**3.3. RTK-mediated immune suppression**

involved in glycolysis, angiogenesis, invasion and epithelial-mesenchymal transition (EMT), which are critical for tumor growth and progression, and likely cooperate and activate other aberrant RTK signaling pathways [36, 37]. We and other reports showed that, in response to hypoxia condition, significantly increased activity of EGFR, as well as its mutant protein EGFRvIII, which further promoted activation of convergent downstream signaling pathways including Ras/MAPK, PI3K/Akt, JAK2/STAT3, and NF-κB signaling, and enhanced malignant

behaviors in GBM cells in vitro, and most likely to act in the same way in vivo [1, 38].

**3.2. RTK and ECM/integrin signaling**

36 Glioma - Contemporary Diagnostic and Therapeutic Approaches

level is greatly correlated with poor clinical prognosis [51, 52].

In addition to VEGF, supplementary proangiogenic factors including FGF, PDGF, placentalike growth factor (PLGF), integrins, HGF/scatter factor, ephrins, angiopoietins (ANGPT), and interleukin-8 (IL-8), matrix-metalloproteinase (MMP)-2, MMP-9, collagen type I α1 (COLIA1), endothelial markers CD34, Tenascin-C, neuron-glial antigen 2 (NG-2) on pericytes, insulin-like growth factor (IGF), and EGF present in GBM [39–42]. Interestingly, many of these factors are RTK ligands and may bind to respective RTK on vascular endothelial cells or GBM cells, act in autocrine or paracrine manner to stimulate the events of neo-angiogenesis. Activation of these proangiogenic factors interacts with a number of signaling pathways include activation of Ras/ Raf/MAPK [41, 42], PTEN/PI3K/AKT [40], PLC-γ/protein kinase C (PKC) [40], nitric oxide (NO) [43], PDGFB [44], and Notch1 [45]. GBMs are diagnosed at the advanced stages when they show hypoxia and leaky vasculatures [35]. The critical role that VEGF and these pro-angiogenic factors play in angiogenesis has rendered them appealing targets to exploit in cancer therapeutics [43].

ECM/ integrins are key components mediating the dialog between cells and the microenvironment. Integrins are composed of two noncovalently associated α and β subunits, which are featured by a large extracellular domain, a short transmembrane domain and a small intracellular noncatalytic cytoplasmic tail [46]. These receptors play a role in the regulation of cell adhesion to ECM proteins or cell surface proteins [47]. Binding of ECM to integrins result in cell adhesion and activation of focal adhesion (FA)-associated signaling pathways [48] and thereafter cascades of intensive activation of downstream signaling that involved in cell proliferation and invasion [49]. Within FA complexes, further auto-phosphorylation of focal adhesion kinase (FAK) leads to its binding to SRC kinase and formation of activated FAK-SRC complexes. Consequently, FAK-SRC complexes activate cascades of downstream pathways including the Ras/Raf/MAPK, RAF/JNK, Rho/Rac/PAK and PI3K/Akt/mTOR [50]. Notably, FAK protein is overexpressed in many tumors including GBM, and its expression

ECM dysregulation is essential for establishing and maintaining a functional tumor microenvironment. ECM in GBM is stiffer and more cross-linked than that in the normal brain tissue, inducing abnormal cell behaviors such as aggressive cell invasion [35]. Dysfunction of ECM and its cognate receptor integrin may lead to aberrant signaling transduction pathways including Ras/Raf/MAPK, Raf/JNK, Rho/Rac/PAK and PI3K/Akt/mTOR, shaping a tumor microenvironment to promote tumor survival, angiogenesis, and invasion [31]. Importantly, many of these cellular signaling pathways are convergent with downstream signaling pathways of RTKs, implicating interaction and cross-talk of RTK- and ECM/integrin-mediated function in GBM GBM patients show marked intratumoral and systemic immunosuppression. The tumor microenvironment contains multiple immunosuppressive factors including TGFβ2, prostaglandin-E2, IL-10; and receptor molecules B7-H1, Fas-ligand, etc. [63]. The tumor is heavily infiltrated by microglia/macrophages, which can represent up to 30% of viable cells in the tumor mass, but lymphocytes infiltration is not common [64]. These monocytes/macrophages in the tumor environment interact with GBM cells and develop immunosuppressive myeloidderived suppressor cells (MDSCs). Systemic immunosuppression in GBM patients shows that total T-cell counts are greatly decreased, especially CD4+ T cell counts [65]. Furthermore, T-cell function is markedly abnormal [66]. Besides other factors that may underlie T-cell dysfunction, increases in circulating cell populations such as regulatory T cells (Tregs) and MDSCs may be more important [67, 68].

