This is the peer reviewed version of the following article: EGFR-dependent Mechanisms in Glioblastoma: Towards a Better Therapeutic Strategy Cristina Zahonero, Pilar Sánchez-Gómez Cell Mol Life Sci . 2014 Sep;71(18):3465-88 which has been published in final form at https://doi.org/10.1007/s00018-014-1608-1 60 61 62 63 64 65 1 5 33 1 EGFR-DEPENDENT MECHANISMS IN GLIOBLASTOMA: TOWARDS A BETTER THERAPEUTIC 2 STRATEGY 3 4 AUTHORS: Cristina Zahonero1, Pilar Sánchez-Gómez PhD1,2 6 7 8 1 Neuro-oncology Unit, Instituto de Salud Carlos III-UFIEC, Madrid. 9 10 2 Corresponding autor (psanchezg@isciii.es; Ph: +34 918223265; Fax: +34 918223269) 11 12 13 14 15 16 17 18 Abstract 19 20 Glioblastoma is a particularly resilient cancer: therapies have to be able to reach the 21 22 brain crossing the Blood Brain Barrier (BBB) and they have to deal with a highly invasive tumor 23 that is very resistant to the damage in the DNA. It seems clear that in order to kill aggressive 24 glioma cells, more efficiently and with fewer side effects on normal tissue, there has to be a 25 26 shift from classical cytotoxic chemotherapy to targeted therapy. Since the epidermal growth 27 factor receptor (EGFR) is altered in almost 50% of glioblastomas, it represents one of the most 28 promising targets. In fact it has been associated with several steps in tumorigenesis, from 29 30 tumor initiation to tumor growth and survival, and also with the regulation of cell migration 31 and angiogenesis. However, EGFR kinase inhibitors have produced poor results in clinical trials 32 in this type of cancer, with no clear explanation for the tumor resistance observed. Here we 34 will revise what we know about the molecular function of EGFR in cancer, and in particular in 35 gliomas. We hope to come out with an operational definition of EGFR addiction in certain 36 37 glioblastomas that could improve the design of future therapies. 38 39 40 41 42 43 Keywords: Glioblastoma, EGFR signaling pathway, EGFR stability, therapy resistance 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 11 22 31 46 1 Introduction 2 3 4 Glial tumors are primary tumors that resemble astrocytes and/or oligodendrocytes. 5 Grade IV astrocytomas (known as glioblastomas (GBM)) are the most common glial tumors and 6 they have a terrible prognosis with a median survival rate of 12 to 15 months. The main features 7 8 of this type of cancer include high levels of mitosis, diffuse infiltration, a tendency for necrosis, 9 significant angiogenesis, resistance to apoptosis, and widespread genomic aberrations (1). 10 Current standard treatment consists of surgery followed by radiotherapy and cytotoxic 12 chemotherapy with the alkylating agent Temozolomide (2) although treatment remains 13 palliative for most patients. Another important feature of GBM is the high degree of intra and 14 15 intertumoral heterogeneity. For decades they have been classified as primary GBM (occurring 16 without evidence of a pre-existing, less malignant lesion) that comprises more than 90% of the 17 cases, and secondary GBM (occurring through the progression from a lower grade glioma) that 18 19 generally affects younger patients (3). However there are not histological differences between 20 these two entities. More recently large scale expression profiles of glioblastomas have provided 21 a molecular classification into three (4) or four (5) main subtypes, with potential implications for 23 patient prognosis and management. 24 25 Multiple alterations in the expression level of genes and/or proteins have been 26 identified in glioblastomas, including activation of oncogenes and/or silencing of tumor- 27 28 suppressor genes. Based on copy number and expression analyses as well as DNA sequencing, 29 researches have confirmed three signaling pathways commonly disrupted in GBM: (i) receptor 30 tyrosine kinases (RTK)/Ras/phosphoinositide 3-kinase (PI3K) pathway (which includes alterations 32 in EGFR (amplified and/or mutated in 40% of cases), the PI3K inhibitor: phosphatase and tensin 33 homolog (PTEN) (inactivated in 36% of cases) and the Ras inhibitor neurofribromatosis 1 (NF1) 34 35 (inactivated in 23% of cases); (ii) the p53 pathway, where the gene TP53 is mutated in 35% of 36 cases; (iii) the Rb pathway, where the cell-cycle inhibitors CDKN2A (p16INK4A) and CDKN2B are 37 alternatively inactivated in about 50% of cases (6;7). A relatively high frequency of mutations 38 39 was also found in the isocitrate dehydrogenase 1 and 2 (IDH1/2). However these mutations are 40 preferentially associated with secondary GBM (80% of the cases) with a significant pathogenic 41 role (3). By contrast primary GBMs are characterized by a high proportion of mutations and/or 42 43 overexpression of EGFR gene. EGFR codes for a 170kDa glycosylated receptor with tyrosine 44 kinase activity. Altered EGFR function has been associated with GBM tumor initiation and 45 growth, as well as with cell invasion, angiogenesis and resistance to chemo- and radiotherapy 47 (8;9). However, although EGFR kinase inhibitors are useful in treating other types of tumors, 48 they offer poor outcomes in GBM patients. Moreover, contrary to what could be expected, 49 50 there is some controversy about the correlation between the EGFR amplification and 51 overexpression, and the clinical response to EGFR kinase inhibitors in GBM patients (10-13). 52 These results underline the special nature of the EGFR oncogenic network in these neoplasms. 53 54 In a simplified view EGFR activates the mitogen activated protein kinase (MAPK) and PI3K 55 signaling cascades, resulting in changes in cell growth, survival, migration and angiogenesis. 56 However the first one responds to many other molecules present in GBMs, and the second one 57 58 is usually activated by mutations in these tumors. Moreover accumulating evidences suggest 59 that EGFR and its mutant forms regulates other aspects of cancer cell biology like cell 61 62 63 64 65 3 11 31 42 46 1 metabolism and response to DNA damage, directly linked to the survival of the cells in the GBM 2 tumorigenic context, that could explain the tumorigenic potential of EGFR in these tumors. 3 4 Recent evidences also suggest that in some cases EGFR actions do not depend on its kinase 5 activity. This atypical mode of EGFR signaling could contribute to the failure of the majority of 6 EGFR-targeted agents designed to inhibit its kinase activity. 7 8 9 Here we will try to draw a comprehensive picture of EGFR signaling (in particular those 10 aspects linked to cell proliferation and survival), based on classical models and more recent 12 findings, in order to explain its oncogenic action in aggressive gliomas. We hope that this review 13 will shed some light into the development of targeted therapies for EGFR-dependent GBMs and 14 15 will suggest better synergistic approaches and/or possible predictive markers. 16 17 18 19 EGFR structure and mutations in GBM 20 21 Epidermal growth factor (EGF) was identified by embryologist Stanley Cohen in the early 22 1960s and its receptor (EGFR), which is also known as HER-1 or c-erbB-1, was identified a decade 23 24 later (14). EGFR is one of the four transmembrane growth factor receptor proteins (c-erbB) that 25 share similarities in structure and function. Other members of the c-erbB group include HER2 (c- 26 erbB-2), HER3 (c-erbB-3), and HER4 (c-erbB-4). EGFR is the receptor for members of the EGF- 27 28 family of extracellular protein including EGF, transforming growth factor- (TGF-); 29 amphiregulin (AR), betacellulin , epiregulin and Heparin-binding EGF-like growth factor (HB- 30 EGF)(15). EGFR consists of a single polypeptide chain of 1186 amino acids with three main 32 regions: an extracellular (EC) receptor domain, a transmembrane region (TM), and an 33 intracellular domain (IC) with tyrosine kinase (TK) function (Figure 1). The EC amino-terminal 34 35 end can be divided into four domains with the L1 and L3 responsible for ligand binding. 36 Cysteine-rich (CR) domains 1 and 2 contain N-linked glycosylation sites and disulfide bonds that 37 determine the tertiary conformation of the external portion of the molecule. A large loop that 38 39 protrudes from the back of CR2 makes a molecular contact with the respective domain of the 40 other receptor (16). EGFR can form homo- and hetero-dimers with other members of the c-erbB 41 family, resulting in differences in ligand affinity and downstream signaling (15). Dimer formation 43 between two EGFR molecules in the phosphorylation of the tyrosine (Y) residues in the C- 44 terminal tail segment, which serve as docking sites for signaling molecules that contain Src 45 homology domain 2 (SH2) or phospho-tyrosine binding (PTB) domains and are responsible for 47 onward transmission of the signal. The kinase activity of EGFR is stimulated by ligand 48 engagement in a manner that depends on intermolecular interactions (17). In contrast to other 49 50 kinases the trans-phosphorylation of the activation loop is not a critical event for EGFR 51 activation (18). In fact, recent structural studies have revealed that the EGFR tyrosine-kinase 52 domain has two different conformations. In the inactive one it is able to inhibit its own activity 53 54 but after EGF induced dimerization, the increase in the local concentration of the kinase domain 55 provokes an allosteric change that forces the activation of EGFR (19;20). 56 57 Ligand binding also results in rapid internalization of activated receptors and targeting of 58 59 internalized EGF-receptor complexes to lysosomes for degradation. The customary EGFR signal 60 attenuator is the ubiquitin-ligase CBL, which binds to the regulatory domain (REG) at the 61 62 63 64 65 4 7 16 27 31 51 59 1 receptors´ s tail. When bound; CBL attaches mono-and di-ubiquitin moieties to multiple lysine 2 residues of EGFR, thus tagging the receptor for lysosomal degradation. Acceleration of 3 4 internalization and lysosomal targeting leads to receptor down-regulation which serves to 5 decrease the number of activated receptors in the cell and prevent excessive signaling (see 6 below). 8 9 There are several mechanisms that could justify the activation of EGFR signaling 10 pathway in GBM. Overexpression on its own could provoke a local accumulation of the kinase 11 domain that would lead to its activation. Moreover high expression of EGFR ligands has been 12 13 reported in high grade gliomas (21-23) and there are reports of TGF amplifications, mainly in 14 recurrent gliomas (24). However it is also well known that many of the GBMs with EGFR 15 amplification have EGFR mutations (16;25). The most common EGFR mutations found in GBMs 17 are deletion mutations (EGFRvI to EGFRvV), and in-frame deletion of regions of the extracellular 18 domain like EGFRvIII (present in 30 to 40% of GBMs). A recent massive sequencing of the 19 20 receptor in GBM tumors has uncovered several oncogenic missense point mutations in the 21 extracellular domain of the receptor that in some cases have been shown to confer oncogenic 22 potential (26), presumably by promoting receptor dimerization. Another common mutation is a 23 24 truncation of the intracellular region at amino acid 958, EGFRvV, present in 15% of GBMs with 25 EGFR amplification. This mutant receptor is internalization-deficient and therefore displays 26 increased ligand-dependent kinase activity (16;25). Mutations of the intracellular portion of 28 EGFR are more common in other neoplasms. In fact, the tyrosine kinase domain mutations that 29 have been shown recently to be responsive to specific inhibition of EGFR in lung cancer (27), 30 have not been found in GBMs (28). Of note, multiple mutations are sometimes seen in the same 32 amplified EGFR gene, a finding unique to GBM (29). A recent genomic study has revealed the 33 presence of recurrent in frame fusions involving EGFR (in 7.6% of GBMs), with the most 34 35 recurrent partners being septin 14 (SEPT14) and phosphoserin phosphatase (PSPH). Interestingly 36 the EGFR-SEPT14 fusions confer mitogen-independent growth, constitutively activate signal 37 transducer and activator of transcription 3 (STAT3) signaling and impart sensitivity to EGFR 38 39 kinase inhibitors (30). 