1

Table of Contents
ABSTRACT ....................................................................................................................................2
Introduction ....................................................................................................................................2
location .........................................................................................................................................3
Types .........................................................................................................................................3
Causes ......................................................................................................................................4
symptoms ..............................................................................................................................4
Diagnosis ............................................................................................................................4
Genetic mutations in Globlastoma Multiforme ..........................................................................5
mutated genes and their function .................................................................................................5
Mechanism of IDHI mutation ..................................................................................................7
Basic mechanism of glioblastoma invasion and proliferation..................................................12

Scherer’s structure.......................................................................................................................12

Glioblastoma stem cells induced invasion..................................................................................13

WNT signaling pathway ..............................................................................................................14

PI3/AKT signaling pathway (Phosphotidyl inositol 3 kinases) ...............................................16

Hedgehog signaling pathway: ....................................................................................................18

Glutamate induced invasion........................................................................................................20

Role of microRNAs: ....................................................................................................................22

Current and future treatment: ..................................................................................................25

Limitations ...................................................................................................................................26
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Glioblastoma multiforme, genetic mutations and therapeutic approach

Abstract: Glioblastoma, a grade IV Glioma presents itself in the form of lesions in

microenvironment of the brain. Glioblastoma is an invasive and resistant malignant tumor of the
brain. The Pathogenesis of glioblastoma includes the over-expression of activator genes and the
down regulation or inactivation of suppressor genes like retinoblastoma, p 53, growth pathway
deregulation and PTEN gene. Current method of glioblastoma treatment includes radiotherapy,
chemotherapy (with TMZ as first line treatment) and surgical removal of the tumor from the
brain. These current treatments present some limitations in term of unavailability of localized
treatment, ineffectiveness of available treatment and resistance of the tumor cells against
chemotherapy. It's the ultimate necessity of this era to develop new therapeutic approach to treat
glioblastoma. Modern techniques that are under clinical trials include IDH receptor inhibitors,
EGHF inhibitors, monoclonal antibodies, anti-angiogenic compounds to arrest blood vessels
growth, electrical fields, proton beam therapy and dendritic cells to deliver the drug locally to the
target site.

Introduction: Glioblastoma Multiforme is the most hostile malignant brain tumor that is
specified by the angiogenic, invasive and therapy resistant glial cells (Wen & Kesari, 2008, p.
492-507). It arises from astrocytes, subtype of glial cells, so it is also known as astrocytic glial
cells. GBM is characterized by such specific gene alterations that allow cells to grow, proliferate
and infiltrate even in an oxygen deficient microenvironment (Furnari & Fenton, 2007, p. 2683-
2710). Pathological view of GBMs indicates the appearance of abnormal arrangement of leaky,
highly proliferative blood vessels and necrotic areas (Wen & Kesari, 2008, p. 492-507).

Its high invasive nature makes it difficult to control locally. It is treated by either by surgery,
radiotherapy and chemotherapy by utilizing temozolomide medicinal agent but this treatment
only increases the patient’s lifespan of 12-14 months after diagnosis (Stupp & Hegi, 2009, p.
459-466). Despite of the fact that GBM requires aggressive surgical intervention, tumor can
occur again even at the original place (Wen & Kesari, 2008, p. 492-507). Basically it is due to its
very high infiltration potential that makes it hard to eradicate tumor completely. Radiations are
also used to remove GBM but it has certain limitations as tumor has oxygen deficient areas that
reduce the effectiveness of IR radiation due to decreased release of DNA damaging free radicals.
The medicinal agents that are used to treat GBM can pass the blood-brain barrier and enters the
 3

glioma cells. Due to enhanced leaky vasculature, a hydrostatic pressure is generated inside the
tumor that reduces drug delivery to the site of action. To overcome this, absorbable
chemotherapy wafers (Gliadel®) are put into the tumorous cells (Perry & Chambers 2007, p.
189). Although the TMZ, IR and wafers combination therapy is also used to treat GBM but still
some cells can survive and produces a large number of undifferentiated therapy resistant cells.

In this article we will discuss Glioblastoma multiforme, involved genetic mutations and
treatment methodologies of this Grade IV malignant tumor.

Location: Glioblastoma Multiforme normally appears in cerebral hemispheres with a major

portion occurring in supratentorial region. Small number of the tumors also develops in spinal
cord, cerebellum and brain stem (Nakada, 2011, p. 3242-3278). It grows rapidly in to the
endothelial cells with glomeruloid structure. Glioblastoma Multiforme has its own blood supply
that enables them to develop and infiltrate surrounding brain tissues.
A distinctive trait of GBM is its diversity in appearance from one area to another. Some areas are
spongy due to tissue necrosis, some of them are hard and white while some areas shows
noticeable hemorrhage and cystic degeneration (Robbins & Angell, 1981, p. 694). Normally
GBM is outsized, distinct irregular shaped abrasion occurring in white matter (Nelson & Cha,
2003, p. 134-145).

Types: On the basis of pathological attribute, Glioblastoma multiforme is divided in to primary

and secondary GBM. Primary glioblastoma arises on its own with no previous lesions or
abrasions while secondary glioblastoma requires precursor astrocytoma for its growth and
progression (Ohgaki & Kleihues, 2007, p. 1445-1453).
WHO classifies brain tumor on the basis of its histological similarity to the evident cancerous
cells. Grading has been done by relating histological features to its aggressive behavior such as
mitotic count, necrosis and vascular endothelial hyperplasia. WHO classification comprises of
four grades on the basis of metastasis of cancerous cells; Grade I glioma have less infiltration
ability and can easily be removed by surgery while Grade II to IV gliomas are highly proliferated
and infiltered. Glioblastoma Multiforme is the most malignant, aggressive and undifferentiated
tumor with Grade IV (Louis, 2007, p. 97-109; Jovcevska, 2013, p. 935-941).
 4

Causes of Glioblastoma multiforme: Glioblastoma multiforme is mainly caused by mutation is

genes especially in those genes that are responsible for programmed cell death, proliferation,
cellular multiplication and angiogenesis. Genetic alterations occur by various factors such as;
introduction of ionizing radiations, abnormal inherited DNA fragments and excessive exposure
of toxic chemicals and carcinogens. In GBM, expression of cancer causing genes is caused by
various mechanisms like hypermethylation of DNA at their CpG island promoters and alterations
in the location of histone variants. Some environmental factors can also contribute towards the
uncontrolled cell division that ultimately leads to brain cancer (Nagarajan & Costello, 2009).

Symptoms: Glioblastoma matures rapidly that put pressure on the brain; as a result brain
produces initial symptoms. Depending on location some of them are; continuous headache,
weakness, aphasia, shortness of breath, vomiting, seizures, troubled mind, dizziness, difficulty in
swallowing, and decreased motor activity (IJzerman-Korevaar, 2018, p. 485-496).

