Glioblastoma Multiforme (GBM) is biologically aggressive neoplasms which have an elevated, often aberrant, prolifetraive capacity with a diffuse pattern of brain invasion. It is the most malignant Astrocytic tumor, composed of poorly differentiated neoplastic astrocytes (Ref.1). The World Health Organization (WHO) grading system classifies Gliomas into Grades I-IV based on the degree of malignancy, as determined by histopathological criteria. In the Central Nervous System (CNS), Grade-I Gliomas generally behave in a benign fashion and might even be circumscribed, whereas Grade-II, Grade-III and Grade-IV Gliomas are malignant and diffusely infiltrate throughout the brain. Glioblastoma Multiforme falls under the Grade-IV category. Glioblastoma Multiforme or the WHO Grade-IV Astrocytoma is divided into two subtypes based on clinical characteristics: Primary and Secondary GBM. Primary GBM/Primary Glioblastoma Multiforme arises as a de novo process, in the absence of a pre-existing low-grade lesion, whereas, Secondary GBM/Secondary Glioblastoma Multiforme develops progressively from Low-Grade Astrocytoma, generally over a period of 5-10 years. Although most neurological tumors are of Glial-lineage origin, however it is unclear whether tumor cells result from the transformation of an immature precursor or from the dedifferentiation of a mature glial cell. Several genetic pathways are involved in the initiation and progression of these neoplasms, particularly during the manifestation of Secondary GBMs (Ref.2).
Normal cells require growth signals for survival and/or proliferation. Many growth signals are mediated by diffusible growth factors that are transmitted into the cell by a group of transmembrane proteins with intrinsic tyrosine kinase activity RTKs (Receptor Tyrosine Kinases). About fifty to sixty percentages of GBMs have over-expression and amplification RTKs like of the EGFR (Epidermal Growth Factor Receptor), PDGFR (Platelet-Derived Growth-Factor Receptor) and IGF1R (Insulin-Like Growth Factor-I Receptor). After binding to their respective growth factor ligands, like EGF (Epidermal Growth Factor), PDGF (Platelet-Derived Growth Factor) and IGF (Insulin-Like Growth Factor), these RTKs undergo receptor dimerization, autophosphorylation and recruitment of adaptor proteins (such as GRB2 (Growth Factor Receptor-Bound Protein-2), SHC (Src Homology-2 Domain Containing Transforming Protein) and SOS (Son of Sevenless)) that interact with and activate various downstream effectors. The small GTP-binding protein, Ras, is an important downstream effector of the growth factor-RTK signaling pathway, and activates at least three downstream cascades: Raf (v-Raf Murine Sarcoma Viral Oncogene Homolog)-MEK (MAPK/ERK Kinase)-MAPK (Mitogen-Activated Protein Kinase)/ERK (Extracellular Signal-Regulated Kinase), PI3K (Phosphatidylinositol-3-Kinase)-Akt (v-Akt Murine Thymoma Viral Oncogene Homolog) and CDC42 (Cell Division Cycle-42)-Rac-Rho. Deregulation of these signaling routes that are vital for gene expressions which control cell growth, cell proliferation, cell survival, regulation of cytoskeleton, apoptosis, cell migration, cell transformation and invasion, ultimately leads to manifestation of Glioblastoma Multiforme or Oncogenesis. Ras signaling is negatively controlled by NF (Neurofibromin) proteins. The NF1 (Neurofibromin-1) gene encodes the protein Neurofibromin, which shares homology with the RasGTPase-Activating Protein (GAP) family and catalyze the conversion of activated Ras-GTP to inactive Ras-GDP thereby negatively regulating cellular Ras activity. Similarly, NF2 interaction with GRB2/SHC/SOS complex blocks Rac1 (Ras-Related C3 Botulinum Toxin Substrate-1) and inhibits the subsequent downstream functions. Disruption of NF1 function causes Neurofibromatosis Type-I, a familial cancer syndrome which involves development of multiple benign and malignant tumors of the CNS and Peripheral Nervous System (PNS). Ras mediated signaling is involved in the initiation of Astrocytoma development and the growth factor-RTK-Ras signaling cascade is one of the most frequently targeted genetic pathways in human cancers, possibly because activating mutations render cancer cells independent of exogenous growth factors. Elevated expression of growth factors and their cognate RTK receptors, including PDGF/PDGFR occur in every grade of Astrocytoma. Furthermore, PDGF and PDGFR are often co-expressed in the same tumor cells, indicating that Astrocytoma cells establish an autocrine stimulatory loop (Ref.1 & 3). The over-expression and amplification of the EGFR contribute to the malignant phenotype of human Glioblastomas. In addition, malignant Glioma cells have higher levels of PKC-Delta (Protein Kinase-C-Delta) than non-neoplastic astrocytes, which reflect the fact that excessive PKC-Delta activity may significantly contribute to astroglial tumorigenicity (Ref.1).