40 41 EGFRvIII accounts for 60 to 70% of EGFR mutations in GBM and involves exons 2 to 7 of 42 the extracellular domain (Figure 1). Regions of multiple Alu repeats in introns 1 and 7 may play a 43 44 role in mediating susceptibility to this specific gene rearrangement (29). This mutated gene 45 encodes for a receptor that lacks amino acids 6 to 273 and creates a novel tertiary conformation 46 of the extracellular domain. This mutated form resembles the viral EGFR homologue, v-erbB, 47 48 which exists primarily in dimers, leading to constitutive kinase activation and subsequent 49 oncogenicity (31). The study of several mouse glioma models has demonstrated that the vIII 50 variant is more tumorigenic that the wild type receptor (see below). Although EGFRvIII is unable 52 to bind to its ligands it has been proposed that its oncogenic action is due to a constitutively 53 active kinase activity. In fact the altered kinetics of EGFRvIII could result in a distinct set of 54 55 downstream signals compared to the wild type EGFR. There are reports of selective and/or 56 constitutive activation of several pathways: PI3K pathway (32;33), Ras (34), c-jun N-terminal 57 kinase (JNK) (35), Src family kinases (SFK) (36), urokinase-type plasminogen activator receptor 8 (uPAR) (37) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) (38). 60 Moreover EGFRvIII confers drug resistance to GBM cells through the modulation of B-cell 61 62 63 64 65 5 11 25 36 40 56 60 1 lymphoma-extralarge (Bcl-XL) and caspase 3-like proteases (39). Some recent transcriptional and 2 biochemical studies have reinforced the different signaling networks activated by wild type 3 4 EGFR (EGFRwt) and its variant EGFRvIII (33;40;41) which could explain the differences in the 5 tumorigenic potential of both receptors. Intriguingly, the oncogenic capacity of EGFRvIII may 6 propagate into non-expressing cells by means of cell-to-cell transfer of microvesicles (also called 7 8 exoxomes) (42). Exosomes are membrane-enclosed vesicles, of 30 to 100 nm in diameter, that 9 are derived from endosomes during the formation of multivesicular bodies (43). In GBM it has 10 been shown that exosomes can mediate the horizontal transfer of EGFR (both protein and 12 mRNA) and alter the proliferation of receptor cells (44). Additionally it has been reported that 13 EGFR ligands can accumulate in exoxomes from cancer patients which could contribute to the 14 15 dialogue between the different tumor cells, and also between the tumor cells and their niche 16 (45). This exoxome-mediated communication could add more complexity to the EGFR-related 17 gliomas. 18 19 20 21 EGFR kinase-dependent downstream signaling 22 23 EGFR signaling is activated by a three step mechanism. First, the binding of any of the 24 specific ligands to the receptor induces dimerization of the ligand-binding domains. Second, the 26 dimerization results in the auto-phosphorylation of five specific tyrosine residues in the carboxy- 27 terminal end of the intracellular part of EGFR: Y992, Y1045, Y1068, Y1148 and Y1173, with 28 29 Y1173 as the major auto-phosphorylation site (46;47). Third, activated EGFR recruits several 30 signaling molecules that associate with the phosphorylated tyrosines through their SH2 and PTB 31 domains and in many cases become phosphorylated by the receptor. These are the main 32 33 cascades linked to this EGFR tyrosine kinase activation (Figure 2): 34 35 MAPK cascade. EGFR has been classically associated with cell proliferation control through the 37 recruitment of Growth factor receptor bound protein 2 (Grb2) and the activation of the 38 MAPK/ERK (Extracellular signal-regulated kinase). Grb2 recruits the guanine nucleotide 39 exchange factor son of sevenless (SOS) via its SH3 domain, and promotes binding of GTP to Ras, 41 than then binds and activates the RAF kinase. Activated RAF in turn binds to and phosphorylates 42 MEK, which then phosphorylates ERK1/2. Upon activation, ERK kinases can translocate to the 43 44 nucleus and activate several transcription factors (TFs) including Elk-1 (ETS domain-containing 45 protein), peroxisome-proliferator-activated receptor γ (PPARγ), signal transducer and activator 46 of transcription 1 and 3 (STAT1 and STAT3), C-myc and activating protein-1 (AP-1). Activation of 47 48 these factors leads to an increased transcription of genes involved mainly in cellular 49 proliferation (48). 50 51 Expression of MAPK and activated phospho-MAPK was significantly correlated with 52 53 proliferation and shorter survival time in gliomas (49). However, while constitutively activated, 54 mutated forms of Ras are found in almost 50% of all human tumors, few Ras mutations have 55 been found in gliomas. However the GTPase activating NF1, which inhibits Ras, is inactivated in 57 23% of cases (7). Nevertheless, even in NF1 wild type tumors, high levels of active Ras-GTP are 58 found (50) suggesting that the main mechanism for MAPK-dependent mitogenic signaling in 59 GBM must be mediated by the inappropriate activation of EGFR and/or other membrane 61 62 63 64 65 6 16 20 23 27 43 54 1 molecules: receptor tyrosine kinases (RTKs), integrins, vascular endothelial growth factor 2 (VEGF). 3 4 5 6 PI3K signaling. EGFR can modulate the balance between senescence and apoptosis through the 7 recruitment of the p85 subunit of PI3K, and the subsequent activation of the p110 subunit. PI3K 8 9 phosphorylation of phosphatidylinositol-4, 5-bisphosphate (PIP2) yields the second messenger 10 phosphatidylinositol (3, 4, 5)-triphosphate (PIP3). PIP3 serves as a membrane-docking site for the 11 serine/threonine protein kinase AKT, which binds to PIP with high affinity through its pleckstrin 12 13 homology (PH) domain. PIP3 is dephosphorylated to yield PIP2 by the tumor suppressor protein 14 PTEN, which attenuates AKT signaling. Phosphorylated AKT appears to be able to prevent 15 programmed cell death through targeted inhibition (phosphorylation) of Bad (a pro-apoptotic 17 member of the Bcl-2 family) and caspase-9, and activation of murine double minute 2 homolog 18 (MDM2) and Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKK) (51). The activated 19 IKK, in turn, phosphorylates inhibitor of B (IB), targeting it for ubiquitination and 21 proteosomal degradation and leading to the activation and nuclear translocation of NF-kB. NF- 22 B plays and important role in inflammation and cancer and it can induce pro-survival genes like 24 Bcl-XL or caspase inhibitors (52). Activated AKT also promotes cell growth through the activation 25 of mammalian target of rapamycin (mTOR), a master integrator of growth factors signals and 26 nutrient and ATP sensing (53). The downstream effectors of mTOR are eukaryotic initiation 28 factor 4E (eIF4E) and the ribosomal protein S6 kinase (S6K1/2). By the first mechanism, eIF4E 29 binding protein 1 (4E-BP1) suppressor protein factor is phosphorylated and inactivated, whereas 30 31 by the second mechanism, translation of mRNA by phosphorylation of S6 is enhanced (54). 32 33 Elevated phosho AKT levels have been observed in up to 85% of GBM cell lines and 34 patient samples (55). Activation of the PI3K pathway is significantly associated with increasing 35 36 tumor grade, decreased levels of apoptosis, and with adverse clinical outcome in human gliomas 37 (56). In fact AKT activation correlates with EGFR amplification (57). However mutations in the 38 PI3K/AKT signaling pathway are frequent in GBM suggesting that in many tumors AKT activity 39 40 would not be sensitive to EGFR inhibitors (see below). Alterations in this pathway include 41 activating mutations and amplifications of p110 (6;58;59), and p110 (60), and also gain of 42 function mutations in the p85 regulatory subunit (6;61). Moreover, as we have already 44 mentioned, the PTEN gene is lost, mutated or epigenetically silenced in 40% -50% of gliomas, 45 resulting in high levels of PI3K activity and downstream signaling (6;7;51;51). Interestingly PTEN 46 47 mutations, like EGFR amplifications, are found almost exclusively in primary GBMs and there is a 48 frequent association between the EGFR amplification and loss of 10q (where the PTEN 49 suppressor gene is located) in GBMs (62). In fact 10q losses are part of the primary GBM genetic 50 51 signature (3). However there was no significant correlation between the presence of EGFR 52 amplification and PTEN mutations (63). In any case there are some important clinical 53 implications as the presence or absence of functional PTEN could influence the effectiveness of 55 certain EGFR-targeted molecular therapies (see below). 56 57 58 STAT3 activation. STAT3 is a latent transcription factor found in the cytoplasm of cells. It is 59 60 activated by tyrosine phosphorylation, leading to dimerization, nuclear translocation, DNA 61 62 63 64 65 7 11 16 29 32 36 50 58 1 binding, and gene activation. STAT3 is transiently activated in normal cells but constitutively 2 activated in a wide variety of hematologic and epithelial primary tumors, and also in 3 4 astrocytomas (64). STAT3 tyrosine phosphorylation is induced by stimulated EGFR although it 5 can also be induced by stimulation of other upstream receptor and/or no receptor kinases 6 including PDGFR, Src and JAK2 (Janus kinase 2) (65). Interestingly, a recent work by Lee and co- 7 8 workers, has indicated that in GBM STAT3 is further activated by enhancer of Zeste homolog 2 9 (EZH2)-mediated methylation. This alternative mechanism is induced by AKT phosphorylation of 10 EZH2 suggesting RTK-independent activation of STAT3 in PI3K pathway- mutated GBM (66). 12 13 Similarly to what it happens to MAPK activation, there is no reports of STAT3 gain-of- 14 function mutations in GBM; rather, the activation of STAT-3 is thought to be a consequence of 15 either deregulation of upstream kinases or loss of endogenous inhibitors (67;68). It has been 17 associated with the control of cell cycle progression, apoptosis and immunosuppression in GBM 18 (67;69) and many studies indicate the anti-neoplastic potential of STAT3 inhibitors in GBMs (70). 19 20 Regarding EGFR-mediated activation some groups have reported that STAT3 constitutive 21 activation coexists with EGFR expression in almost 30% of high-grade gliomas and that targeting 22 STAT3/JAK2 sensitizes these tumors to anti-EGFR agents (68). However other authors have 23 24 reported that STAT-3 phosphorylation correlates only with the presence of EGFRvIII (57) 25 suggesting that is specifically activated in the presence of such mutation. These findings could 26 be related to the increased gliomagenesis potential of this mutant receptor form (see below). 27 28 PLC-PKC signaling. The protein kinase C (PKC) pathway also plays an important role in 30 mediating the effects of activated growth factor receptors including EGFR. Phospho-lipase C 31 (PLC-) is recruited and phosphorylated by EGFR. Activated PLC in turn interacts with the 33 plasma membrane where it cleaves PIP2 to inositol triphosphate (IP3) and diacyglycerol (DAG). 