Diagnosis: GBM doesn’t spread outside of the brain and occupy large portion of cerebral lobe.
Thus diagnostic tools are employed to diagnose tumors just inside the brain and can be identified
using computerized tomography technique. Scans represent tumor as irregular masses of tumor
cells, edema, and necrotic hemorrhage. The major disadvantage of CT scan is, small minor
tumors can be missed.

MRI (Magnetic resonance imaging) is more sensitive technique for scanning than CT scan but
can detect invasion outside the tumor cells as well.

Positron emission tomography is done for the differentiation of tumor from the scars formed
after operative removal of tumor but PET cannot be used for primary diagnosis of tumor.

Glioblastoma multiforme exists in various astrocytic variations; hence biopsies are of no value to
diagnose GBM.

The diagnostic features for GBM include swelling in cerebral area, hemorrhage, inflammation,
stroke, tumor of meninges, and accidental death of brain cells.
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Gene mutations in Glioblastoma:

Genetic mutation of oncogene and tumor suppressor gene promotes tumor production. After
activation, oncogene inhibits cell apoptosis and promotes cell proliferation. It has been observed
that there are three main pathways that lead to the formation of Glioblastoma Multiforme;
Retinoblastoma (RB) pathways and p53 inactivation, the PI3K pathway activation, and growth
factor (receptor tyrosine kinase—RTK) signaling pathway de-regulation. Mostly glioblastoma
occurs due to alteration in TP53 signaling pathway that has an effect on MDM2 (murine double
minute-2), MDM4, p53, and cyclin-dependent kinase (CDK) N2A genes. Around 78% of the
GBMs arise due to interruption in RB signaling with commonly mutated genes; CDK4, CDK6,
RB1, CCND2 and CDKN2 (cyclin-dependent kinase inhibitor 2). At the end, activation of PI3K
pathway with affecting genes PIK3R1, NF1, AND PIK3CA can also lead to GBMs formation
(Richterova & Kolarovszki, 2016, p. 83).

Some of the involved genes along with their functions are mentioned in a table.

Table 1: Commonly altered genes and their functions

Genes Function of responsible genes

V-erb-b2-erythroblastic leukemia viral Control signaling pathways of cell and

oncogene homolog 2 (ERBB2) promote its replication and growth

Epidermal growth factor receptor (EGFR) Control signaling pathways of cell and
promote its replication and growth

Neurofibromin 1 (NF1) Control signaling pathways of cell and

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promote its replication and growth

Phosphoinositide-3-kinase regulatory 1 Control signaling pathways of cell and

(PIK3R1) promote its replication and growth

Phosphatase and tensin homolog (PTEN) Control signaling pathways of cell and
promote its replication and growth

Phosphoinositide-3-kinase catalytic alpha

Control signaling pathways of cell and
(PIK3CA)
promote its replication and growth

Isocitrate dehydrogenase 1(IDH1) Produce NADPH

Tumor protein p53 (TPF3) Involved in regulating programmed cell death

(apoptosis)

Protein tyrosine phosphatase receptor type D Control signaling pathways of cell and
(PTPRD) promote its replication and growth

Retinoblastoma 1(RB1) cell cycle regulator

MDM2 (murine double minute-2) Promotes cell growth

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Though in glioblastoma, specific genes get mutated but some familiar cancer gene such as TP53
and PTEN alteration is also very common (Parsons & Jones, 2008, p. 1807-1812). Epidermal
growth factor receptor (EGFR) alteration is also observed in glioblastoma. Extracellular part of
mutant receptor EGFRvIII lacks 267 aminoacids as a result receptor become much more
activated and become independent of its EGF ligand for further signaling (Cancer Genome Atlas
Research Network, 2008), p. 1061). Alteration in some cancer genes such as BRAF and RAS
genes is not commonly observed in glioblastoma (Cancer Genome Atlas Research Network,
2008), p. 1061). Alteration in PIK3CA and PIK3R1 genes, which coded for the regulatory
subunit (P85α) and catalytic subunit (p110α) of PI3K, is also seen in glioblastoma

Mutated genes are different for developing primary and secondary glioblastoma. Phosphatase
and tensin homolog deprivation, enhanced expression of EGFR, and Loss of Heterozygosity of
chromosome 10 results in primary glioblastoma whereas; p53 loss, alteration in ATP-dependent
helicase (ATRX), and Loss of Heterozygosity of chromosome no 19 leads to secondary
glioblastoma.

Mutation in one of the most important gene that distinguishes primary and secondary
glioblastoma is Isocitrate dehydrogenase 1(IDHI) that is responsible for generation of ATP in
the body (energy metabolism). IDHI mutation is majorly observed is secondary glioblastoma
(Cancer Genome Atlas Research Network, 2008; Parsons & Jones, 2008, p. 1807-1812).

Mechanism involved in tumor formation caused by IDHI mutation:

Isocitrate dehydrogenase 1(IDH1) is an enzyme involved in energy metabolism. It is responsible

for the conversion of isocitrate into α-ketoglutarate by utilizing NADP+ as a precursor of
NADPH. This NADP+ accepts an electron and converts in to NADPH which reduces cell
damage by oxidation and involved in lipid production (Geisbrecht & Liang, 1999, p. 25814-
25820).

Alpha-ketoglutarate is a crucial intermediary metabolite of kreb’s cycle. In the presence of low

oxygen, IDHI mediate inverse reaction by converting α-KG to isocitrate, which is then converted
into acetyl-CoA, involved in many organic reactions.
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Acetyl-CoA

Isocitrate
NADP+

Prevent oxidative cell IDHI

damage NADPH

Lipid production Alpha- Ketoglutarate

NADP+ Altered
IDHI
NADPH

2-HydroxyGlutarate

Alpha-ketoglutarate dependent deoxygenases

TET2 Histone Collagen propyl 4 HLF propyl 4

hydroxylase demythylase hydroxylase hydroxylase

Increased DNA Increased histone Decreased collagen Increased Hypoxia

hypermethylation tail demethylation hydroxylation induced factor

Tumor production

Figure 1: pathways involved in tumorigenesis caused by IDHI gene alteration. Mutation in IDHI enzyme causes the
production of 2-hydroxyglutarate that is an onco-metabolite. This onco-metabolite inhibits Alpha-ketoglutarate
dependent deoxygenases that increases histone demethylation and DNA hypermethylation. It further causes over-
expression of Hypoxia induced factor. Collectively it leads to on-cogenesis.

Alteration in IDHI gene is responsible for tumor production (Dang & White, 2009, p.739-744).
Mutated gene causes conversion of α-Keto Glutarate in to its enantiomer, 2-hydroxyglutarate
which is an oncometabolite (Pietrak & Zhao, 2011, p. 4804-4812). As a result of mutation,
affinity of active site of IDHI gene for its substrate (isocitrate) is reduced whereas it concurrently
 9

increases for NADPH and α-KG. Reduction in affinity arises due to changes in the binding site
that is responsible for hydrogen bonding between alpha and beta groups of isocitrate (Pietrak &
Zhao, 2011, p. 4804-4812). Hence the alpha-ketoglutarate conversion in to 2-HG is increased
resulting into tumergenisis.