In wild type of glial cells PKC-Delta downstream signaling is regulated by growth factor-RTK-Ras signaling in which GRB2/SHC/SOS complex activates c-Src (v-Src Avian Sacroma (Schmidt-Ruppin A-2)Viral Oncogene) and PI3K, activating PLC (Phospholipase-C) and Akt, respectively. PLC cleaves PIP2 (Phosphatidylinositol 4,5-bisphosphate) to generate DAG (Diacylglycerol) and IP3 (Inositol 1,4,5-trisphosphate). DAG in turn activates PKC-Delta. Further IP3 modulates release of Ca2+ (Calcium ions) through IP3R (IP3 Receptor) enhancing PKC-Delta activation. PKC-Delta and c-Src are involved in transactivation of the EGFR during development of Glioblastomas. The non-receptor tyrosine kinase c-Src phosphorylate the EGFR at Tyr1068 (Tyrosine-1068) and lead to an increase in EGFR kinase activity to activate the Ras/Raf/MEK/MAPK pathway in Glioblastoma cells through PKC-Delta. Activation of PKC-Delta again leads to the phosphorylation of several proteins that are involved in the regulation of cell growth, differentiation, and apoptosis. The existence of cross-talk between PKC-Delta activation and EGFR, which is over-expressed in fifty percent of Primary GBMs, provides a novel signaling pathway that is altered in Astrocytic tumors and that may provide a useful therapeutic target. Therefore, gene silencing of PKC-Delta and c-Src with siRNA (Small interfering RNAs) and pharmacological inhibition with BIM (Bisindolylmaleimide) and Rottlerin attenuate this process of EGFR/c-Src/PKC-Delta induced cell proliferation in Glioblastoma cells (Ref.1). PI3K is capable of activating Akt through phosphorylation of PIP2 to PIP3 (Phosphatidylinositol 3,4,5-trisphosphate), a mechanism inhibited by the phosphatase, PTEN (Phosphatase and Tensin Homolog). Cancer phenomics mainly involve mutations in PTEN which in turn lead to enhancement of Oncogenesis. Akt works indirectly on mTOR through the actions of the TSC1/TSC2 (Tuberous Sclerosis Complex). The physical association of the proteins TSC1 (Hamartin) and TSC2 (Tuberin) produces a functional complex that inhibits mTOR. Akt may also phosphorylate mTOR directly. mTOR phosphorylation and ERK activation in turn controls activation of p70S6K (p70 Ribosomal-S6 Kinase), a major regulator of cell growth (Ref.4). Akt also regulate the growth inhibitory effects of FkhR (Forkhead In Rhabdomyosarcoma) (controls Oncogenesis) and p27(KIP1)) (through down regulation of CDK2 (Cyclin-Dependent Kinase-2) and CcnE (Cyclin-E)). Apart from CDK2 and CcnE, gene expression by CcnD1 and CDK4/6 are critical for the regulated progression through the cell cycle as they maintain the Retinoblastoma protein, Rb levels in the hypophosphorylated state, which prevents the transcription factors, E2Fs from inducing the aberrant expression of genes required for G1-S phase transition (Ref.3).