34 IP3 can bind its receptors on the endoplasmic reticulum (ER) to induce calcium (Ca 2+) influx into 35 the cell. Ca2+ and DAG can activate the serine/threonine kinase activity of PKC that in turn 37 phosphorylates a plethora of substrates regulating proliferation, apoptosis, cell survival and cell 38 migration (71). In GBM, survival in patients with tumors expressing PKC or PLC was significantly 39 40 shorter (49). A novel study has indicated that PLC signaling, in response to GBM EGFR 41 activation, induces IKK and leads to NFB translocation (72). As we just mentioned NFB 42 43 activity has been linked to the suppression of apoptotic signals. In fact this transcription factor 44 cooperates with EGFR in breast (73) and lung cancer (74) and aberrant constitutive activation of 45 NFB has been observed in glioblastomas (75). Bredel and co-workers have recently 46 47 demonstrated that NF I1A, the gene that codes for the NFB inhibitor (IB, is often deleted 48 in these tumors. They have further showed that deletion of IB has a similar effect to that of 49 EGFR amplification in the pathogenesis of GBM and is associated with comparatively short 51 survival (76). These results suggest that activation of NFB is another fundamental pathway for 52 glioma progression and that it can be achieved either by genetic deletion of its inhibitor, or by 53 54 EGFR amplification. 55 56 Apart from its role in apoptosis suppression, nuclear NFB also cooperates with other 57 transcription factors like Hypoxia-inducible factor 1 (HIF1). This interaction can lead to an 59 overexpression of pyruvate kinase M2 (PKM2) (72). PKM2 catalyzes the last step within 60 glycolysis, the dephosphorylation of phosphoenolpyruvate to pyruvate, and is responsible for 61 62 63 64 65 8 11 16 19 23 39 59 1 net ATP production and the accumulation of lactate within the glycolytic sequence (77). Tumor 2 cells have elevated rates of glucose uptake and higher lactate production, even in the presence 3 4 of oxygen. This phenomenon, known as aerobic glycolysis or the Warburg effect, supports tumor 5 cell growth. Accordingly PKM2 expression is increased and facilitates lactate production in 6 cancer cells (78;79) and a higher expression of PKM2 has been shown in GBM compared to 7 8 lower grade gliomas and normal tissue (80). In fact Yang and co-workers have postulated PKM2 9 upregulation as the key step for EGFR-promoted glycolysis, demonstrating a good correlation 10 between PKM2 expression and EGFR and IKK activity in GBM samples (72). 12 13 PKM2 is also located in the nucleus, in fact it has been shown to function as a co-factor 14 for HIF-1, facilitating the transcription of hypoxia responsive genes and further promoting 15 glucose uptake and lactate production (81). Therefore PLC-PKC signaling, through the activation 17 of NFB and the overexpression of PKM2, links EGFR activation to the regulation of glycolysis 18 and the hypoxia response, contributing to the survival of the GBM cells in their harsh 20 tumorigenic niche. Furthermore it has been shown that PKM2 is required for EGFR activation- 21 induced -catenin transactivation (82). These authors postulate that brain tumor development 22 promoted by EGFR requires PKM2-modulated -catenin transactivation, suggesting a crosstalk 24 between EGFR and Wnt signaling in the regulation of glioma cell growth. 25 26 EGFR-Src interaction. Although the major tyrosine sites of the EGFR C-terminal domain seem to 27 28 be auto-phosphorylated, some tyrosine residues are phosphorylated by other intracellular 29 tyrosine kinases. For example, EGFR Y845 is phosphorylated by Src, also known as tyrosine- 30 protein kinase CSK. Src and EGFR form an EGF–dependent heterocomplex in vivo; this 31 32 interaction is mediated by Src's SH2 domain, which can directly bind to phospho Y891, Y920 and 33 Y1101 sites (83;84). The non-receptor tyrosine kinase Src was one of the first oncogenes 34 identified, and the SFKs collectively regulates a variety of cellular functions in many cancer types 35 36 including proliferation, invasion, motility, survival, differentiation, and angiogenesis (85). Co- 37 overexpression of EGFR and Src frequently occurs in human tumors and is linked to enhanced 38 tumor growth. Src is capable of potentiating receptor-mediated tumorigenesis, causing 40 synergistic increases in EGF-induced DNA synthesis, soft agar colony growth, and tumor 41 formation in nude mice (86-88). Recent work indicates that SFKs are frequently coactivated 42 43 with EGFR in GBM cell lines and patients (89) and it has been shown that dasatinib (a SFK 44 inhibitor) enhances the efficacy of EGFR-targeted therapies in these tumors (36).Moreover it has 45 been proposed that Src-dependent EGFR activation is induced by IR in glioma cells (90). All the 46 47 above provide a rationale for combined anti-EGFR and anti-SFK therapies 48 49 One of the proposed binding targets for EGFR phospho-Y845 is STAT5b (a transcription 50 factor known to be overexpressed in several tumors) that could mediate the synergism between 51 52 Src and EGFR (91). In GBM STAT5 seems to be preferentially activated by EGFRvIII (92) and the 53 STAT5b isoform has been shown to be overexpressed compared to normal tissue or lower grade 54 gliomas. Moreover it has been linked to the control of GBM cell growth, cell cycle progression, 55 56 invasion and migration through regulation of gene expression (93). 57 58 Another pathway that appears to be regulated through Y845 following EGF stimulation 60 is that mediated by the cytochrome-c oxidase subunit II (CoxII)-related trafficking of EGFR to 60 61 62 63 64 65 9 11 22 26 41 52 56 1 mitochondria, the central organelles that produce energy and initiate apoptosis (94). The 2 catalytic activity of EGFR and Src, as well as endocytosis and a mitochondrial localization signal 3 4 are required for these events. Rapamycin, apoptosis inducers, and EGFR inhibition can further 5 enhance EGFR mitochondrial transport (95;96). Once in the mitochondria CoxII can be 6 phosphorylated by both EGFR and c-Src, reducing Cox activity and cellular ATP. These findings 7 8 suggests EGFR plays a novel role in modulating mitochondrial function via its association with, 9 and modification of CoxII and contributing to regulate cell survival (94;97). Although the 10 relevance of the EGFR-CoxII interaction in GBM remains to be determined, the Src induced 12 localization of EGFRvIII is stimulated in conditions of low glucose and this mutant EGFR reduced 13 glucose dependency by stimulating mitochondrial oxidative metabolism (98). Interestingly the 14 15 amount of mitochondrial EGFR seems to be fine-tuned by the balance between autophagy and 16 apoptosis, inhibition of the first or induction of the second provokes an accumulation of EGFR in 17 this organelle as a pro-survival mechanism (96). The study by Cao and coworkers indicated that 18 19 both wild type and vIII EGFR can translocate to the mitochondria when induced by apoptosis- 20 inducing agents and EGFR kinase inhibitors, and that tumor cells with accumulated 21 mitochondrial EGFR are resistant to apoptosis induced by these agents (95). Taken together 23 these studies suggest that tumor cells reprogram their intracellular trafficking of EGFR/EGFRvIII 24 by increasing its mitochondrial accumulation, as a mechanism for escape from therapy- and 25 stress-induced apoptosis and growth suppression. Moreover they suggest that targeting 27 mitochondrial translocation could synergize with EGFR inhibitors and classical therapies. 28 29 30 Nuclear EGFR signaling. Throughout the previous paragraphs we have mentioned that most of 31 the EGFR-activated signaling pathways end up in the nuclear translocation of secondary 32 messengers and in the modulation of several transcription factors activity. However EGFR itself 33 34 has been often detected in the nuclei of cancer cells, primary tumor specimens, and other highly 35 proliferative tissues (99). Increased nuclear EGFR localization correlates with poor clinical 36 outcome in several types of cancer (100). Although this analysis has not been performed in 37 38 gliomas both, EGFRwt and EGFRvIII, have been detected in the nucleus of normal glial cells and 39 primary GBM specimens, where they cooperate with STAT3 function (101;102). 40 Recent reports have characterized a novel nuclear localization sequence (NLS) 42 comprising amino acids 645 to 657, adjacent to the transmembrane domain in EGFR and its 43 family members (103). This 13 amino acids sequence has a dual role: it mediates EGFR allosteric 44 45 conformational change and dimer stabilization, which are indispensable for the receptor 46 activation, but it also allows nuclear translocation via binding to importin  (104). Furthermore 47 cumulative evidence indicates that EGFR internalization serves to transport the receptor from 48 49 the cell surface to different compartments within cells, like the mitochondria and the nucleus. 50 Whereas further studies are required to determine if EGFR is integrated into the mitochondrial 51 membrane through endosomal membrane fusion, it has been demonstrated that disruption of 53 receptor internalization suppressed nuclear entry of EGFR (105). Once in the nucleus EGFR still 54 functions as a tyrosine kinase, phosphorylating and stabilizing PCNA and thus enhancing the 55 proliferative potential of cancer cells (106). This could explain the strong correlation between 57 nuclear localization of EGFR and the highly proliferative status of tissues (99). It remains to be 58 determined if there are other possible substrates for EGFR and/or EGFRvIII in the nucleus. 59 60 61 62 63 64 65 10 11 22 36 40 51 56 1 Nuclear EGFR and DNA damage regulation. EGFR overexpression has been implicated in 2 radioresistance in a variety of human cancers, including GBM. Moreover it has been correlated 3 4 with poor radiographic response to radiation therapy in some patients with this tumor (9;107). 5 EGFR itself can be activated by radiation in a ligand-independent way, promoting cancer cells 6 survival and proliferation. Furthermore, EGFR and EGFRvIII have been linked, by several authors, 7 8 to the repair of double-strand breaks (DSB) (the most lethal DNA lesions induced by ionizing 9 radiation (9;108). In fact the use of EGFR inhibitors in GBM cell lines and intracranial xenografts 10 caused tumor regression when combined with radiotherapy (109;110). It appears that both PI3K 12 and ERK pathways mediate the signals downstream of the receptor in order to activate DNA- 13 dependent serine/threonine protein kinase (DNA-PK), which is required for non-homologous 14 15 end joining (NHEJ) of DSBs (111;112). Moreover, the disruption of PI3K/AKT pathway signaling 16 by small-molecule inhibitors blocks DSB repair in GBM, whereas PTEN loss promotes it, resulting 17 in radioresistance (113). More recent discoveries indicate that nuclear EGFR can influence DNA 18 19 repair directly via physical interaction with DNA-PK. The EGFR antibody, cetuximab, decreased 20 nuclear DNA-PK protein and kinase activity by reducing its physical interaction with the receptor 21 (114). Moreover cetuximab blocks nuclear shuttling of EGFR and prevents phosphorylation of 23 DNA-PK and DSB repair (115;116). Altogether these suggest that DNA-PK inhibitors and/or EGFR 24 inhibitors may be an effective therapeutic strategy for radiosensitizing GBM tumors. 25 26 27 28 EGFR expression in gliomas 29 30 The frequent amplification of the EGFR gene in GBM was initially reported in 1985 by 31 Libermann and coworkers (117) and it has been confirmed in many subsequent studies. It has 32 33 been estimated that 30 to 40% of GBM exhibit EGFR gene amplification and nearly 50% of them 34 overexpress the receptor (1;6) (7). Although it is not well understood, high levels of EGFR mRNA 35 are also present in less malignant astrocytomas and oligodendrogliomas without the underlying 37 gene amplification (118). These observations underlie the relevant function of EGFR in glial cells 38 and suggest that other oncogenic events may lead to increased transcription of this gene. 39 Amplification of EGFR has been reported in only 3% of anaplastic (grade III) astrocytomas (119) 41 and is infrequent in secondary GBMs (only 8%) whereas 60% of primary GBMs show EGFR 42 overexpression and 40% of them contain EGFR amplifications. In consonance with this EGFR- 43 44 amplified GBM are rare in patients younger than age 35. With increasing patient age, EGFR 45 amplification becomes more common and the median age of GBM patients with this alteration 46 is 62 years (3). Pediatric GBMs are rare tumors and show genetic differences compared with 47 48 adult GBMs, including a much lower prevalence of EGFR amplification (0%-5%) and EGFR protein 49 overexpression (25%) (120). From the histopathological point of view EGFR gene amplification is 50 relatively common in small cell GBMs (69%) but rare in gliosarcomas (0%) and giant cell GBMs 52 (6%) (107). 