Research suggests that almost 5.6% of primary glioblastoma occurs due to IDHI mutation
whereas it accounts for 76% of secondary glioblastomas.

One of the most important genes that are commonly mutated in tumor cells is p53 gene, tumor
suppressor gene. Its location is 17p13.1 (Iacob & Dinca, 2009, p. 386-393). Protein p53 limits
cell multiplication, restores DNA damage, arrest cell in G1/S phase of cell cycle, and promotes
programmed cell death (apoptosis). It has pluripotent inhibition effect and restricts stem cells
regrowth (Zhao, 2010, p. 170-175). In glioma cells TP53 induced-programmed cell death is
absent. Impaired TP53 functioning interrupt p14ARF pathway as a result retards apoptosis and
promotes unstableness in genome (Krakstad & Chekenya, 2010, p. 135-149). Normally
mutations occur in binding domain of p53 especially in 175, 248, and 273 codons

Alteration in MDM2 (murine double minute-2) oncogene is also seen in primary

Glioblastomas. MDM2 gene location is 12q14.3-q15. E3 ubiquitin ligase is its protein product
that is majorly involved in transcription. This enzyme can occur in five forms and only two of
them can interact with p53. This protein is involved in inhibition of cell cycle arrest and
apoptosis by having a negative effect on p53 gene and promotes cellular growth (Thomasova &
Mulay, 1012, p. 1097-1101). Up regulation of MDM2 oncogene eliminates effect of p53 on cell
cycle and plays a significant role in low grade glioma formation (Wang & Liu, 2011, p. 2530-
2533).

Retinoblastoma gene has RB protein that act as a cell cycle regulator and controls cell cycle
progression by limiting entry of cells into S-phase. Its location on chromosome is 13q14.2. RB
protein binds with non-phosphorylated E2F (transcription factor) and produces RB-E2F
complex. Following phospshorylation of complex releases E2F and promotes CDK2 entry into
S-phase of cell cycle. Further phosphorylation also inhibits RB-E2F complex formation thus
promoting CDK2 entry into synthesis phase of cell-cycle (Goldhoff & Clarke, 2012, p. 83-89).
Mutation of RB gene and amplification of CDK4/CDK6 have same impact but these alterations
 10

cannot occur simultaneously as a result tumorgenisis proceeds by one of these pathways (Chow
& Endersby, 2011, p. 305-316). Mutated RB gene causes enhanced infiltration of glioma cells
and reduces cell survival.

Amplification and mutation of tumor suppressor gene PIK3R1 ((phosphatidylinositol-3-kinase

regulatory subunit 1) is also seen in GBMS. Its location is 5q13.1. PIK3R1 binds with catalytic
subunit and restricts its activity (Sun MHP & Hofmann, 2010, p. 547). Alteration of PIK3R1
gene results in no inhibition of catalytic subunit hence produces large number of cancerous cells
that grows and multiplies (Weber & Parat, 2012, p. 833-849).

PTEN gene (Phosphatase and tensin homolog) is a tumor suppressor gene located on 10q23.3.
PTEN protein, a lipid phospatase is the product of PTEN gene that inhibits PI3K signaling
pathway by causing inverse conversion of PIP3 to PIP2 (Lee & Kim, 2011, p. 666-667). Singly
copy (monosomy) of chromosome number 10 in PTEN gene results in glioblastoma formation
(Hede & Nazarenko, 2011).

GBM invasion: Invasion of glioma cells occur through both mechanisms, biophysical and
biochemical processes that are involved in the maintenance of the shape, movement and
arrangement of matrix between the cells.

Invasion of glioblastoma at molecular level

Adhesion Molecules

Mechanism of invasion of brain tumor cells involves detachment of tumors from the neighboring
molecules, and then these tumor cells migrate to the distant areas and get themselves attached
there. Here the molecules NCAM and cadherins play important role in this metastasis. Another
immunoglobulin molecule intercellular adhesion molecule-1 (ICAM1) also metastasize the
tumor cells. Intracellular adhesion molecules are also involved in inflammation, signal
transduction pathways, adhesion of antigen presenting cells, and activity of leucocytes.

Integrins act as a barrier between tumor cells and normal cells. They perform their function
through ITGs cluster (through intracellular signaling pathway) and by activation of kinase
 11

enzymes. These two pathways allow the integrins to enhance cell division, their survival,
movement, and invasion to neighboring cells.

Extracellular matrix composition: Composition of extracellular matrix in the normal cells and
in tumor cells is entirely different. Hyaluronic acid is found in large amount in tumor cells as
compared to the normal non-tumor cells, that upregulates NFκB production and thus promotes
the invasion of the cells. In the same way, extracellular matrix rich in glycosylated chondroitin
sulfate proteoglycans is linked with non-migrant non-invasive lesions.

Another strategy of glioma cells is to degrade extracellular matrix in order to remove their
invasion hindrance. They perform these functions by employing proteases, metalloproteinase,
and cysteine proteases. MMP-2 & 9 are associated with high capacity to move tumor cells to the
neighboring non-tumor areas.

Biochemical modifications:

Epithelial to Mesenchymal Transition (EMT): Cytoskeleton is involved in the maintenance of

the cellular shape and maintains its integrity. In cancerous cells, polarized epithelial cells are
rearranged to non-polarized mesenchymal type. Mesenchymal cells are loosely attached and are
not compactly arranged, thus allow the flow of cells from tumor to non-tumor area.

EMT transition is mainly regulated by Snail and Slug, Twist1/2 and the zinc-finger E-box-
binding homeobox (ZEB)1/2. These modulators modulate the transcription of vimentin,
fibronectin, and N-cadherin genes and suppress claudins, cytokeratins, and E-cadherins. All
these factors result in increased proliferation of the cells, and cells invasion.

Cytoskeletal Remodeling and Cell Motility: Remodeling of Cytoskeleton results in formation

of invadopodia and lamellipodia. These are involved in cell movement. Tumor cells can invade
easily through mesenchymal cell type. Tumor cells change their shape from non-polarized to
polarized edges that lead to outward extension of the cells. Tumor cells contract them by actin-
myosin contraction movement, by the recycling of integrin and thus invade to the neighboring
cells.
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Cross-Talk with Host Cells and Immune Modulation: Tumor cells integrate themselves with
the stromal cells that secrete growth factors and milieu for the proliferation and division of
neoplastic cells.

Thus microenvironment of tumor cells is considered to play regulatory role during tumor
therapy. Local immune system changes and vessels networking play a key role in developing
tumor in patients with immunosuppressant or immune-modulation therapy.

Basic mechanism of glioblastoma multiforme cell proliferation and invasion:

Glioblastoma multiforme is a grade IV malignant tumor with various complex molecular
pathways of invasion and proliferation. Molecular pathways are interlinked with extracellular
matrix, cancer cells and white matter. These interactions are controlled by 140 genes (Park &
Agnihotri, 2014, p. 9382). Molecular pathways for glioma cell invasion includes; g, Sonic
hedgehog, PI3K/Akt, micro RNAs, and Wnt pathways. These pathways have very high tendency
to invade and proliferate as a result it is difficult to control glioblastoma locally (Paw &
Carpenter, 2015, p. 1-7). On the basis of difference in their genetic profile, GBMs can be further
subdivided into pro-neural, neural, mesenchymal and classic types. Knowledge of these
pathways helps us to understand the invasive power of GBMs however these pathways are hard
to understand completely.

