**3. Proliferative and antiproliferative pathways and their roles in gliomas**

As more and more detailed studies into intracellular signaling cascades and modulators that regulate these pathways are published, intricacy of the fine-balance between celluar survival and death is revealed. A revolution in the signaling cascades has provided near complete resolution of how physiologically important signaling proteins interact with extracellular cues to trigger proliferation. More detailed understanding of the regulatory and activation

processes of uncontrolled cellular proliferation is proving to be a key in identification of newer approaches to improve the efficacy of existing therapeutics. The homeostasis of mitogenic signaling is tactfully controlled by multiple mechanisms. The past several years have seen a dramatic leap in our understanding of how Receptortyrosine kinase (RTK) mediated signaling is rewired during tumorigenesis to support the transformed phenotype. Activation of RTK results in receptor dimerization and autophosphorylation. More importantly, docking sites are created for different adaptor protein complexes such as Grb2/SOS. Mutant Ras is report‐ edly involved in 50% of all human tumors. There are direct pieces of evidence emphasizing on role of mutant Ras in gliomas. High Ras-GTP levels in advanced astrocytomas have been reported [8, 9].

Epidermal growth factor receptor (EGFR), coded as a cell-surface-bound receptor, is another important molecule involved in cell proliferation with potential effects on clinical prognosis of GBM. It is known that approximately <10% of secondary GBMs and 50% of primary GBMs have EGFR mutations [10]. The presence of EGFR variant III mutation (EGFRvIII) is known to upregulate mitogenic signaling pathways. There is a deletion of the regulator N-terminal domain (6–273) of EGFR in this pattern of EGFR. About 10–60% of the patients with GBM have EGFRvIII which can be detected in the peripheral blood of brain tumors. The detection of this mutation in brain cancer patients has great importance for anti-EGFRvIII therapies and patients can be monitored to track their response to these therapies [11,12]. Better and deeper knowledge of mechanistic insights that cause EGFR heterogeneity in GBM will prove to be helpful in identification of drugs with maximum efficacy. *EGFRvIII* mutation to identify patients fortreatments such as erlotinib therapy for non-small cell lung cancer orRNA-directed treatments and vaccine therapies [13]. As of now, we still have a limited knowledge about downstream signaling pathways for EGFR and whether *EGFR* mutations affect these down‐ stream signaling pathways, such as AKT, MAPK, and STAT3. On the other hand, it has been suggested that the clinical utility of this biomarker and its use for targeted treatment are complicated [1].

Platelet-derived growth factor receptor (PDGFR), a cell-surface tyrosine kinase, plays role in GBM proliferation and stem cell renewal. There are multiple isoforms of PDGFR, mutated in up to 30% of GBMs. One of them, the most significant one regarding GBM, is PDGFRA. The other isoform is also PDGFRAD (with a deletion of exons 8 and 9), seen in 40% of GBMs, and leads to constitutive activation [14,15]. According to the Cancer Genome Atlas, PDGFRA has a crucial role in the proneural subtype of GBM; however, no changes were observed in prognosis of the evaluated patients [16].

The phosphatidylinositol 3-kinase (PI3K)/AKT (PI3K/AKT) pathway is known as a crucial intracellular signaling pathway, taking role in regulating cell proliferation, migration, quiescence, proliferation, cancer, and longevity [17]. PI3K is an enzyme which phosphory‐ lates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphos‐ phate (PIP3) at the cell inner membrane. This activation process leads to recruit and upregulate various downstream pathways including AKT, a molecule localized in the plasma mem‐ brane [18].

Phosphatase and tensin homolog (PTEN) is known as a natural inhibitor of PI3K/AKT pathway. It has been shown to inhibit transduction of signals to downstream effectors via dephosphorylation of PIP3 to PIP2 [19]. It has been reported that an increased PI3K/Akt/mTOR signaling is seen in ~88% of all glioblastomas [20,21]. All of these biological pathways has been related to genetic alterations of key regulatory molecules involved in mitogenic signaling in RTKs and also in the PI3K-PTEN-Akt signaling axis.