53 54 There have also been several correlation studies between the EGFR status and changes 55 in other common GBM pathways. In general there is a tendency for mutual exclusion between 57 EGFR alterations and mutations in the tumor suppressor p53 (120-124). Indeed they are 58 considered as hallmarks of primary and secondary GBM respectively (3). On the other hand the 59 RB1 pathway seems to be important in both primary and secondary GBM. However, 60 61 62 63 64 65 11 16 50 1 homozygous deletions of the INK4A-ARF locus, which encodes tow gene products (p16INK4A 2 and p19ARF) involved in cell-cycle arrest and apoptosis, are more frequent in primary than in 3 4 secondary tumors (3). Moreover there is a frequent association of INK4A/ARF loss of function 5 and activation of EGFR in GBM (125), raising the possibility that critical functional interactions 6 between these mutations are necessary for cellular transformation as it was lately corroborated 7 8 in several in vivo mouse models (see below). 9 10 From the neuropathology point of view, identification of EGFR amplification or the 11 presence of EGFRvIII constitute strong evidence that the tumor is a GBM, or at least that it 12 13 should be treated like one, even in the absence of necrosis and microvascular proliferation in 14 the biopsy (126). On the other hands, in patients with EGFR amplification, multivariate analysis 15 revealed that EGFRvIII overexpression was an independent, significant, poor prognostic factor 17 for overall survival (127). However, although nobody doubts about the diagnostic value of EGFR 18 analysis in GBM, there are some discrepancies regarding the prognostic value of EGFR 19 20 amplification/overexpression, especially when patients of all ages are analyzed together (16). In 21 fact EGFR amplification has been associated with a worse prognosis in younger patients but with 22 a better prognosis among older patients (124;127-129). Moreover, among the younger patients, 23 24 the EGFR amplification predicted worse prognosis only in those with tumors without p53 25 mutations, suggesting that the oncogenic potential of the receptor could be overcome by 26 alterations in the tumor-suppressor pathway (130). EGFR amplification is also present (26%) in 27 28 long term GBM survivors (patients surviving longer than 3 years) (131) suggesting that this 29 oncogenic pathway is not much more aggressive than other GBM related alterations. 30 31 32 33 34 EGFR and GBM molecular profiling 35 36 During the last years, the availability of high-throughput profiling techniques has 37 allowed the identification of molecular subclasses of an otherwise apparently uniform disease. 38 39 As a proof of principle specific expression profiles were found to distinguish robustly primary 40 from secondary GBM (132;133) demonstrating that they are two different entities. More 41 recently The Cancer Genome Atlas (TCGA), using unsupervised clustering of global 42 43 transcriptional data, designated four GBM subclasses: proneural, neural, classical and 44 mesenchymal (5). An earlier transcriptional study analyzing a set of grade III and IV gliomas, 45 established three molecular variants of malignant glioma with at least two of them (also name 46 47 proneural and mesenchymal) being very similar to those of the TCGA analysis (4;134). 48 Interestingly the proneural subgroup has a better prognosis and this gene expression signature 49 is enriched in anaplastic astroctyomas and also in tumors with oligodendroglioma histology 51 (135). In accordance with these observations proneural tumors are more common in younger 52 patients. In fact the beneficial effect of younger age in patients diagnosed with GBM is entirely 53 54 due to the higher proportion of proneural tumors among them (136). Moreover, the gene 55 signatures of the different subgroups correlated best with different cell lineages. Neural tumors 56 correlated best with mature neurons, proneural tumors with oligodendrocytes, and 57 58 mesenchymal and classical tumors with astrocytes (5), suggesting that the different GBM 59 subtypes could have different cells of origin. In fact, when GBM cells are grown in mouse 61 62 63 64 65 12 11 22 26 46 1 xenografts or in cell culture they retain their molecular differences, reinforcing the notion that 2 there are different GBM tumor-initiating-cells (4;5). However it is important to keep in mind that 3 4 the GBM molecular subclasses are not homogenous and that many tumors may be composed of 5 different subpopulations. 6 7 8 The different GBM groups correlate with defined genomic abnormalities. More 9 specifically, proneural tumors have been strongly associated with alterations in platelet-derived 10 growth factor receptor alpha (PDGFRA) and IDH1,2, and mesenchymal tumors with mutations in 12 NF1 (5). Regarding EGFR, the TCGA analysis indicates that gene amplification, and in particular 13 the presence of the vIII isoform, are enriched in the classical GBMs, although it is also present in 14 15 the other subtypes (5). On the other hand Phillips and coworkers suggested that chromosome 7 16 (where EGFR gene is located) amplifications are more frequent in the mesenchymal subtype (4). 17 Apart from the genomic studies, a proteomic analysis further supports three basic subdivisions 18 19 in malignant glioma characterized by NF1 expression, upregulated PDGF signaling, or 20 upregulated EGF signaling, highly reminiscent of the genomic abnormalities enriched in 21 mesenchymal, proneural and classical GBMs respectively (137). Regarding the classification of 23 GBM cells, grown in vitro as primary cultures, most groups define only two subtypes, 24 characterized by differential expression of markers like CD133 and Olig2 in one group, and CD44 25 in the other. They have been so called as proneural and mesenchymal GBM cells based on their 27 expression profiles (138-141) although EGFR amplification have been described in both groups 28 (139). Another study, however, have suggested the existence of three different subgroups of 29 30 GBM cells, with EGFR being amplified and expressed in neurospheres expressing the signature 31 of the classical subtype and MET (encoding a protein known as hepatocyte growth factor 32 receptor, HGFR) being present in the mesenchymal and proneural subtypes (142). Likewise it 33 34 has been suggested that the gene expression profiles of glioma subtypes overexpressing EGFR 35 are distinct from the rest (143) suggesting that EGFR alterations drive a specific tumor 36 developmental program and that EGFR addicted GBM might behave differently from other 37 38 aggressive gliomas. 39 40 41 42 EGFR and GBM tumor initiation: mouse models of GBM related to EGFR 43 44 The extraordinary presence of EGFR alterations in primary GBMs suggest that this 45 receptor participates not only in tumor growth but also during tumor initiation. In fact 47 numerous investigations carried out using animal models, including transgenic mice, have 48 indicated that the enhanced expression of EGFRwt and especially truncated EGFRvIII, in neural 49 50 stem cells (NSCs) or more committed neuronal or glial precursor cells, can cooperate with other 51 genetic alterations to induce primary brain cancer initiation and progression (Table 1). Moreover 52 these mouse models provide insight into the participation of EGFR in tumor maintenance and 53 54 also in the resistance to therapeutic intervention, either to unspecific cytotoxic agents or to 55 EGFR tyrosine kinase inhibitors. 56 57 In accordance with the strong correlation between EGFR gene alterations and loss of the 58 59 INK4A-ARF locus (125), most of the glioma models based on overexpression of wild type or vIII 60 EGFR have been obtained in cells that are deficient for this tumor suppressor. The first model 61 62 63 64 65 13 11 22 26 46 1 described made use of avian retroviral vectors to transfer EGFRvIII into mice expressing tv-a, a 2 gene encoding the retrovirus receptor, TVA, under the control of brain cell type-specific 3 4 promoters, in an Inka-Arf null background (144). Interestingly the astrocytic lesions observed 5 where much more frequent when the nestin promoter (progenitor-specific) was used than when 6 the astrocyte-specific glial fibrillary acidic protein (GFAP)-tv-a mice were injected. In contrast 7 8 EGFRvIII appeared incapable of generating gliomas in a p53-deficient background, unless CDK4 9 was also overproduced (145) suggesting an explanation for the mutual exclusivity of EGFR and 10 P53 mutations found in GBM (see above). A different approach was used by Bachoo and 12 coworkers, as they used retroviral vectors to overexpress EGFR in vitro, in well-defined astrocyte 13 or NSCs cultures from Ink4a-Arf deficient mice. Those cells were then reintroduced into the 14 15 brains of SCID mice. Their results showed that both compartments are equally permissive for 16 the generation of high-grade gliomas (146). With a similar approach, in vitro EGFRvIII expression 17 in PTEN deficient NSCs was able to synergistically induce chromosomal instability and form 18 19 astrocytic tumors (147). These data suggest that deregulation of specific genetic pathways, 20 rather than the cell-of-origin, dictates the emergence and phenotype of high-grade gliomas. 21 However, the analysis of a transgenic mouse model that express the v-erbB oncogene under the 23 control of S100b promoter, demonstrated the appearance of low grade oligodendrogliomas in 24 20% of the mice. These lesions were more aggressive and the penetrance was higher in the 25 context of p53 or Ink4-Arf heterozygous mice (148). S100b is expressed by oligodendroglia and 27 astrocytes during early brain development although it is also present in NSCs so it is difficult to 28 conclude which ones where the cell of origin of the tumors observed (149). However, the 29 30 differences with the previous models suggest that oligodendrocytes are more readily 31 transformed by v-erbB, at least during early neural development. 32 33 Other groups have demonstrated that EGFRvIII overexpression can cooperate also with 34 35 oncogenic Ras mutations. In this case the Ras astrocytoma-prone model (RasB8 mice: GFAP- 36 V12Ha-ras transgenic mice) was used (150). Ding and coworkers made double transgenics to 37 express EGFRvIII and mutated Ras in the same cells and they observed an increase in the 38 39 penetrance of the tumors (compared to the single Ras transgenic mice). However they also 40 reported a change on the phenotype as overexpression of EGFRvIII leaded to the appearance of 41 oligodendrogial and mixed oligoastrocytoma (151). Interestingly the same authors 42 43 demonstrated that GFAP-EGFRvIII astrocytes, forced to express mutated Ras in vitro, generated 44 oligodendroglioma-like tumors when inoculated back into immunodeficient brains. By contrast 45 injection of adenovirus expressing EGFRvIII in adult RasB8 brains was able to induce low grade 47 and high grade astrocytomas with a high penetrance (152). These results, together with the 48 studies in the Ink4-Arf-/- background confirm that expression of EGFRvIII is not sufficient to 49 50 initiate gliomagenesis although it cooperates with other genetic alterations to induce glioma 51 formation. They also suggest that the same EGFR mutation can generate tumors with astrocytic 52 or oligodendrocytic phenotype, depending on the cell type and the developmental stage in 53 54 which the alteration takes place. However it is still not known if EGFRvIII activates different 55 signaling pathways in the germline and in the somatic context that could explain the changes in 56 the phenotypic outcomes. 