Scherer’s structures:

Glioma cells proliferate along certain structures called as secondary structures that include
subarchnoid space, surrounding blood vessels, white matter and parenchymal cells of brain.
These secondary structures are also called as scherer’s structure on the name of scientist who
discovered them (Medyouf & Ghysdael, 2008, p. 297–303). Scherer also stated that these glioma
cells don’t have any proper shape or appearance and their shape depend on their following
pathway of proliferation. This statement complies with the modern invasion theory of glioma
cells (Cuddapah & Robel, 2014, p. 455).

Movement of invasive glioma cells is same as normal cell movement during embryonic
development (Cuddapah & Robel, 2014, p. 455). Firstly extracellular membrane gets polarized
and protruded (Paw & Carpenter, 2015, p. 1-7). Glioma cells chemically interact with the
extracellular membrane, stick to it and start moving forward. These invasive cells also effect
 13

stromal cells thus lead to change the structure of extracellular membrane (Bonnans & Chou,
2014, p. 786). By changing their shape, glioma cells can move through membrane holes and
spaces. Migration of glioma cells is controlled by various chemokines and growth hormones.
Extracellular matrix is the main obstruction for the glioma cells to multiply and control cell
machinery. Metalloproteinases (MMP) degrade extracellular matrix and these enzymes have
enhanced expression for glioma cells in comparison to their normal cells (Paw & Carpenter,
2015, p. 1-7). Some other enzymes such as cathepsin D, plasmin, cathepsin B and heparanase
also facilitate degradation of extracellular matrix by glioma cells (Fonovic & Turk, 2014, p.
2560-2570). Glioma cells also possess amoeboid movement by ROCK (Rho/Rhzo kinase)
activation and mesenchymal movement by Rac1 activation (Frame & Brunton, 2002, p. 36-43).

Glioblastoma stem cells and their invasive potential: GBM cells consist of a large number of
GBM stem cells that possesses all the effects of somatic stem cells (Beier & Hau, 2007, p. 4010-
4015). These GBM stem cells also known as GSCs (Glioblastoma stem cells) or GICs (Glioma
initiating cells) have very high potential for the tumor progression (Li Z & Bao S, 2009, p. 501-
513). Their function is explained on the basis of their ability to replicate and invade surrounding
tissues, and potential for tumorigenisis (Singh& Hawkins, 2004, p. 396-401). GSCs have very
high tendency for tumor propagation as compared to other non-stem cancerous cells. These stem
cells promote new blood vessels production by increasing expression of vascular endothelial
growth factor (VEGF) thus encouraging tumor genesis (Bao, Wu & Sathornsumetee, 2006,
p.7843-7848).

GSCs also increase expression of pro-angiogenic growth factor’s ligand, SDF-1 (stromal-derived
factor-1 or CXCL12) (Folkins & Shaked, 2009, p. 7243-7251). The main reason behind their
increased tumor infiltration potential is the enhanced expression of few invasive proteins and
genes in GBMs comparable to non-stem tumor cells. These includes; ADAMTS1 (a disintegrin
and a metalloprotease with thrombospondin motifs 1), ADAMTS9, ADAMTS2, MMP16
(Matrix metalloproteinase 16), L1CAM (cell adhesion molecule), MMP14, and SEMA3C
(Semaphorin3C). MMP16 and LICAM surface molecule plays a major role in enhancing
proliferation and infiltration of glioma cells (Xia & Qi, 2009, p. 158-165). ADAMTS1 increases
cell migration by causing breakdown of SEMA3C (Semaphorin3C) gene (Raveh & Gavert,
 14

2009, p. 137-145). These proteins also have a significant role in determining the mechanism of
GICs invasion.

Wnt signaling pathway in Glioblastoma multiforme:

Wingless/Int1 signaling pathway plays an integral role in the maturation and self-renewal of cells
in central nervous system CNS comprises of neural stem cells (NSCs) that are present in the
hippocampus and post natal sub ventricular zone (Kalani & Cheshier, 2008, p. 16970–16975).
This signaling pathway is responsible for the growth and maturation of neural stem cells and any
abnormality in this pathway leads to the production of cancerous cells (Holland, 2011, p. 7280–
7290).WNT pathway comprises of WNT signaling proteins that regulate cell maturation, their
movement and interactions with other cells (Nusse & Varmus, 1982; p. 99–109). Any alteration
in this pathway causes growth defects however it’s over activation leads to tumorigenesis. There
are three types of WNT pathways; canonical (β-catenin- dependent) pathways, non- canonical
(β-catenin- independent) and WNT/Ca++ pathway (Clevers, 2006, p. 469-480).

Canonical pathway Non-Canonical pathway

WNT target Ligand

Frizzled receptor Extracellula
(FZD) r

DVI Intracellular
DVI
Axin
Daam1
GSK3β
Rac Inward
APC movement of
β- catenin Rho calcium
β- catenin
JNK
β- catenin
FoxM1 ROCK PK
C
NFAT
Re-organization of
cytoskeleton

Translocation of Nucleus AP NFAT

β- catenin I
TCF/LEF
Nucleus
s
Cyclin D1 and c-myc expression
 15

Figure 2: Canonical, non-canonical and WNT/Ca2+ signaling pathways

In the canonical pathway, WNT proteins attach themselves to the low-density lipoprotein
receptor-related protein/alpha 2-macroglobulin receptor (LRP) and frizzled (FZD) cell surface
receptor. After binding with the surface receptors these proteins, by activating numerous
integrants in cytoplasm transfer signals to β-catenin. β-catenin than moves in to the nucleus and
causes transcription of WNT intended genes by forming a complex with T-cell factor (TCF). β-
catenin target genes comprises of MMPs, cyclin D1, and c-Myc (Kaur & Chettiar,2013, p. 44-
57). MMPs are involved in enhancing the infiltration capacity of Glioblastoma cells whereas
cyclin D1 and c-Myc are associated with cellular multiplication. Wnt1, Wnt 3a and Wnt 7a

catenin that leads to the production of glioma cells (Kamino & Kishida, 2011, p. 540-548).

Non-canonical / β-catenin independent pathway, exert influence on planar cell polarity and is
involved in neuronal cellular migration (Semenov & Habas, 2007, p. 1378). WNT factors such as
WNT4, WNT5A, and WNT11, initiate the PCP pathway by binding with the FZD cell surface
receptor and causes activation of Dv1 (Dishevelled (Dvl) and Dvl-associated activator of
morphogenesis 1 (Daam1). This complex causes activation of Rho GTPases and Rac to initiate
cell migration and arrangement of cytoskeleton. Over expression of WNT ligands especially
WNT5A enhances MMP2 activity that enhances production of glioma cells.