processes of uncontrolled cellular proliferation is proving to be a key in identification of newer approaches to improve the efficacy of existing therapeutics. The homeostasis of mitogenic signaling is tactfully controlled by multiple mechanisms. The past several years have seen a dramatic leap in our understanding of how Receptortyrosine kinase (RTK) mediated signaling is rewired during tumorigenesis to support the transformed phenotype. Activation of RTK results in receptor dimerization and autophosphorylation. More importantly, docking sites are created for different adaptor protein complexes such as Grb2/SOS. Mutant Ras is report‐ edly involved in 50% of all human tumors. There are direct pieces of evidence emphasizing on role of mutant Ras in gliomas. High Ras-GTP levels in advanced astrocytomas have been

Epidermal growth factor receptor (EGFR), coded as a cell-surface-bound receptor, is another important molecule involved in cell proliferation with potential effects on clinical prognosis of GBM. It is known that approximately <10% of secondary GBMs and 50% of primary GBMs have EGFR mutations [10]. The presence of EGFR variant III mutation (EGFRvIII) is known to upregulate mitogenic signaling pathways. There is a deletion of the regulator N-terminal domain (6–273) of EGFR in this pattern of EGFR. About 10–60% of the patients with GBM have EGFRvIII which can be detected in the peripheral blood of brain tumors. The detection of this mutation in brain cancer patients has great importance for anti-EGFRvIII therapies and patients can be monitored to track their response to these therapies [11,12]. Better and deeper knowledge of mechanistic insights that cause EGFR heterogeneity in GBM will prove to be helpful in identification of drugs with maximum efficacy. *EGFRvIII* mutation to identify patients fortreatments such as erlotinib therapy for non-small cell lung cancer orRNA-directed treatments and vaccine therapies [13]. As of now, we still have a limited knowledge about downstream signaling pathways for EGFR and whether *EGFR* mutations affect these down‐ stream signaling pathways, such as AKT, MAPK, and STAT3. On the other hand, it has been suggested that the clinical utility of this biomarker and its use for targeted treatment are

Platelet-derived growth factor receptor (PDGFR), a cell-surface tyrosine kinase, plays role in GBM proliferation and stem cell renewal. There are multiple isoforms of PDGFR, mutated in up to 30% of GBMs. One of them, the most significant one regarding GBM, is PDGFRA. The other isoform is also PDGFRAD (with a deletion of exons 8 and 9), seen in 40% of GBMs, and leads to constitutive activation [14,15]. According to the Cancer Genome Atlas, PDGFRA has a crucial role in the proneural subtype of GBM; however, no changes were observed in

The phosphatidylinositol 3-kinase (PI3K)/AKT (PI3K/AKT) pathway is known as a crucial intracellular signaling pathway, taking role in regulating cell proliferation, migration, quiescence, proliferation, cancer, and longevity [17]. PI3K is an enzyme which phosphory‐ lates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphos‐ phate (PIP3) at the cell inner membrane. This activation process leads to recruit and upregulate various downstream pathways including AKT, a molecule localized in the plasma mem‐

reported [8, 9].

148 Neurooncology - Newer Developments

complicated [1].

brane [18].

prognosis of the evaluated patients [16].