57 58 59 An important corollary from these mouse models is that EGFRwt cannot substitute for 60 EGFRvIII in driving infiltrative glioma formation in genetically engineered mice (151) or in Ink4- 61 62 63 64 65 14 11 22 28 32 43 48 52 1 Arf-/- cells (145;146). One possible explanation for these phenomena would be that a sustained 2 EGFR signaling is necessary for glial transformation. The mitogenic effect of EGFRvIII have been 3 4 explained by a low but constitutively active kinase activity, amplified by failure to attenuate 5 signaling by receptor down-regulation (153). However, overexpression of self-activating EGFRwt 6 levels might not be sufficient to induce cell transformation due to its constant lysosomal 7 8 targeting. The results of Zhu and coworkers are in agreement with this hypothesis. They used a 9 conditional transgenic model based on somatic induction of vIII or EGFRwt in adult animals. In 10 these models expression of EGFR is triggered by stereotactic injection of an adenovirus 12 expressing Cre recombinase. In consonance with the results of the previous groups they 13 reported that expression of EGFRvIII, concomitant with loss of the cdkn2a and/or PTEN locus, 14 15 promotes the formation of aggressive gliomas (33). Interestingly, they also showed that 16 overexpression of EGFRwt, to levels comparable to those observed in human GBMS, is very 17 inefficient at forming tumors under the same conditions (33). Later on, the same group used 18 19 bicistronic lentiviral vectors designed to express TGF and Cre recombinase so that they can 20 induce EGFR expression in Ink4-Arf-/- and/or PTEN deficient mice. They demonstrated that 21 somatic, ligand-mediated activation of EGFR was necessary for gliomagenesis (154;155) further 23 supporting that persistent EGFR signaling is a necessary oncogenic event. These results are in 24 agreement with the clinical observation of common overexpression of EGFR ligands in receptor- 25 26 amplified GBM (see above). 27 Although many of these mouse models demonstrate the greater biological activity of 29 the truncated vIII variant of EGFR, in patients this mutation occurs almost exclusively together 30 with EGFR amplifications, suggesting a crosstalk between the mutant and the wild type 31 receptors in human GBM cells. This cooperation could be explain by a cell-autonomous manner 33 as the vIII isoform can be a substrate for EGFRwt and this phosphorylation triggers nuclear entry 34 of EGFRvIII and STAT3 activation (156). However autocrine and/or paracrine crosstalks have also 35 36 been proposed. It has been shown that EGFRvIII induces expression of EGFR ligands (HB-EGF) 37 whereas EGFRwt activates the mutant isoform by facilitating EGFRvIII dimerization (157), 38 suggesting a feed-forward loop that regulates the oncogenic action of the receptors. More 39 40 recently it has been reported that the EGFRvIII-expressing cells release cytokines (like interleukin 41 6 (IL6) and leukemia inhibitory factor (LIF)) that activate neighboring EGFRwt-expressing cells, 42 favoring the formation of heterogeneous gliomas in mice (158). These non-cell autonomous 44 effects could explain the coexistence of the mutation with gene amplification in the same tumor 45 but not always in the same cells. 46 47 Animal models can also provide opportunities to determine whether EGFR activation 49 generates a specific GBM subclass. Although it has been described the formation of 50 oligodendroglial histological tumors in transgenic models expressing vErbB, the rest of the EGFR- 51 related mouse studies reported the appearance of astrocytic tumors (Table 2), which could be 53 related with the absence of EGFR alterations in oligodendrogliomas. In fact Jun and coworkers 54 have shown that the TGF-EGFRwt; Ink4-Arf-/- tumors resemble molecularly the classical GBM 55 56 subtype (155), the one that presents a higher frequency of EGFR alterations (5). However their 57 results also indicate that the same combination of mutations generates mesenchymal-like GBM 58 in the absence of PTEN expression (155). It remains to be confirmed if a similar combination of 59 60 mutations occurs in human tumors classified in this subgroup. 59 60 61 62 63 64 65 15 6 1 Table 1. GBM mouse modeling linked to EGFR 2 3 Driving mutations Method for EGFR 4 overexpression Target cells Observations Reference 5 EGFRvIII RCAS viral vectors Ink4-Arf -/- 7 Nestin + GFAP + Astrocytomas (145) Much higher penetrance in Nestin + cells 8 EGFRvIII 9 Cdk4 RCAS viral vectors Nestin + Astrocytomas Higher penetrance in the absence of p53 (145) 10 EGFRvIII 11 Ink4-Arf -/- 12 EGFRvIII In vitro transduction Ink4-Arf -/- Ast. and NSCs High grade Astrocytomas and mixed tumors (146) 13 In vitro transduction PTEN -/- NSCs Astrocytomas (147) 14 15 16 17 18 19 20 21 22 23 24 EGFRvIII; V12Ha-Ras Adenoviral 25 in vivo injections 26 EGFRwt RasB8 brains (GFAP +) Low grade and High grade Astroctyomas (152) 27 Ink4-Arf -/- 28 PTEN loss 29 30 EGFRwt +EGFRvIII Cond. Exp. (AdCre) in vivo injections Cond. Exp. (AdCre) Ink4-Arf -/- PTEN 2Lox brains Ink4-Arf -/- Highly aggressive gliomas Very low penetrance (33) 31 Ink4-Arf -/- 32 PTEN loss in vivo injections PTEN 2Lox brains Higher penetrance than EGFRwt alone (33) 33 EGFRvIII 34 Ink4-Arf -/- 35 PTEN loss 36 Cond. Exp. (AdCre) in vivo injections Cond. Exp. (lenti-TGF- Ink4-Arf -/- PTEN 2Lox brains Lower latency than in the presence of (33) EGFRwt 37 EGFRwt-TGF 38 Ink4-Arf-/- 39 Cre) in vivo injections Ink4-Arf -/- brains High grade astroctyomas (154) 40 EGFRwt-TGF 41 PTEN loss 42 Cond. Exp. (lenti-TGF- Cre) in vivo injections PTEN 2Lox brains Reduced penetrance and longer latency (155) than in the Ink4-Arf-/- mice 43 EGFRwt-TGF 44 Ink4-Arf-/- 45 PTEN loss 46 EGFRwt + EGFRvIII Cond. Exp. (lenti-TGF- Cre) in vivo injections Ink4-Arf -/- PTEN 2Lox brains Reduced penetrance and longer latency (155) than in Ink4-Arf-/- or PTEN loss mice EGFRwt and EGFRvIII expressed by 47 Ink4-Arf -/- In vitro transduction Ink4-Arf -/- Ast. 48 49 RCAS: Replication-Competent ALV Splice acceptor 50 51 Ast.: Astrocytes 52 53 NSCs: Neural Stem Cells 54 RasB8 mice: glioma-prone mice expressing Ha-Ras (V12Ha- 55 56 Ras) in GFAP positive (+) cells 57 58 different cells (158) Con. Exp.: Conditional EGFR Expression using the lox- stop-lox EGFR WT transgenic mouse strain AdCre: Adenovirus transducing Cre recombinase PTEN 2LOX : Conditional PTEN loss lenti-TGF-Cre: Lentiviral bicistronic vector transducing TGF and Cre recombinase PTEN loss v-erbB Transgenic mice S100b + Low Grade Oligodendrogliomas (148) v-erbB; Ink4-Arf -/- Transgenic mice S100b + High grade Oligodendrogliomas (148) v-erbB; p53-/- Transgenic mice S100b + High grade Oligodendrogliomas (33) GFAP + in RasB8 EGFRvIII; V12Ha-Ras Transgenic mice mice Oligodendrogliomas (151) EGFRvIII; V12Ha-Ras Transgenic mice GFAP + in vitro transduced Ras Oligodendrogliomas (151) 59 60 61 62 63 64 65 16 23 33 39 50 54 1 Targeting EGFR in GBMs 2 3 After the discovery that EGFR was implicated in the development of a variety of 4 epithelial cancers the receptor began to be considered as an interesting therapeutic target. 5 6 Several anti-EGFR based therapeutic strategies have been assessed as monotherapy, or in 7 combination with radio and conventional chemotherapy, in pre-clinical and clinical trials. Here 8 we review the most promising results and ongoing clinical trials in GBM patients (Table 2). 9 10 Antibodies against EGFR. Anti-EGFR monoclonal antibodies (mAbs) have been tested in cancer, 11 12 in an attempt to block the binding of EGF to its receptor and therefore to avoid the activation 13 of the downstream signal transduction pathways. The mAb225 (C225), also known as 14 cetuximab (Erbitux® [Bristol-Myers Squibb, ImClone Systems]) is currently in phase I/II to study 15 16 the efficacy of its combination with radiotherapy and TMZ to treat patients with primary GBM, 17 and also as a second line treatment in combination with bevacizumab (anti VEGF receptor, 18 VEGFR, antibody) and irinotecan (159;160)) (reviewed in (161)). More recently, a randomized 19 20 phase III study using the humanized mAb nimotuzumab (hR3, Theraloc®; YM BioSciences Inc.) 21 in first line GBM treatment combined with chemo and radiotherapy, has shown some survival 22 benefit (162). Furthermore several antibodies targeting specifically the truncated receptor 24 have been developed. mAb806, for example, binds to the short cysteine loop of the 25 extracellular domain that is always exposed in EGFR vIII and attenuates receptor 26 27 autophosphorylation. Due to mAb806 tolerance, great biodistribution and specificity for its 28 target in GBM patients, several clinical trials with a humanized version of mAb806 (ABT-806; 29 Abbott) are being currently performed (163;164). 30 31 The strongest reason for the use of EGFR directed antibodies in GBM patients is that 32 they provoke fewer side effects than the traditional chemotherapy treatments. However it is 34 still not known if their capacity to go through the BBB will be enough to improve the results 35 obtained with small molecules and the cost-effectiveness ratio is still very high. 36 37 Vaccination against EGFR vIII. Immunotherapy is also an attractive alternative to cytotoxic 38 strategies as they could eliminate tumor cells with reduced toxicity. The current strategies in 40 GBM are focused on targeting the vIII-truncated form of EGFR. The generation of a new amino 41 acid sequence (PEPvIII) with immunogenic capacity, due to the loss of exons 2-7 in the 42 43 truncated EGFRvIII and the fusion of two distant regions of the wild type molecule, makes it an 44 appropriate target for peptide-based vaccination (165). Rindopepimut (CDX-110, Celldex) is an 45 experimental vaccine containing the amino acid sequence PEP-vIII linked to the carrier protein 46 47 keyhole limpet hemocyanin (KLH), to generate both humoral and cellular immune responses. 48 This approach has shown ability to eliminate the EGFRvIII-expressing cells as a high proportion 49 of the relapsing tumors after rindopepimut treatment show no EGFR vIII reactivity. Moreover 51 it has been reported that the combination of rindopepimut with TMZ improves both 52 progression free survival (14.2 months vs. 7.3 months) and median survival (26 months vs. 15. 53 2 months) in GBM patients (166). However, one of the main caveats of this approach is the 55 intratumoral heterogeneity of GBMs as the antigen is being expressed only by a subgroup of 56 tumor cells. Therefore it is still not known if this immunotherapy will induce long-term 57 58 reduction of the tumors. 59 60 61 62 63 64 65 17 17 32 1 Small-molecule tyrosine kinase inhibitors (TKIs). The development of small-molecule TKIs was 2 simultaneous to the generation and improvement of the anti-EGFR mAbs. However they are 3 4 nowadays the most advanced EGFR-based therapies in the clinic. The best-studied TKIs are 5 quinazoline-derived synthetic molecules with low molecular weight, which are able to block 6 the magnesium-ATP- binding pocket of the intracellular TK domain. This union prevents the 7 8 activation of the kinase domain by ligand-induced auto-phosphorylation and downstream 9 activation of survival signaling pathways (167). 