In the WNT/ Ca2+ pathway, Wnt ligands attach itself to the FZD receptor and promote G-
protein-coupled Ca2+ release. This intracellular calcium actuate the calmodulin-dependent
protein kinase 2 and protein kinase C. high level of Calcium activate the calcineurin (Ca2+-
dependent serine/threonine phosphatase) causing activation of T cells in the nucleus (Medyouf
& Ghysdael, 2003, p. 2205–2232).Tumor malignancy is the major contributor of death caused by
cancer (Smit & Peeper, 2011, p. 3735–3744). Tumor cells are delocalized from epithelium to
other organ by a specialized process called as Epithelial–mesenchymal transition (EMT). Wnt
signaling pathway is linked with EMT and tumor infiltration.

WNT signaling pathways have three main roles; to increase cellular invasion and proliferation, to
regulate GSCs and to generate drug resistance. Wnt gene expression regulator (such as
Evi/Gpr177, PLAGL2, FoxM1, ASCL1 and FoxM1) initiates activation of Wnt regulated
pathway and promote cellular multiplication. Over activation of Wnt signaling pathway causes
 16

enhanced expression of genes of EMT (N-cadherin, SLUG, ZEB1, TWIST, MMP, and SNAIL)
as a result increases proliferation of GBM cells (Han & Kim, 2011, p. 489-496). Over activation
of β-catenin, also enhances the ZEB1 expression in GBM cells thus promotes migration of
glioma cells (Kahlert & Maciaczyk, 2012, p. 42-53).

Infiltration potential Glioblastoma stem cells

Low High
PLAGL2 ASCL1
FoxM1

WNT pathway activation

Expression of WNT pathway

Increased expression of ZEB1, SNAIL,

TWIST, SLUG, MMP, and N-cadherin Maintenance of Stem cells

Figure 3: Over expression of WNT signaling increases the glioma cells infiltration and proliferation potential.

PI3/AKT signaling pathway (Phosphotidyl inositol 3 kinases)

PI3Ks are group of kinases that cause the phosphorylation phosphatidyl-inositols and
phosphoinositides (Aziz, 2009, p. 3029-3036). Class I-a has two catalytic units, p85 and p110
(Franke, 2008, p. 6473-6488; Marone, 2008, p. 159-185). PI3/AKT signaling pathway accounts
for 63% of brain tumors of grade III and IV (Kita D, 2007, p. 295-302). This pathway comprises
of four genes, epidermal growth factor receptor, PIK3R, PIK3CA and PTEN.

PI3K pathway is activated by a number of substrates; each substrate binding has its specific
consequence. When growth factors bind to the binding site of EGF receptor, it phosphorylates
the tyrosine kinase part of the receptor (Ogiso, 2002, p. 775-787). This phosphorylation leads to
the activation of the receptor and binding of tyrosine to p85. This binding has two impacts; it
results in conformational change in the p85 and release of p 110 (Li X, 2016, p. 33440-33450).
 17

P110 has the ability to phosphorylate PIP2 to PIP3 by adding inorganic phosphate group. PIP3 in
turn phosphorylates AKT by PDK1, serine–threonine kinase. AKT positively regulates the
multiplication of the cells and their persistence. AKT has multiple functions; it suppresses
apoptosis and promotes proliferation of the cells by activating anti apoptotic factors (NFkB &
Bcl-2) and inhibiting proapoptotic factors (casepase 3/8, Bad/Bax). AKT is also involved in drug
resistance through the activation of MDR1 pathway. PI3K/AKT signaling pathway increases
invasion of tumor cells in normal parenchymal cells of the brain by activating MMP2 and MMP3
activity. AKT causes the inhibitory phosphorylation of GSK3β; thereby increasing the level of
cyclinD1, which in turn promotes cell cycle (Woodgett, 2005, p. 150-157).

AKT is further involved in the phosphorylation of apoptotic signal kinase (ASK1), leukemia of
B-cells, MDM2 protein, X-linked apoptosis inhibitor, cAMP response element binding protein,
mammalian target of rapamycin (mTOR) and IkB kinase. Phosphorylation of this protein causes
the movement of NFkB from the cytoplasm into the nucleus which facilitates cell survival and its
proliferation (Madhunapantula, 2009, p. 400-419) (Manning, 2007, p. 1261-1274)

Diagrammatic representation:

Receptor
Tyrosine
kinase

P85
PIP2 PIP
P11 3 PIP2
0
PI3
K

AKT
MDM2

GSK3β
Pro-apoptotic Anti-apoptotic
factors factors
P53

NFkB
Cyclin D1

Cell proliferation
Cell division
Cell survival
Drug MDR1
Resistance

Cell cycle
 18

Figure 4: PIK3/AKT signaling pathway: PIP3 induced AKT phosphorylation activates group of cascades with the
ultimate consequence of cell proliferation, cell division and cell survival. This pathway is also involved in drug
resistance through GSK3β.

Check points: PTEN, protein phosphatase 2A (PP2A) and leucine rich phosphatase (PHLLP)
are PI3K antagonists. PTEN causes dephosphorylation of PIP3 to PIP2 and PHLLP removes
phosphate group from T308 and S473. PTEN also suppresses the invasion of the tumor cells by
suppressing MMP2 (Koul D, 2012, p. 184-195).

PI3K pathway in melanoma: PI3K mutation, over expression of this pathway and inhibition of
feedback mechanism (PTEN) protein accounts for the over expression of PI3K pathway resulting
in increased proliferation and cell division.

Hedgehog signaling pathway:

Hedgehog, a molecule present inside the cell has three homologous forms, Indian Hedgehog
(Ihh), sonic Hedgehog (Shh) and desert Hedgehog with sonic Hedgehog a strong oncogene. Shh
is involved in central nervous system related mechanisms. DHh pathway is involved in the
regulation of spermatogenesis in the males. Hedgehog pathway is the highly complicated and
sophisticated pathway involved in cell proliferation, differentiation of the cells and tumor genesis
(Dlugosz AA, 2009, p. 1202-1205) (Yoo YA, 2011, p. 7061-7070).Hh pathway is involved in
embryonic development in embryos. Aberrant perseverance of this pathway results in a number
of human cancers including glioblastoma. Hh signaling pathway is activated by two mechanisms,
canonical Hh signaling (Gli family is involved in this signaling), non-canonical (through Gli
independent mechanisms). In Hedgehog signaling pathway, two molecules are of prime
importance, Patched (Ptch) and G protein coupled receptor resembling protein (GPCR Class F)
smoothened (Smo). Smo is a seven trans-membrane protein. Gli (Glioma associated oncogene),
transcription factor is available in three forms, Gli1, Gli2 and Gli3. Gli1and Gli2 are the
activators in Hedgehog pathway while their third homologous isoform GLi3 is a suppressor.