Genetic pathways that are specifically disrupted in high-grade but not low-grade Astrocytoma are considered to be involved in tumor progression. To maintain tissue homeostasis, normal cells have several mechanisms to regulate cell-cycle progression and to prevent uncontrolled proliferation. One of these regulatory stages takes place at the G1/S-phase checkpoint. The tumor suppressor Rb is a key regulator of the G1/S checkpoint. A hallmark of high-grade Astrocytomas is high mitotic activity. It is, therefore, not surprising that the Rb-CDK-CKI (Cyclin-Dependent Kinase Inhibitor) regulatory circuit is frequently disrupted in these tumors (Ref.3). Loss of p16(INK4A), also known as p14(ARF) are encoded by the CDKN2A (Cyclin Dependent Kinase Inhibitor-2A) gene, is detected in forty-fifty seven percent of GBMs. p16(INK4A) acts on the Rb pathway by inhibiting CDK4/6 and CcnD, whereas, p14(ARF) acts on the p53 pathway by interacting with MDM2 (Mouse Double Minute-2), a downstream target of Akt; thereby blocking degradation of p53 and resulting in p53 stabilization and activation. The binding of p14(ARF) to MDM2 results in sequestration of MDM2 in the nucleolus. This prevents shuttling of MDM2 between nucleus and cytoplasm (to where it must export p53 for degradation), thereby leaving p53 free within the nucleus to regulate the transcription of p53 responsive genes. The p21(CIP1) is also up regulated by the tumor suppressor protein p53, inhibiting CDK4/6 and CcnD1. The tumor suppressor, p53 is activated under stress conditions like DNA damage, however DNA repair proteins like MGMT (O6-Methylguanine-DNA Methyltransferase) blocks this damage and renders the cell resistant to oncogenic agents. MGMT directly changes damaged bases rather than removing the bases, and most of the GBMs have hypermethylated MGMT. Similarly, CDK4 amplification occurs in twelve-fourteen percentages of GBMs, and loss of Rb is a persistent factor in fourteen-thirty three percentages of GBMs. Over-expression of CDK6 and CcnD1 occur in small number of GBMs, whereas, under-expression of E2F1 and over-expression of CcnE occur in some high-grade Gliomas. In total, mutations in INK4A/CDK4/Rb are detected in more than eighty percentages of GBMs and in fifty percentages of Anaplastic Astrocytomas. By contrast, such mutations are rare in Low-Grade Astrocytomas. The remaining twenty percentages of GBMs that lack detectable INK4A/CDK4/Rb mutations might harbor mutations in other components of this pathway (Ref.3 & 5).
Coincident with proliferation and differentiation during development are processes that shape and mould the individual structures in the nervous system. Proteins important for the patterning of the developing nervous system also act as tumor suppressors. The WNT (Wingless-Type MMTV Integration Site Family Member)/Fzd (Frizzled) signaling that features during tumor suppression in the cerebellum plays a vital role in controlling brain tumors. In the absence of WNT signaling, Ctnn-Beta (Catenin-Beta) is associated with a cytoplasmic complex containing CK1Alpha (Casein Kinase-1-Alpha), GSK3Beta (Glycogen Synthase Kinase-3-Beta), AXIN (Axis Inhibitor) and the APC (Adenomatous Polyposis Coli) protein. This promotes phosphorylation of Ctnn-Beta and its interaction with Beta-TRCP (Beta-Transducin Repeat-Containing Protein), leading to the ubiquitination of Ctnn-Beta and its degradation by the Proteasomes. Mutations in AXIN, Ctnn-Beta gene or germline mutations of APC predispose the glial cells towards development of GBMs. Under such conditions, Ctnn-Beta translocates to the nucleus and interacts with LEF1 (Lymphoid Enhancer-Binding Factor-1) and TCF3/4, transcription factors, to induce c-Myc and CcnD1 gene expressions leading to manifestation of brain tumors (Ref.5). Although primary and secondary GBMs have similar histopathological characteristics and clinical outcomes, the kinetics of tumor development in these two subtypes is dramatically different. Primary GBMs arise rapidly (less than three months) without clinical or histological evidence of pre-existing low-grade lesions, which makes it difficult to distinguish between genetic alterations that contribute to the initiation of Primary GBMs and those that are associated with the progression of Primary GBMs. However, mutational analysis indicates the fact that the same genetic pathways are dismantled in both Primary and Secondary GBMs-namely p53, growth factors-RTKs-Ras and Rb-mediated pathways (Ref.3). Understanding the key effectors that allow differential interpretation of a signaling pathway, such as regulation of cell size versus cell number and the induction of cell death versus tumorigenic growth, may provide insights for therapeutic targets. Further progress and understanding depends on the refinement of the model systems that are used for analysis. Useful avenues of investigation may involve the generation of more advanced mouse technologies, comparisons with other model organisms, stem-cell biology and the use of unbiased large-scale screening techniques, such as gene-expression arrays and proteomics approaches, combined with bioinformatics. The challenge for the future is to exploit such experimental systems and knowledge of pathological conditions to identify the crucial regulatory steps that determine when disruption of tumor-suppressor activity results in cancer (Ref.5 & 6).