10 11 The first EGFR-specific TKIs used in the clinic for the treatment of newly diagnosed and 12 recurrent gliomas were gefitinib (Iressa®, ZD1839; AstraZeneca), erlotinib (Tarceva®, OSI774; 13 14 Genentech), which are EGFR/HER1 specific, and lapatinib (Tykerb/Tyverb; GSK), with ability to 15 block both EGFR/HER1 and HER2. However, in spite of the promising pre-clinical results, 16 obtained both in vitro and in vivo with these first generation TKIs, they have not accomplished 18 the expectatives. In two recent phase II clinical trials for GBM therapy, erlotinib was well 19 tolerated, but showed no clinically meaningful results, only a modest effect over placebo 20 21 (168;169). Regarding gefitinib, a phase II clinical trial showed that it was well tolerated and 22 displayed anti-tumor activity, but the median overall survival time in GBM patients was only 23 38.4 weeks from treatment initiation, which supposed a very modest clinical benefit (10). 24 25 These findings led to the design of second generation TKIs, which are able to bind irreversibly 26 the ATP binding site of various HER receptors (pan-HER inhibitors) such as afatinib (Gilotrif®, 27 Boehringer Ingelheim), which binds EGFR/HER1 and HER2, and dacomitinib (PF-0299804, 28 29 Pfizer) that bind to EGFR/HER1, HER2 and HER4, among other TKIs. Most of these second 30 generation TKIs have proven some efficacy in other tumors and they are currently in clinical 31 testing with GBM recurrent patients (Table 2). However while we wait for the results of these 33 next generation TKIs we need to reconsider the possible explanations for the lack of 34 therapeutic response observed until now with EGFR-directed strategies in GBM patients. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 44 45 46 47 48 49 18 Inc. 8 16 21 23 37 1 2 3 4 5 6 7 Agent Brand name Company Target Class Selected references/ Trial identifier 9 10 Cetuximab 11 (C225) 12 Nimotuzumab Erbitux ImClone Systems EGFR/HER1 Mouse-human chimeric antibody (159;160) 13 14 Monoclonal 15 antibodies (h-R3) TheraCIM YM Biosciences EGFR/HER1 Human antibody (162) Pantitumumab Vectibix Amgen EGFR/HER1 Human antibody NCT01017653 17 18 125I-MAb 425 19 Fox Chase Cancer EGFR Center Radiolabeled murine (170) antibody 20 mAb 806 ABT-806 Abbott EGFR vIII Human antibody (164) 22 Gefitinib 24 (ZD1839) Astra Zeneca EGFR/HER1 Pharmaceuticals Aniliquinazoline-based (10) reversible inhibitor 25 Erlotinib 26 (OSI-774) 27 Genentech Inc. EGFR/HER1 Aniliquinazoline-based reversible inhibitor (168;169) 28 29 Small- Lapatinib (GW572016) Tykerb GlaxoSmithKline EGFR/HER1, Thiazolylquinazoline based reversible inhibitor (171) 30 molecule 31 tyrosine 32 kinase Afatinib Gilotrif Boehringer Ingelheim EGFR/HER1, HER2, HER4 Anilinoquinazoline based irreversible inhibitor 33 inhibitors 34 Dacomitinib (PF-00299804) Pfizer EGFR/HER1, HER2, HER4 Anilinoquinazoline based irreversible inhibitor NCT01520870 35 Vandetanib 36 (ZD6474) AstraZeneca Pharmaceuticals EGFR/HER1, VEGFR Aniliquinazoline-based (172) inhibitor 38 Pelitinib 39 (EKB-569) 40 41 Vaccines Rindopepimut 42 (CDX-110) 43 Wyeth Pharmaceuticals Celldex Therapeutics EGFR/HER1 Cyanoquinoline-based irreversible inhibitor EGFRvIII Peptide vaccination ACT VI Iressa   60 61 62 63 64 65 19 23 29 44 50 54 1 Conclusions from TKI studies in GBM: synergistic approaches and predictive markers 2 3 There are several explanations for the EGFR-targeted therapy failure in GBM patients. 4 Deficient tumor drug penetration and TKIs systemic availability reduction by enzyme inducing 5 6 antiepileptic drugs, which are used in most GBM clinical trials, could be some of the reasons 7 behind the lack of success of these compounds (8;161;167). However, beyond the drug delivery 8 limitations, some GBM molecular features could be responsible for the limited benefit provided 9 10 by these therapeutic agents. 11 12 EGFR vIII mutation. It has been proposed that EGFR vIII-positive cells are resistant to gefitinib 13 because they need greater amounts of the drug and a longer exposition to it to reduce EGFR 14 downstream signaling. Moreover tumors bearing EGFRvIII have a worse response to cetuximab 15 16 (173). It has been argued that the strong activation of the PI3K/AKT signaling pathway by EGFR 17 vIII cannot be reduced by gefitinib treatment, leading to the persistence of tumor cell 18 proliferation and survival signals in the presence of this TKI (167;174;175). By contrast 19 20 Mellinghoff and coworkers described that expression of EGFRvIII, in a wild type PTEN context, is 21 associated with GBM responsiveness to EGFR kinase inhibitors (176). However these findings 22 could not be confirmed in subsequent trials, including a randomized phase II trial (168). The 24 results of the new upcoming trials with next generation TKIs will help to understand the 25 relevance of the presence of the vIII isoform as a predictive marker. 26 27 PTEN deletion and phospho-AKT levels. AKT basal activation levels (due to activation by other 28 RTKs or by PTEN deletion) seem to be one of the causes that could explain EGFR inhibitors 30 failure in several types of cancer. It was reported, for example, that PTEN restoration in a PTEN 31 deficient line augments the response to EGFR TKIs, through the induction of higher levels of 32 33 apoptosis (177). In GBMs it was proposed that patients carrying wild type PTEN tumors or low 34 levels of phosphorylated PKB/AKT would have a better outcome in response to anti-EGFR 35 treatments (12;176). PTEN function in tumors can be altered by post-traductional modifications 36 37 such as oxidation, phosphorylation, acetylation and ubiquitination, leading to malignant 38 transformation (178). It has been also shown that tyrosine phosphorylation of PTEN by SFKs and 39 fibroblast growth factor receptor (FGFR) can modulate its function. In particular, PTEN 40 41 phosphorylation at Y240 by FGFR is linked to EGFR-TKI resistance and reduced survival in GBM 42 patients (179). These results suggest that checking PTEN status (both at the genetic and at the 43 protein level) might be an important surrogate marker for EGFR directed clinical trials. Moreover 45 they indicate that targeting PI3K-AKT pathway could add a beneficial effect to EGFR TKIs. 46 47 Different EGFR conformations in lung cancer and GBM. The EGFR mutations present in GBM and 48 non-small cell lung cancer (NSCLC) have oncogenic transforming potential and promote high 49 levels of basal phosphorylation of the receptor in vitro. However, the different location of the 51 lung and brain tumor mutations has been associated with the diverse response of these cancers 52 to EGFR inhibitors. Crystallography studies have indicated that EGFR TKIs bind to different 53 configuration of the receptors. When coupled with gefitinb and erlotinib the receptor shows an 55 active conformation, also known as “type I” conformation, which is related to mutations in the 56 intracellular kinase domain and frequent in NSCLC such as the deletion of exons 19 and 21 and 57 58 point mutations in the exons 18-21, which sensitize tumor cells to EGFR targeted therapies 59 (27;28;180). In complex with lapatinib, by contrast, EGFR is in an inactive configuration, also 59 60 61 62 63 64 65 20 7 11 17 32 43 48 52 1 called “type II” conformation, which is typical of the ectodomain mutations found in GBM 2 samples, including missense and in frame deletions such as the EGFR vIII variant. It is not 3 4 surprising then that glioma cells carrying extracellular EGFR mutants were poorly inhibited by 5 erlotinib, whereas type II inhibitors induced cell death in the same cells (180). Furthermore 6 Barkovitz and coworkers confirmed that EGFRvIII releases erlotinib more rapidly than wild type 8 or lung cancer mutants and the kinase-site occupancy was correlated directly with cell-cycle 9 arrest (181). The tropism of the TKIs for a particular receptor conformational state could explain 10 why some agents are more effective in lung cancer than in GBM patients and supports the use 12 of Type II EGFR inhibitors for GBM, although they should have a better brain penetrance than 13 lapatinib. 14 15 Redundancy of receptor tyrosine kinase (RTK) signals. Treatment of GBM cells in vitro with 16 gefitinib resulted in dephosphorylation of the EGFR, but also of most of the pathway regulators 18 that have been mentioned above (Figure 2). However, analysis of the in vivo effect in 19 established GBM xenografts or in the tissue of treated patients demonstrated that gefitinib 20 21 efficiently dephosphorylates its target without significant effect on pathway constituents (182). 22 These data suggest that there are compensatory mechanisms in vivo, probably mediated by 23 other RTKs that share parts of their signaling pathways and show redundant regulatory circuit 24 25 (183). One RTK that is ubiquitously expressed in cancer cells is the insulin-like growth factor 26 receptor (IGF-1R). There is a functional cross-stalk between EGFR and this receptor as IGF-1R- 27 deficient cells are resistant to transformation by EGFR (184). IGF-1R has been linked to GBM 28 29 resistance to gefitinib through increased signaling of PI3K/AKT and the ribosomal protein S6 30 kinase. In addition, the pharmacological inhibition of IGF-R1 results in the sensitization of tumor 31 cells to EGFR-TKIs treatment (185). Another RTK that is particularly expressed in GBMs is PDGFR. 33 Interestingly the results from two different groups indicate that there is genetic heterogeneity in 34 aggressive gliomas, with EGFR and PDGFR being amplified and activated simultaneously in 35 36 adjacent intermingled cells (186;187). These results could suggest that combination of different 37 inhibitors could be more efficient than EGFR-TKIs alone. However some recent evidences 38 suggest that this is not necessary the case, as the addition of sunitinib (with capacity to inhibit 39 40 several RTK including PDGFR and VEGFR) to gefitinib only improved the anti-GBM efficacy in 41 vitro, but not in xenograft models (188). However Akhavan and coworkers have reported 42 recently that inhibition of EGFR signaling de-represses the transcription of PDGFR, and that 44 combined inhibition of both receptors potently suppresses tumor growth in vivo (189). 45 Therefore there is still space for a synergistic approach targeting both signaling pathways. 46 47 Crosstalk EGFR-MET. The MET RTK is amplified in 5% of GBM although it is overexpressed in 49 30% of these tumors, being a bad prognosis factor (190). Moreover, MET is activated in GBM 50 cells with increased levels of EGFR/EGFRvIII (183; 191). In fact there are evidences of autocrine 51 and paracrine cross-talks between both signaling pathways (192). In line with these results 53 resistance to EGFR inhibition can be overcome by using MET small molecule inhibitors (183;191) 54 or neutralizing antibodies to hepatocyte growth factor (HGF), the MET ligand (192;193). Jun and 55 56 coworkers have demonstrated that treatment of EGFR amplified mouse GBM cells with EGFR 57 TKIs induces a cytostatic response, characterized, among other changes, by an increase in MET 58 expression. Moreover they confirmed that pharmacological inhibition of MET overcomes the 59 60 61 62 63 64 65 21 7 17 37 48 57 1 resistance to EGFR inhibition by inducing a cytotoxic response (155). Interestingly they have 2 shown that the MET positive cells are preferentially located in the vascular niches and they are 3 4 resistant to radiation and highly tumorigenic (194). Altogether these results underline the 5 importance of MET status evaluation in EGFR-directed approaches and support the necessity for 6 synergistic therapies. 8 Hypoxia and cell metabolism. As we have mentioned above there is an important relationship 9 10 between EGFR and tumor cell metabolism, which could be particularly relevant in the GBM 11 niche context. A histopathological hallmark of GBM (particularly the primary ones) is the 12 presence of large fields of necrosis, which are related to a worse outcome (195). Interestingly 13 14 Steinbach and coworkers have shown that EGFR inhibitors have a protective effect against cell 15 death induced by acute hypoxia, opposite to their pro-apoptotic effects under normoxia (196). 