Normally in the absence of the Hedgehog molecule, Patched (Ptch) inhibits the trans membrane
protein Smo, which in turn inhibits the entry of Gli1/Gli2 fused complex into the nucleus.
Moreover, it activates Gli3.Canonical pathway is initiated in the presence of Hh molecule, Hh
ligand binds to PTCH receptor, Patched no longer inhibits Smo. Smo smarts intracellular
 19

signaling,It modulates kinase fused protein(Fu), suppressor of Fused (SuFu) and kinesin related
protein costal 2 (Cos2). Smo activation results in the increased expression of the transcription
factors Gli1 and Gli2, and their translocation from the cytoplasm into the nucleus. this further
degrades Gli3(F. Shuang, 2015)

Activation of Hh signaling pathway results in the tumerogenesis and blockade of this cascade
blocks cancer genesis, proliferation and its metastasis. (Takezaki T, 2011, p. 1306-1312)

Non-canonical pathways: Non-canonical pathways of hedgehog signaling are of two types,

Type 1 which depends on PTCH and type II which depends on Smo.

SMO
PTCH

Gli 1
Gli 3
Gli 2
Proteolysis

Hh Target genes
OFF

SMO
PTCH

Gli 3
Gli 1
Gli 2

Proteolysis

Hh Target genes ON
 20

Figure 5: The off and on states of the Hedgehog (Hh) signaling pathway. In off state, Patched inhibits the activity of
Smo which causes the proteolysis of Gli 1 AND Gli2, and has positive effect on Gli3 which is a suppressor molecule
thus Turning off the Hh target gene. In on state, Patched effect on Smo is blocked by Hh ligand molecule, resulting
in the proteolysis of Gli3 and translocation of Gli1 and Gli2 into the nucleus turning on the Hh gene, increased
proliferation and cell division

Type 1 non-canonical pathway (Autocrine/juxtacrine mode) is involved in programmed cell

death and cell division and depends on PTCH1 and Hh ligands for its activity. It performs its
apoptotic function by binding with caspase9 which is a pro-apoptotic body. Hh ligand presence
facilitate the linkage between PTCH1 and G-protein receptor kinase-2 (GRK2), this process
translocate the cyclin B1 into the nucleus and induces cell division and its survival (Robbins,
2012; Dahmane, 1999, p. 3089–3100)

Type 2 non-canonical hedgehog pathway activates RhoA and Rac1, which in turn causes the
rearrangement of cyto-skeleton. Activation of Smo- PLC-γ activates IP3 which increases the
concentration of calcium ions inside the cell facilitating the processes like cell division,
programmed cell death and movement (Robbins, 2012; Teperino, 2014, p. 81-92).

Β-catenin induced degeneration pathway

if the Wnt signals are not present then β –catenin attaches itself to the glycogen synthase kinase-
3β (GSK-β) that phosphorylates the β –catenin. This phosphorylated product is then destroyed by
proteasome (Furnari & Fenton, 2007, p. 2683-2710). This pathway is regulated by many factors;
EGFR disrupts the interaction between α-catenin and β –catenin that in turns stimulate the β –
catenin (Silber & Jacobsen, 2012, p. 33844). FRAT-1 suppresses the activity of GSK- β that
further reduces the breakdown of β-catenin (Brennan & Verhaak, 2014, p. 753). As a result high
stimulation of β-catenin occurs that promotes invasion and proliferation of GBM. Whereas
inhibition of transcription factor Lef-1 reduces the GBM induced cellular proliferation (Li &
Guessous, 2007, p. 7569-7576).

Glutamate stimulated GBMs proliferation and invasion:

Glutamate is an important excitatory neurotransmitter of the brain. It is discharged by the nerve

cells and is involved in sending impulses either within the cell (autocrine) or from one brain cell
 21

to another (paracrine) (Stepulak & Rola, 2004, p. 933-944). Glutamate causes excitation in the
brain by binding with its two ionotropic; α-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid (AMPA)/kainate and N-methyl-d-aspartate (NMDA) receptor, and one metabotropic
receptor (mGluR) receptor (Hollmann, M., & Heinemann, 1994, p. 31-108). Both AMPA and
NMDA receptors are involved in Ca2+ and Na+ influx by utilizing separate mechanisms
(Prickett & Samuels, 2012, p. 4240-4246). Increased Ca2+ level within the cell is linked with the
activation of various multiplication pathways for example PKA (Danbolt, 2001, p. 1-105), Akt
(Ishiuchi & Yoshida, 2007, p. 7987-8001) and ERK/MAP kinase pathway (Wiegert & Bading,
2011, p. 296-305). GBMs have the potential to increase the glutamate release. Glutamate then
binds with AMPA receptors and causes the high intracellular release of Ca2+ thus allowing
glioma cells to invade and proliferate through Akt pathway. This high Ca2+ concentration is also
linked with the activation of Calcium-dependent ATP-gated receptor P2X7 that causes external
release of Ca2+ in the extracellular matrix (Strong & Indart, 2018, p. 53-62). GBMs also causes
up-regulation of cystine/glutamate antiporter xc (-), that exchanged glutamate of the cell with
extracellular cystine (Massie & Boillee, 2015, p. 1062-1079). Over expression of AMPA
receptors, enhanced ATP concentration, and upregulation of cystine/glutamate transporter favors
Glioma cells to grow, multiply and invade surrounding cells.

Over expression of metabotropic glutamate receptor (mGluR) is also associated with glioma cells
induced invasion. This receptor is located in pre- and post synaptic areas of brain. Metabotropic
glutamate receptor (mGluR) is comprises of 8 sub-types that are further divided into three
categories on the basis of their G-protein binding ability and homogeneity (Niswender, 2010, p.
295-322). Following activation, mGluRs transfer signals by stimulating second messengers like
adenylate cyclase pathway inhibition and phospholipase C/inositol triphosphate/diacylglycerol
pathway activation (Nakanishi, 1994, p. 1031-1037). The mGluRs also have a potential to
regulate glutamate postsynaptic effect, stimulate performance of AMPA and NMDA receptors
and promote cellular growth, invasion, and proliferation (Pin & Duvoisin, 1995, p. 1-26).
Particularly group 1 mGluRs includes mGluR1 and mGluR5 that binds with Gαq and stimulate
protein kinase C (PKC) and Phospholipase C beta (PLCβ) further causing downstream
phosphorylation. Group II comprises of mGluR2 and mGluR3, and group III, consists of
mGluR4 and mGluR6–8 are linked with Gαi/o and inhibit adenylyl cyclase thus promotes
 22

activation of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase

(MAPK) signaling pathways (Iacovelli & Bruno, 2002, p. 216-223).

Role of microRNAs: MicroRNAs are a polymeric chain of 20 to 25 nucleotides that are

responsible for the suppression of the stem cells regulatory genes (Paw I, 2015, p. 1-7). It
terminates the translation of mRNA into protein and increases the breakdown of mRNA. After
transcription from DNA, primary miRNAs (pri-miRNAs) have a short stem-loop structure
(SLSs), and is converted in to premature miRNAs (pre-miRNAs), further processing converts it
into mature miRNAs. The main binding site of miRNAs is on the 3′ untranslated region (3′-
UTR) of mRNA. MiRNAs causes the dysregulation of gene expression and thus act as an
oncogene and tumor suppressor gene (Lu & Getz, 2005, p. 834-838).