Some regulatory and effector molecules play important role in classical cell death networks of both extrinsic (death receptor-mediated) and intrinsic (mitochondria-dependent) apoptosis signaling pathways [22]. Since the discovery of TNF (Tumor Necrosis Factor) family mem‐ bers, a new milestone in apoptosis-inducing cancer therapies has emerged. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a protein reportedly involved in selec‐ tive killing of cancer cells while leaving normal cells intact. The major biological role of this 281-amino acid-type II transmembrane protein is apoptosis induction after interacting with its receptors to trigger extrinsically and intrinsically controlled pathways. Four different homologous human TRAIL receptors have been categorized into TRAIL-R1/DR4, TRAIL-R2/ DR5 also known as Killer, TRAIL-R3 or DcR1, and TRAIL-R4 or DcR2. Substantial fraction of information has been added into the existing pool of knowledge related to TRAIL biology and known that different cancers are resistant to TRAIL-based therapeutics. Mechanistically it has been shown that downregulation of death receptors considerably impaired TRAIL-induced apoptosis in cancer cells. In the upcoming section, we briefly summarize advancements in our understanding related to the underlying mechanisms of resistance against TRAIL-induced apoptosis. We also discuss how TRAIL has shown as a potent anticancer agent in xenograft‐ ed mice. Natural products have also added more options in the armory against brain tumor. Detailed mechanistic insights have provided a near complete resolution of protein network TRAIL-resistant glioblastoma and increasingly it is being realized that imbalance of stoichio‐ metric ratios of proapoptotic and antiapoptotic proteins modulates response of cancer cells to TRAIL. Previously, it has been convincingly revealed that Znf domain of A20 E3 ligase ubiquitinated RIP1 through a K63-linked polyUB chain that structurally interacted with p18 domain of caspase-8 and blocked its dimerization and cleavage. Functionally inactive caspase-8 was unable to proteolytically process downstream effectors that resulted in impairment of TRAIL-induced apoptosis in glioblastoma [23]. Adeno-associated virus (AAV) vectors are being used to efficiently deliver secreted, soluble TRAIL in different preclinical studies. Additionally, these are also used in combination with TRAIL-sensitizing cardiac glycoside, lanatoside C (lan C). Tumor growth was considerably reduced in intracranial U87 tumor-bearing mice treated with AAV-sTRAIL and lanatoside C [24]. Mesenchymal stem cells (MSCs) have the ability to migrate toward intracranial glioma xenografts. Experimental‐ ly verified data indicated that MSCs expressing firefly luciferase (fluc) injected into the left hemisphere migrated rapidly toward right, tumor-bearing region ofthe brain. Results revealed that 11% of implanted MSCs were noted to be localized in right hemisphere within 2 hours after MSC inoculation. Coculture of GBM43 and U87 glioma cells with MSCs-TRAIL dis‐ played notable rise in caspase-3 activity. Survival rate of tumor-bearing mice was enhanced intranasally delivered with MSCs-TRAIL [25]. Carbenoxolone (CBX), a derivative of 18 glycyrrhetinic acid, has been shown to effectively enhance killing activity of TRAIL-express‐ ing MSCs. CBX considerably upregulated cell-surface expression of DR5 in CBX-treated ΔGli36 and U87MG cells. CBX also inhibited gap junction (GJ) communication via modula‐ tion of connexin (Cx43). CBX remarkably reduced expression levels of Cx43 in U87MG and ΔGli36 cells after 72 hours. Results revealed that TRAIL-induced apoptosis was markedly higher in cells transfected with Cx43-siRNA [26] .

Antibody-based anticancer therapies have attracted considerable attention and different structural variations are being tested for efficacies which involve smaller antibody frag‐ ments such as ScFvs, Fabs, and nanobodies. Single-chain Fv fragment (scFv) consists of a variable light-chain (VL) and variable heavy-chain (VH) domains, which contains whole antigen-binding site.

Multidrug resistance protein 3 (MRP3) is frequently overexpressed in glioblastoma multi‐ forme cells. scFvM58-sTRAIL is an engineered protein formed by fusion of MRP3-specific scFv antibody M58 with N-terminus of soluble TRAIL. scFvM58-sTRAIL was effective against MRP3-positive GBM cells. Expectedly, scFvM58-sTRAIL did not show significant activity against MRP3-negative Jurkat cells. These results indicated that scFvM58-sTRAIL was effective against MRP3-positive cancer cells [27].

Various bivalent EGFR-targeting nanobodies (ENbs) have been designed and noted to be effective. Neural stem cells (NSC) are potent agents to deliver ENbs. Preclinical study revealed that tumor regression was significantly higher in xenografted mice treated with NSC-ENb2- TRAIL. Xenografted mice survived for 51 days upon treatment with control NSC-ENb2 and 80% of mice survived for 80 days after treatment with NSC-ENb2-TRAIL. These findings indicated that tumoritropic NSC-releasing ENb2 inhibited growth of glioblastoma and effectiveness of ENb2-based therapy was markedly improved by NSC-releasing ENb2- TRAIL [28].

Diethylamino-curcumin mimics with substituted triazolyl groups have previously been synthesized and reported to effectively sensitize resistant CRT-MG astroglioma cells to TRAIL [29].

Gingerol, a major bioactive component of ginger, has been shown to trigger expression of DR5 in a p53-dependent manner in U87 glioblastoma cells. Digitoxin (DT), a clinically approved cardiac glycoside, has been observedto overcome resistance againstTRAILin resistantU87MG glioblastoma cells. Digitoxin effectively enhanced DR5 expression on cell surface of resistant cancer cells [30].