16 In hypoxic conditions cells generate metabolic adaptative responses such as reduced synthesis 18 and increased nutrient catabolism. The mTOR protein kinase, present in the EGFR downstream 19 signaling pathway, is known to act as a nutrient and energy sensor, and the S6 ribosomal protein 20 21 functions as an effector of altered translation of genes implicated in metabolic control. It has 22 been proposed that under low oxygen conditions, EGFR inhibition reduces glucose intake, delays 23 ATP exhaustion and maintains the integrity of the mitochondrial membrane potential, probably 24 25 throw the desphosphorylation of ribosomal protein S6. The authors hypothesize that EGFR 26 inhibition may simulate a nutrient deprivation situation, preparing cells to the low oxygen and 27 starving conditions (196). According to these authours EGFR-TKIs could be counteractive in a 28 29 highly necrotic context. However, despite the relevance of this hypothesis, a possible correlation 30 between hypoxic and/or necrotic markers and lack of response to EGFR inhibitors is still missing. 31 32 33 34 Kinase independent functions of EGFR 35 36 Studies on EGFR have been focused mainly on the conventional signal transduction 38 pathways. However, it has long been known that many functions of EGFR require other 39 mechanism besides those early transient responses. In fact compelling evidence indicates that 40 41 EGFR can mediate cellular processes independent of its kinase activity in several types of cancer 42 (100). For example, the expression of a mutant EGFR receptor (D813A), with no kinase activity, 43 is able to induce MAPK activation and DNA synthesis (197). Moreover another kinase dead EGFR 44 45 mutant (K721M) can activate survival signals through the interaction with other proteins like 46 HER2 (198;199). These kinase-independent functions of EGFR could be and additional 47 explanation for the failure of the TKI strategies as alternatively downstream signal transducers 49 could be regulated in a phosphorylation independent manner. Here we summarize some of the 50 non-catalytic actions of EGFR in GBM and other cancers (Figure 3). 51 52 EGFR and glucose uptake. Weihua and coworkers have found that the receptor prevents 53 54 autophagic cell death by maintaining intracellular glucose levels through interaction and 55 stabilization of the sodium/glucose cotransporter 1 (SGLT1) (200). Interestingly EGFR-SGLT1 56 interaction does not respond to EGF stimulation or EGFR tyrosine kinase inhibition (201). SGLTs 58 are capable to take up glucose into the tumor cell even against a high chemical gradient (202) 60 61 62 63 64 65 22 7 11 16 31 51 55 1 and this seems to protect the cells from apoptosis inducers. In line with these results it has been 2 proposed that knocking down EGFR, but not inhibiting its tyrosine kinase activity, sensitizes 3 4 prostate cancer cells to the apoptosis inducer adriamycin (203). Moreover siRNA mediated 5 downregulation of EGFR also stimulated the apoptotic effect of adriamycin in liver cancer cells 6 (204). Interestingly this apoptosis could be inhibited by increased extracellular glucose level, 8 further supporting that intracellular glucose deficiency is a key mediator of the apoptosis 9 sensitization induced by downregulation of EGFR (203). These same authors have demonstrated 10 that EGFR and SGLT1 co-localized in prostate cancer tissues, and that inhibition of SGLT1 12 sensitized prostate cancer cells to EGFR inhibitors (gefitinib and erlotinib) (201) providing an 13 alternative synergistic approach to cure EGFR-addicted cancers. 14 15 SGLTs are overexpressed in several tumor entities (205). Moreover, in oral squamous cell 17 carcinoma the expression of SGLT1 has been correlated with EGFR expression (206). In these 18 tumors an irradiation-stimulated and EGFR-mediated increase in SGLT1-generated glucose 19 20 uptake, has been proposed to be required for the survival of genotoxically stressed tumor cells 21 (207). Furthermore it has been proposed that the interaction between SGLT1 and EGFR is 22 induced by radiation in lung cancer and that the subsequent increase in glucose uptake 23 24 counteracts the ATP crisis in tumor cells due to chromatin remodeling (205). Importantly, the 25 blockade of recovery from ATP crisis by SGLT1 inhibition may radio-sensitize tumor cells, as it 26 has been reported in lung adenocarcinoma and head and neck squamous carcinoma cell lines 27 28 (205;207). Although there are no reports of SGLT1 expression in gliomas (with or without EGFR 29 amplification) this is one of the cancers with higher glucose consumption so one would expect 30 to have an important expression of glucose transporters. In fact Flavahan and coworkers have 32 recently shown that there is a metabolic reprogramming in GBM with more aggressive cells 33 being able to express GLUT3, the high affinity neuronal glucose transporter allowing them to 34 35 survive in nutrient restrictive environments (208). It will be interesting to test whether EGFR 36 modulates GLUT3 stability in glioma cells. In any case it is tempting to propose a synergistic 37 effect of an increase in glucose uptake (mediated by the stabilization of SGLT1 and/or other 38 39 glucose transporters) with the PKM2-mediated glycolytic upregulation in EGFR amplified and/or 40 mutated high-grade gliomas (72). These would reinforce EGFR addiction in those GBM and 41 would argue for the synergistic inhibition of both, EGFR tyrosine kinase activity and receptor 42 43 over-expression (or increased protein stability). 44 45 Lipid rafts activation of EGFR. TKIs targeting EGFR have also failed in breast cancer, even if the 46 cells still depend on EGFR expression for growth. Interestingly the receptor was found to be 47 48 localized to plasma membrane lipid rafts in the TKI-resistant cell lines. Lipid rafts are specialized 49 membrane microdomains enriched in cholesterol, sphingolipids and proteins. Moreover 50 interfering with cholesterol biosynthesis or lowering cholesterol levels synergized with gefitinib 52 (209). These authors postulated that lipid rafts provide a platform to facilitate the interaction of 53 EGFR, c-Src and PI3K, leading to AKT activation and pro-survival signals, independently of EGFR 54 kinase activity (210). Furthermore in colorectal cancer there are evidences that HIF1,2 direct 56 transcriptional activation of CAV1, an essential structural constituent of caveolae (specialized 57 lipid raft microdomains), leads to increased dimerization and signaling of EGFR (211). However 58 59 there have been no reports of lipid rafts-related activation of EGFR signaling in GBM. In fact 60 61 62 63 64 65 23 7 11 16 39 54 1 caveolin1 (CAV1) acts as a tumor suppressor for GBM cells. Moreover it has been proposed that 2 caveolae-enriched cellular fractions sequesters EGFR and blocks signaling through this receptor 3 4 (212). However it has been reported that EGF-induced phosphorylation of the receptor results in 5 EGFR dissociation from caveolae whereas EGFRvIII is predominantly cytoplasmic and does not 6 associate with CAV1 unless cells are exposed to TKIs (213). Therefore although CAV1 is 8 overexpressed in GBM cells it seems to act as a tumor suppressor for EGFR-dependent cells 9 (214). However it is still possible that lipid rafts (other than caveolae) activate EGFR and that 10 cholesterol modulation of EGFR localization in the different membrane subdomains could have 12 different survival outcomes depending on the tumor cell type or in the presence of different 13 EGFR isoforms. 14 15 Mitochondrial apoptosis inhibition. Another kinase-independent role for EGFR in GBM survival is 17 related to the mitochondrial control of apoptosis. Both EGFR and EGFRvIII associate with p53- 18 upregulated modulator of apoptosis (PUMA), a pro-apoptotic member of the Bcl-2 family of 19 20 proteins primarily located on the mitochondria (215). PUMA strongly induces apoptosis in 21 colorectal cancer, malignant gliomas and in adult stem cells (216). EGFR-PUMA interaction is 22 independent of EGF stimulation or kinase activity and induces PUMA sequestration in the 23 24 cytoplasm, where it cannot initiate apoptosis. These observations are in agreement with the co- 25 expression of PUMA with EGFR/EGFRvIII in cell lines and patient samples and with the high 26 resistance to apoptosis inducting agents of GBMs (215). 27 28 29 Transcriptional activity of EGFR. Once in the nucleus EGFR can act as a modulator of 30 transcription of several genes. In fact it has been previously described that a kinase-dead EGFR 31 32 mutant can stimulate DNA synthesis in a kinase independent manner (217). Later on Lin and 33 coworkers defined nuclear EGFR as a transcriptional co-factor that contains a transactivation 34 domain in its C-terminus and that is able to modulate cyclin D1 gene expression (99). From then 35 36 on several other transcriptional targets of EGFR have been defined, mostly implicated in cell 37 cycle progression and the nitric oxide pathway: nitric oxide synthase (iNOS) (a protein involved 38 in inflammation, tumor progression and metastasis) (218), B-Myb (a protein controlling 40 proliferation) (219), cyclooxygenase-2 (COX-2) (102), aurora kinase A (a protein involved in 41 chromosomal instability)(220), c-Myc (221), and breast cancer resistance protein (BCRP) (222). 42 43 Given the fact that EGFR lacks a DNA-binding domain, mechanisms of EGFR-mediated gene 44 regulation involve direct interaction of EGFR with STAT3 to regulate iNOS and COX2 promoters, 45 with STAT5 for regulation of the Aurora Kinase A promoter, with E2F1 transcription factors for 46 47 regulation of the B-Myb promoter, and with SRC and STAT3 to form and heteromeric complex in 48 the nucleus that contributes to the expression of c-Myc. Constitutive presence of EGFR in the 49 tumor nuclei may be beneficial to the tumors encountering EGFR-targeted antibodies and TKIs. 50 51 In fact it has been shown that cancer cells that have acquired resistance to cetuximab (223) or 52 gefitinib (222) expressed increased levels of nuclear EGFR. These observations provide a 53 rationale for the combinations of inhibitors of this receptor and molecules that could block EGFR 55 nuclear translocation like for example AKT inhibitors, as AKT-mediated EGFR phosphorylation at 56 Ser-229 has been shown to be required for EGFR nuclear entry (222). Dasatinib, a known SRC 57 58 inhibitor, has demonstrated also a synergistic effect with cetuximab by limiting EGFR 59 translocation to the nucleus (223). 60 61 62 63 64 65 24 9 24 39 46 50 1 2 3 4 5 Targeting EGFR stability in GBM 6 7 If kinase-independent functions of EGFR are responsible for tumor maintenance we should 8 look for alternative strategies that could downregulate levels of receptor, alone or in 10 combination with TKIs. Among the different strategies that could be used to regulate EGFR 11 protein levels the use of antisense RNAs against both wild type and mutant sequence of the 12 13 receptor have been tested. This method impairs GBM cells growth in vitro and in vivo (224; 14 225;226), however the absence of efficient and specific siRNA delivery tools hampers the 15 possibility of its immediate application in the clinic. Figure 4 reviews the mechanisms that 16 17 control EGFR downregulation and its implication for the development of new GBM therapies. 