Abnormal expression of miRNAs accounts for the development of glioma cells (Ciafre &
Galardi, 2005, p. 1351-1358). Microarray based mi-RNA analysis suggests some of the miRNAs
that are up regulated in GBMs are; miR-106b, miR-183, miR-21, miR-10b and miR-92b
(Sasayama & Nishihara, 2009, p. 1407-1413) and some of the micro-RNAs that are
downregulated are miR-181a/181b-/181c, miR-379, miR-368-3p , miR-302c, miR-324, miR-
134 , and miR-128 (Ciafre & Galardi, 2005, p. 1351-1358). Some of the micro-RNAs along with
their infiltration potential is discussed below;

Over expression of miR-183 causes activation of hypoxia inducible factor HIF-1α that
promotes angiogenisis, proliferation and infiltration of glioma cells (Tanaka & Sasayama, 2013,
p. 273-283).

MicroRNA-21 over expression causes the inhibition of apoptosis- controlling genes thus
promotes oncogenesis by having an anti-apoptotic potential (Chan & Krichevsky, 2005, p. 6029-
6033).

Down regulation of miR-218 in GBMs; inversely correlates the tumor potential to invade (Peng
& Li, p. 3831-3837). It further suppresses lef1 (Lymphoid Enhancer Binding Factor 1) regulation
that is involved in stimulation of beta-catenin pathway. MiR-218 further causes down-regulation
of NF-kB that promotes cell infiltration by increasing MMP-9. It also acts on hedgehog pathway
transcription factor- GLI1 that causes glioma cells to invade and proliferate (Tu & Gao, 2013, p.
6046-6055).
 23

Tumor suppressor gene MiR-101 is also down-regulated in GBMs. It suppresses the activation
of protein chitinase-3 by acting on KLF-6 (Kruppel-like factor 6) thus disable the PI3 signaling
and MEK1/2 pathway (Yao & Ma, 2015, p. 40-51). KLF-6 (Kruppel-like factor 6) causes
cellular invasion by increased expression. It’s down-regulation reduces the cellular infiltration
and proliferation.

MiR-152 inhibits invasion of the tumor cells by acting on Kruppel-like factor (KLF4) which
causes the inactivation of dPI3K signaling pathway. (Mehran, 2014, p. 2731-2741).

MiR-491 causes the inhibition of protein kinase B (p-AKT1), PCNA, MMP-2 and Cyclin D1
activation. This results in arrest of GBMs stem cells in G0/G1 phase of cell cycle thus decreases
the ability of cellular invasion and infiltration (Pan & Zhan, 2012, p. 871-881).

MiR-125b is over expressed in GBMs. It induces resistance against temozolamide and is a

therapeutic approach for current anticancer treatment (Shi L, 2010, p. 120-126).

MiR-152 is also down-regulated in GBMs and reduces the cell induced apoptosis and invasion.
It targets Kruppel-like factor (KLF4) that causes the down-regulation of galectin-3 (LGALS3)
and thus deregulate the PI3K signaling and MEK ½ pathway (Mehran & Nilsson, 2014, p. 2731-
2741).

Up-regulation of miR-181c causes inhibition of GSCs induced cellular proliferation and

movement. It causes the over expression of E-cadherin but at the same time it down regulates the
vimentin and N-cadherin. Following over expression, this micro-RNA causes inhibition of TGF-
β through TGFBRAP1, TGFBR1 and TGFBR2 down-regulation. Hence miR-181c is majorly
involved in glioma cells proliferation and invasion (He & Liu, 2016, p. 1041-1048).

Table 2: microRNA and their targets

Targets of microRNA in hedgehog signaling pathway

miR-302-367 CXCR4 Shh-Gli-Nanog
miR-125b Smo
miR-326 Smo, Gli2
miR -24-5p Smo, Gli1
 24

miR-214 Sufu

Diagnosis of Glioblastomas using MRI technique or CT:

Glioblastoma presents itself in the form of lesions with abrupt and irregular boundary when MRI
or CT scan technique is used. Brain biopsy is also performed to assess the degree of glioblastoma
penetration. In MRI and CT, GBM appears in the form of ring lesions, these lesions are also
shown by other tumor tissues thus providing non-specific diagnosis.

Biomarkers for the diagnosis of glioblastoma:

Serum biomarkers make it easy to diagnose the glioblastoma with more efficiency and with non
invasive method.

ALA the florescent dye 5-aminolevulinic acid stains the lesions in glioblastoma during tumor
resectioning.

Glial fibrillary acidic protein or GFAP is an intermediate protein produced by mature astrocytes
(Eng, 2000, p. 1439-1451). GFAP protein was first in 1971. It is member of cytoskeleton family
present abundantly in neural stem cells and can be used as diagnostic marker of GBM as its level
increases in case of brain lesions, traumatic brain injury, blood brain barrier dysfunction or
hemorrhage (Brommeland, 2007, p. 380-384; Gallego, 2014, p. 3972-3980). GFAP level is high
in almost 100% cases of GBM and is expressed abundantly in traumatic parts of the brain. GFAP
concentration is extremely high in GBM as matched with other neoplasm.

The genes undergoing aberrant changes in GBM are p65, DNM3 and CD117 which are up-
regulated more than 10 times than their normal regulation. PTEN, p53 and ST14, the genes
responsible for the maintenance or suppressor of cell proliferation are down-regulated in GBM.
GBM presents over-expression of EGFR (activator gene) and deletion of CDKN2A and mutation
of PTEN suppressor molecules (Verhaak, 2010, p. 98-110). Moreover, some pathways like
signaling of tyro- sine kinase pathway, calcium signaling, mTOR singnaling and Wnt/b-catenin,
HER-2, PDGF receptor A, and MET are de-regulated in GBM (Rao, 2004, p. 595-604).
 25

Other biomarkers which distinguish GBM patients from normal ones are C-terminal fragments of
alpha-1-antichymotrypsin, albumin N-terminal residue, osteopontin, and transthyretin. as well as
a N-terminal residue of albumin. Beside EGFR, other receptor tyrosine kinase genes are also
deregulated.

MET plays its role by over expressing or by co-expressing its role with EGFR activation and
PTEN inactivation thus contributing to cell proliferation, division and renewal. These entire
characteristic features make MET a potential biomarker for the diagnosis of glioblastoma stem
cells (Boccaccio, 2013, p. 3193-3199)

Novel therapeutic approaches to treat glioblastoma:

Conventional system of drug delivery has certain limitations to treat glioblastoma, thus alternate
new therapeutic approaches are under investigations that include stem cell therapy, peptides, new
neutraceuticals and antibodies that specifically target glioblastoma.

Peptides: Peptides are novel therapeutic molecules employed for treating glioblastoma. Peptides
are categorized into three categories, cell penetrating peptides, tumor homing peptides, and
peptides that target aberrant cellular signaling pathway.