Because of immunosuppressive features of GBM, in recent years, new therapies such as tumor vaccines and peptides are tested in preclinical and clinical studies [63]. The mutant protein EGFRvIII is a cancer specific antigen bearing a targetable epitope that is almost exclusively present in GBM [69]. Rindopepimut is composed of an EGFRvIII-specific peptide conjugated with an adjuvant protein KLH (keyhole limpet hemocyanin). The vaccination produced active anti-tumor response with significant survival benefit in GBM patients. Furthermore, the underlying immune response is not only effective regarding specific removal of EGFRvIII-positive GBM cells, but also the increase in the titer of anti-EGFRvIII sera in beneficial patients [70]. More importantly, phase II clinical trial with the vaccine confirmed these results [71], and the randomized phase III clinical study is ongoing [63]. EGFRvIII lacks the ligand binding domain and is persistently activated, promoting tumor formation by activating aberrant signaling pathways, epigenetic mechanisms, and metabolic networks, and thus, is a promising cancer target [72]. Further with rindopepimut efficacy, chimeric antigen receptor (CAR) T cells transduced with humanized scFv against EGFRvIII were produced, and the studies are ongoing [73]. Other immunotherapy approaches that are tested or ongoing in GBM clinical studies include the administration of dendritic cells-based therapies; application of check-point inhibitor drugs, and adoptive cell therapy (ACT), etc.

Cancer development, progression, and response to treatment are greatly influenced by cancer cells' intracellular metabolism and the exogenous tumor environment [93]. The metabolic reprograming that cancer cells adapted to take up and utilize nutrients to drive tumor growth rigorously often relies on signaling and epigenetic/transcriptional networks induced by activated oncogenes (e.g., EGFR, RAS, MYC) and deactivated tumor suppressor proteins (e.g., TP53) [94, 95]. In primary GBM, the frequent genetic changes of key components of RTK/PI3K/Akt pathways, one of the three core signaling pathways that significantly altered in GBM [96], may result in constitutive activation of mechanistic target of rapamycin (mTOR) signaling [96, 97]. Recent studies showed cooperation between EGFRvIII signaling and c-Myc, the transcription factor and one of the master regulators of cancer metabolism [96], to reprogram cellular metabolism and promote tumor proliferation via activation of mTOR signaling, resulting in changes in intracellular nutrients levels [98–100]. Moreover, RTK- and Myc-dependent metabolic reprogramming maybe also involved in IDH1-mutant glioma malignant progression [101, 102]. Therefore, targeted therapies against Myc-dependent metabolism may be an effective therapy for patients with high-grade gliomas. Recent data indicated that extracellular nutrients such as glucose or acetate were required to maintain EGFRvIII signaling via activating mTORC2 [96], leading to GBM resistance to molecularly targeted therapies [103]. Nonetheless, the intricate interactions between the oncogenic signaling and cancer metabolism have only been recently revealed, and metabolism in primary GBM is dominantly regulated hypoxia and RTK-dependent c-Myc upregulation to modify cancer metabolome and cause resistance to therapies [93]. Future studies are needed to govern regulations on genetic and epigenetic alterations, oncogenic signaling and cancer metabolic reprograming, and

Receptor Tyrosine Kinase Interaction with the Tumor Microenvironment in Malignant…

http://dx.doi.org/10.5772/intechopen.76873

39

translate these insights into more effective treatments for GBM patients.

gable targets, including EGFR and EGFRvIII, VEGFR and PDGFR family.

Since the phase III trials of Temozolomide in 2005, there have been few successes regarding treatment for patients with malignant glioma [82]. RTKs, which are the most commonly amplified and mutated genes in cancers, are the key targets in cancer research including malignant glioma. Up to date, three RTKs and their family members present the major drug-

Small molecular weight kinase inhibitors include gefitinib (EGFR) and erlotinib (EGFR and EGFRvIII), and these two irreversible inhibitors, unfortunately, have achieved limited success either as a single agent or as combination therapies in numerous Phase I and II trials in patients with newly diagnosed or recurrent GBM [104–107]. The resistance may be driven by a subset of EGFR mutations, activation of alternate signaling pathways and suppression of EGFRvIII on extrachromosomal DNA, etc. [72]. Besides, irreversible inhibitors currently in clinical trials including lapatinib (EGFR, ErbB-2), AEE788 (EGFR, VEGFR), and dacomitinib