18 The downregulation of EGFR signaling entails a variety of cellular processes such as receptor 19 ubiquitination, dephosphorylation, depletion of ligand access, receptor trafficking to the 20 21 lysosome and its subsequent degradation (227). As we have previously mentioned CBL is the 22 primary E3 ubiquitin ligase that is recruited to EGFR after ligand stimulation. CBL can bind 23 directly to phospho-Y1045, or indirectly via Grb2. CBL recruits E2 enzymes to its ring-finger 25 domain to promote EGFR ubiquitination. Ubiquitinated EGFR is recognized by the ubiquitin- 26 binding domains of epsin I, Eps15, Eps15R and Hrs/Hgs proteins, strongly associated with 27 28 chlatrin (228). Clathrin-coated vesicles containing ubiquitinated EGFR fuse with early endosomes 29 and interact with the ESCRT (endosomal-sorting complex required for transport) which guide 30 the ubiquitinated EGF-EGFR complex to the lysosome for degradation. Internalization seems to 31 32 be a kinase-independent process, which is followed by efficient recycling to the plasma 33 membrane (229). In fact the equilibrium between degradation and recycling determines the 34 output of EGFR stimulation. Defective endocytic downregulation of EGFR is associated with 35 36 cancer. Indeed, dominant-negative forms of CBL are found as oncogenes in human myeloid 37 neoplasms (230). No such mutations have been found in GBM although the 19q13 allele 38 containing the CBL sequence is frequently lost in these tumors (231). Another way to 40 manipulate the EGFR network is to maintain the level of activity just below the threshold 41 required for CBL recruitment and receptor degradation. This is the case for several mutant 42 43 forms detected in lung cancer (232) and also for EGFRvIII (153). It is also noteworthy that EGF, 44 but not TGF (frequently overexpressed alongside EGFR) (233) or amphiregulin (234), triggers 45 efficient degradation of EGFR. Interestingly co-expression of TGF drives the tumorigenic 47 potential of EGFR for tumor initiation (154). These results suggest that GBM cells need to block 48 EGFR receptor degradation in order to enhance the downstream signaling and promote cell 49 growth and proliferation. In fact this property has already been used to isolate by flow 51 cytometry the GBM cells with higher capacity to form tumors, which are the ones that express 52 higher levels of EGFR in the plasma membrane (225). 53 54 55 LRIG1. Given the relevance of EGFR signaling pathway it is not surprising that the internalization 56 process involves a variety of positive and negative regulatory loops, which are responsible of the 57 58 maintenance or termination of the intracellular signal cascades triggered by EGF-EGFR 59 interaction and fine-tuned the cellular decisions. Among these regulatory processes, EGFR 60 61 62 63 64 65 25 7 11 22 26 37 41 1 activation is responsible for the transcription of genes like leucine-rich repeats and 2 immunoglobulin-like domains-1 (LRIG1) and mitogen-inducible gene 6 (MIG-6), which code for 3 4 positive inducers of receptor degradation, and Sprouty 2 (SPRY2), which code for an inhibitor of 5 EGFR internalization (235). In fact the transmembrane glycoprotein LRIG1 has been proposed as 6 a tumor suppressor protein due to its role in EGFR degradation. LRIG1 increases the amount of 8 CBL recruited to the EGFR, limiting its downstream signaling (236;237) and it is also involved in 9 EGFRvIII variant degradation in GBM cells in a CBL independent manner (238). Besides, it has 10 been shown an increased EGFR/LRIG1 ratio in gliomas when compared with normal brain tissue, 12 suggesting that LRIG1 downregulation is connected to the tumor progression. The 13 overexpression of LRIG1 in glioma cell cultures leads to EGFR reduction in cell surface, 14 15 independently of its activation status, and triggers cell growth inhibition and impaired invasion 16 (mediated via MAPK and AKT signaling blockade), and enhanced apoptosis through increased 17 caspase-8 levels release (239;240). Johansson and collaborators have recently demonstrated 18 19 that soluble LIGR1 (sLIGR1) has an antitumoral effect both in vitro and in vivo, promoting cell 20 cycle arrest by downregulating MAPK phosphorylation, with no effect on EGFR levels and 21 activation state (240). It is thought that sLRIG1 may act through other RTKs or through an RTK 23 independent way, which makes it a promising RTKs inhibitor with wide antitumoral activity. It 24 has been recently published a similar effect performed by gambogic acid, which activates AMP- 25 activated protein kinase (AMPK) with the subsequent upregulation of LRIG1, having as a result 27 EGFR signaling inhibition, GBM cells increased apoptosis and impaired tumor growth (241). 28 29 30 MIG-6. MIG-6 has been identified as a molecule that is induced after EGF stimulation and 31 enhances EGFR trafficking into late endosomes/lysosomes for its degradation (242). Upon EGFR 32 ligand stimulation, MIG-6 is recruited to the activated receptor and suppresses its downstream 33 34 signaling. During the ligand-stimulated EGFR trafficking, MIG-6 interacts with STX8, a SNARE 35 protein required for late endosomes fusion, originating a complex that leads to EGFR lysosomal 36 degradation (243). Mig-6 knockout mice exhibit hyperactivation of endogenous EGFR, resulting 38 in hyperproliferation and impaired differentiation of epidermal keratinocytes (244). High- 39 resolution genomic profile of GBM allowed the identification of a highly recurrent (13% of 40 tumors) focal 1p36 deletion which contains MIG-6. Moreover the same authors demonstrated 42 that MIG-6 expression is down-regulated in half of the GBMs tested and there is a positive 43 correlation between the existence of and MIG-6 genomic alterations and the presence of EGFR 44 45 amplification and/or the mutant EGFR vIII in GBM samples (242). These results support its role 46 as a tumor suppressor in GBM, especially for EGFR-dependent tumors. 47 48 SPRY2-DYRK1A. It is well known the implication of the protein SPRY-2 in EGFR stability 49 modulation. SPRY-2 is an inducible regulator, which is phosphorylated on a conserved tyrosine 50 51 residue (Y55) after EGFR activation. This phosphotyrosine acts as a docking site for the SH2 52 domain of CBL, and competes with activated EGFR Y1045 phosphorylation. Hence, SPRY-2 53 removes CBL from activated EGFR and blocks CBL-mediated EGFR ubiquitination, endocytosis 54 55 and degradation, which leads to sustained receptor signaling (245;246). Although SPRY2 is a 56 tumor suppressor in different types of cancer, it has a tumor-promoting activity in colon cancer 57 (247). In GBM, several members of the SPRY family are included in a transcriptome module that 58 59 was associated with the EGFR amplification status in GBMs (248) suggesting that they could act 60 61 62 63 64 65 26 7 11 32 44 50 54 1 as oncogenes in at least a subset of glial tumors. In relation to this it has recently been described 2 the role of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1A) in EGFR 3 4 stability regulation in neural progenitor and GBM cells (249;250). Although the mechanisms 5 through which DYRK1A regulates EGFR stability are not fully characterized it has been proposed 6 that DYRK1A may act upstream SPRY-2 modulating EGFR targeting to the lysosomes. Pozo and 8 coworkers have indicated that DYRK1A function avoids EGFR degradation and favors recycling of 9 the receptor to the cell surface, which results in EGFR signaling enhancement and an increase in 10 tumor progression. Moreover the expression of DYRK1A correlated with that of EGFR in GBM 12 suggesting that this kinase is necessary for the oncogenic action of EGFR (250). The 13 downregulation of DYRK1A activity by siRNAs resulted in the reduction of EGFR levels in vitro 14 15 and in vivo, leading to self-renewal and proliferation inhibition, increased apoptosis and delayed 16 tumor growth (250). More interestingly pharmacological inhibition of DYRK1A kinase activity 17 also showed a clear anti-tumor effect, indicating that it could be a good target in order to induce 18 19 EGFR degradation. 20 21 All the previously exposed reinforces the notion that GBM trend to stabilize EGFR in the 22 membrane through inhibition of receptor degradation, either by downregulating positive 23 modulators of internalization or by overexpressing negative effectors of this process. Alterations 24 25 in the expression of these modulators could explain why there is not a linear correlation 26 between EGFR protein levels and the response to anti-EGFR therapy (10-13;168). In fact several 27 studies suggest that EGFR activity and erlotinib sensitivity can be more accurately predicted by 28 29 the ratio of MIG-6/EGFR in different tumors and that resistance to TKIs is associated with an 30 increase in MIG-6 expression and therefore a decrease in EGFR activity (251;252). In fact Mig-6 31 knock-out cells are unusually sensitive to gefitinib (244). Therefore measuring levels of 33 membrane EGFR or analyzing the expression of EGFR turnover modulators might be relevant to 34 predict the response to TKIs. On the other hand it will be interesting to test if targeting EGFR 35 36 could enhance the efficacy of the current strategies focused on inhibiting EGFR activity. As a 37 proof of principle, green tea (-)-epigallocatechin-3-gallate (EGCG), a known DYRK1A inhibitor 38 (253) showed a potent antitumor synergistic effect with erlotinib in head and neck (254) and 39 40 lung (255) cancer. 41 42 43 Concluding remarks 45 EGFR was one of the first tyrosine kinase receptors to be described and linked to 46 47 tumorigenesis. In GBM, alterations in the EGFR gene occur in almost 50% of the cases, 48 particularly in primary tumors. Nevertheless there is a need to revise what we know about EGFR 49 signaling in gliomas in order to fully elucidate the mechanism for its tumorigenic action. Studies 51 on this receptor have been focused mainly on the conventional signal transduction pathways 52 such as MAPK and PI3K, controlling cell proliferation and survival. However accumulating data 53 indicate that this classical view is not enough to explain the complexity of cellular functions that 55 are being associated with activation and/or overexpression of EGFR in astrocytic cells. 56 Compelling evidence links EGFR activity with the regulation of cellular metabolism and with the 57 58 adaptative responses of GBM cells to their hypoxic microenvironment. Moreover the 59 localization of the receptor in different subcellular compartments (mainly the nucleus and the 59 60 61 62 63 64 65 27 7 11 1 mitochondria) seems to be important for the control of DNA damage and apoptotic responses, 2 crucial steps for tumor initiation and survival. Crystallographic analysis has shed some light into 3 4 the nature of GBM-associated EGFR mutations, indicating the prevalence of the receptor 5 inactive state in this tumor cells. Therefore molecules that could bind to this conformation 6 would be preferable to treat aggressive gliomas. All these studies are fundamental to orient the 8 therapeutic targeting of EGFR in GBMs, defining better readouts of the action of EGFR inhibitors 9 and understanding why molecules working in other tumors fail in gliomas. While we wait for the 10 results of clinical trials with second and third generation TKIs we need to anticipate the possible 12 compensatory mechanisms activated by other RTKs or downstream mutations that could 13 suggest bona fide predictive markers as well as more effective synergistic approaches. Finally we 14 15 have to keep in mind that the response to EGFR activation can be independent of its kinase 16 activity, therefore targeting receptor stability could be more effective than TKIs. 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The 47 green tea polyphenol EGCG potentiates the antiproliferative activity of c-Met and epidermal growth factor 48 receptor inhibitors in non-small cell lung cancer cells. Clin Cancer Res 15:4885-4894. 49 50 51 52 53 54 55 56 57 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image