l s . P h a s e I a n d I I s t u d i e s w i t h c o m p o u n d s d i r e c t e d a t T R A I L r e c e p t o r s 79, 81, 82 C u r c u m i n (a n a t u r a l p l a n t c u r c u m i n o i d )T a r g e t s m u l t i p l e p a t h w a y s —E G F R a n d d o w n s t r e a m A k t a n d S T A T s , N F -κB s o n i c h e d g e h o g /G L I s , W n t /β-c a t e n i n , e t c .I n d u c e s a p o p t o s i s a n d i n h i b i t s p r o l i f e r a t i o n a n d i n v a s i o n i n c a n c e r c e l l s i n c l u d i n g c a n c e r s t e m /p r o g e n i t o r c e l l s (C D 133+ G S C s ). R e c e n t l y , a p o l y m e r i c n a n o p a r t i c l e e n c a p s u l a t i n g c u r c u m i n h a s b e e n u s e d f o r b e t t e r d e l i v e r y i n v a r i o u s c a n c e r s i n c l u d i n g g l i o m a s83, 84T a b l e 2. (C o n t i n u e d )。