**4. Lessons from RTK-targeted therapies**

**4.1. Experience with RTK-targeted therapies in GBM**

*4.1.1. EGFR family and EGFRvIII*

EGFR also plays a protruding role in GBM cell invasion and aggressiveness [74]. It was previously showed that microglia cells stimulate GBM invasion via the EGFR signaling [75]. Coniglio et al. [76] demonstrated in vitro that microglia secreted EGF, which may activate EGFR and signaling pathways in GBM cells [77]. For example, EGFR or EGFRvIII may activate the STAT3 pathway [38], which is induced in various immune populations, and mediate immunosuppression potentiated by the GSCs [78]. Moreover, recent data implicated VEGF as a potent mediator of immunosuppression, again via GSC-associated mechanisms [79, 80]. A VEGF inhibitor, aflibercept, was applied in combination with an antitumor vaccine. Delayed tumor progression and survival extension were observed, which confirmed the efficacy of combining antiangiogenic and immunotherapy approaches, as well as the value of delineating tumor microenvironment [81]. Antiangiogenic therapy added to immunotherapeutic approaches toward glioma may show clinical benefits, among which the endogenous microenvironment or vaccineinduced inflammatory responses is importantly subsidiary to its effectiveness [82].

### **3.4. RTK, GSC and tumor metabolism**

GSCs or glioma initiating cells (GICs) are preferentially located in perivascular and around necro/hypoxic zones where they closely react with the microenvironment and, thus, escape from apoptotic stimuli and preserve the capacity of self-renewal [83]. These interactions with microenvironment components, such as stromal cells or extracellular matrix (ECM) etc., seem important for GSC maintenance, possibly via metabolic and/or epigenetic modifications [83, 84]. Besides, GSCs may be protected from external factors via specific survival signals that they receive from the niche [85]. For instance, hypoxia induces VEGF expression, which promotes angiogenesis and supports the GSC tumor-initiating capacity [86, 87].

Besides VEGF/VERGFR signaling, HGF/Met signaling involves in regulating cell growth, motility and has a role in embryogenesis, degenerative disease and wound healing [88]. This RTK-mediated signaling also promotes the acquisition of stem-cell like properties in glioma cells and the formation and malignant progression of GBM [89, 90]; overexpressing of Met in vitro in glioma cells was highly clonogenic [88]. Met expression seems to be associated with genetic features with EGFR and the tumor suppressor PTEN inactivation, indicating cooperation among these RTK-mediated signaling in keeping GSC phenotype in glioma [91, 92]. A recent mouse study showed that EGFR inhibition induces increased c-Met expression and associated proliferation of GSCs expressing pluripotency TFs and displaying multi-lineage potential [89]. There is now the debate as to the long-term safety of anti-EGFR treatments, which may possibly induce MET-driven GSC populations [88, 89]. On the other hand, however, it implies the combination of targeting EGFRvIII and GSC as a new therapeutic approach.

Cancer development, progression, and response to treatment are greatly influenced by cancer cells' intracellular metabolism and the exogenous tumor environment [93]. The metabolic reprograming that cancer cells adapted to take up and utilize nutrients to drive tumor growth rigorously often relies on signaling and epigenetic/transcriptional networks induced by activated oncogenes (e.g., EGFR, RAS, MYC) and deactivated tumor suppressor proteins (e.g., TP53) [94, 95]. In primary GBM, the frequent genetic changes of key components of RTK/PI3K/Akt pathways, one of the three core signaling pathways that significantly altered in GBM [96], may result in constitutive activation of mechanistic target of rapamycin (mTOR) signaling [96, 97].

Recent studies showed cooperation between EGFRvIII signaling and c-Myc, the transcription factor and one of the master regulators of cancer metabolism [96], to reprogram cellular metabolism and promote tumor proliferation via activation of mTOR signaling, resulting in changes in intracellular nutrients levels [98–100]. Moreover, RTK- and Myc-dependent metabolic reprogramming maybe also involved in IDH1-mutant glioma malignant progression [101, 102]. Therefore, targeted therapies against Myc-dependent metabolism may be an effective therapy for patients with high-grade gliomas. Recent data indicated that extracellular nutrients such as glucose or acetate were required to maintain EGFRvIII signaling via activating mTORC2 [96], leading to GBM resistance to molecularly targeted therapies [103]. Nonetheless, the intricate interactions between the oncogenic signaling and cancer metabolism have only been recently revealed, and metabolism in primary GBM is dominantly regulated hypoxia and RTK-dependent c-Myc upregulation to modify cancer metabolome and cause resistance to therapies [93]. Future studies are needed to govern regulations on genetic and epigenetic alterations, oncogenic signaling and cancer metabolic reprograming, and translate these insights into more effective treatments for GBM patients.