Tumor homing peptides bind to the specific target sites on cell surface that are over expressed
in glioblastoma. In glioblastoma, expression of the receptors on cell surface increases thus
increasing cell signaling and endocytosis. One such receptor is LRP (low density lipoprotein
related protein). ANG-PEG-NP-PTX is a complex tumor homing peptide, consisting of
polyethylene based nano-particle encapsulating paclitaxel with two fold increase in efficiency
than paclitaxel alone (Xin, 2011, p. 4293-4305) Another peptide is chlorotoxin, which is 36
amino acid long peptide and has high efficiency to bind with high expressed receptor
metalloproteinase-2 (MMP-2) in glioblastoma. MMP-2 is not expressed normally in brain’s
normal tissues. Peptide-1 linear, a tumor homing peptide binds specifically to Interleukin-13
receptor a2 (IL-13Ra2). Interleukin-13 receptor a2 is extensively expressed in glioblastoma
absent in normal brain tissues (Pandya, 2012, p. 6-18)

Peptides target aberrant cellular signaling pathway: Peptides target and regulate the cellular
signals involved in oncogenesis, they target with specificity in tumor microenvironment with
 26

improved therapeutic effect. In glioblastoma, various cellular signaling pathways are

overexpressed. VDAC1 is a voltage dependent ion channel that protects glioblastoma cells from
death by interacting with anti-apoptotic proteins. VDAC1 based peptides specifically target the
VDAC1 signaling pathway thus inducing apoptosis of the tumor cells. NEMO a polypeptide,
inhibits NFKB.

Cell penetrating peptides: Cell penetrating peptides are of organic origin and penetrate into the
cell by crossing the plasma membrane. These peptides can be used to carry drug molecules and
imaging agents into the cell. Transcriptional activator TAT protein (TAT) are cell penetrating
peptides conjugated with polyamidoamine encapsulating siRNA (Han, 2010, p. 417-426).

Limitations: Treatment of glioblastoma is challenging because of its poor prognosis because of

resistance against drugs, radiotherapy and chemotherapy. Another limitations/ challenges to treat
this disorder are the inability of drugs to cross blood brain barrier and blood barrier tumor barrier
thud raising a need to develop novel prognostic and diagnostic tools to treat this disorder.
Moreover, heterogenic nature of tumor cells makes it difficult for a therapy to target all
glioblastoma cells.

Treatment: The current standard treatment for GBM is surgery followed by the radiation and
chemotherapy (Chou, 2016, p. 14-23). The chemotherapeutic agent providing maximal
therapeutic effect is temozolomide (TMZ) and its combination agents. (National Comprehensive
Cancer Network [NCCN], 2015).

The main limitation in treating glioblastoma is its invasive nature, making the disease recurrent
and not properly cured after surgical removal of tumor.

Chemotherapy: Temozolomide (TMZ) is the first line-drug to treat glioblastoma. TMZ is

preferred because of its less side effects, it produces gastrointestinal disturbances; nausea and
vomiting (anti-emetics are advised with TMZ), low level of platelets, low level of neutrophils
and lymphocytes.Other chemotherapeutic agents treating glioblastoma include DNA alkylating
agents, carmustine, lomutine and nimustine. These chemotherapeutic agents are of less use
because suppress the functioning of bone marrow and kidney dysfunction.
 27

TMZ promotes autophagy of the cells by arresting the cell in G2/M phase and induce apoptosis
of the cells.

Surgery: Removal of the glioblastoma by surgical resection within safety margin delays the
recurrent growth of tumor. A study conducted by Vuorinen to assess the outcomes of the surgical
removal of tumor cells indicated 2.8 fold increase in the survival rate than biopsy (Vuorinen,
2003, p. 5-10). The surgery is aimed at reducing the volume of the tumor first; good resection is
associated with good prognosis even in adults over the age of 60 years. Surgical procedure can
be employed as a diagnostic tool to assess the metastasis of the tumor and contributes to
information to the about the biological markers employed in glioblastoma (mutation of IDH and
methylation status of MGMT)

Radiotherapy: Radio therapeutic treatment involves the insertion of encapsulated Cs-131

operative cavity, with a total dose of 60 Gy 30 fractions of (2Gy) (Stupp, 2005, p. 987-996).This
treatment also known as Gamma Tile, an FDA approved radio-therapeutic agent (Gessler, 2020,
p. 2445-2455).

First line treatment plan: Radiotherapy along with chemotherapy is given to the patient with
newly diagnosed glioblastoma. Radiotherapy is given at a dose of 60Gy in fractions (2Gy per
day for 5days a week). Along with radiotherapy, chemotherapeutic agent TMZ is also
administered daily 75 mg per 1m2 of body surface area per day and the treatment is continued for
7 days a week. After 28 days of this conjunctive treatment, TMZ is again administered to the
patient at the dilution of 200 mg per m2 of total body surface area for continuous five days (for
six months, six cycles with each cycle after 28 days).

Grade IV Glioblastoma

Primary glioblastoma Secondary glioblastoma

Diffused midline Glioma
IDH wild type IDH mutated

Resection Resection Biopsy

Sequential RCT Sequential RCT Sequential RCT

With TMZ and TMZ With TMZ and TMZ With TMZ and TMZ
Or RT Or RT
 28

In geriatric patients, low dose of radiotherapy (40 Gy in 15 sessions) in conjunction with TMZ
75 mg per m2 of body surface area followed by 12 cycles of TMZ improves glioblastoma.

To benefit from TMZ intake, it’s recommended to take the medicine in fasting condition to
increase its absorption. To treat Recurrent glioblastoma second line of treatment is employed
(Nayak, 2017, p. 625-635)

Nonbulky Resection plus

systemic CT

Resection plus
BCNU Wafer
High grade Glioma
(Glioblastoma) TMZ

Clinical Trial
> 4 months after
Bulky/ Diffused end of TMZ
Symptomatic tumor Bevacizumab
with mass effect
< 4 months after
end of TMZ
Symptomatic tumor Nitrosourea
with no mass effect

Clinical Trial

Targeted Therapies

Targeted therapy focuses on the delivery of the drugs to the targeted sites to minimize the
harmful effects of the conventional drug delivery. Examples include IDH inhibitors AG-120
(mIDH1 Inhibitor), AG881, FT-21-2 and IDH305 and inhibitors of EGFR receptors gefitinib,
erlotinib, and afatinib (but clinically they show less success in glioblastoma) (Reardon, 2015, p.
430-439). Regorafenib, a tyrosine kinase receptor inhibitor is under phase II clinical trial for the
treatment against newly and recurrent glioblastoma (Lombardi, 2019, p. 110-119).

Depatuxizumab Mafodotin also known as ABT-414 is a conjugate combination of drug molecule

and antibody that performs its anti-tumor function by attaching with monomethyl auristatin F
and is under phase II clinical trial for EGFR over-expressed glioblastoma.
 29

BCNU-impregnated polymer is the US-FDA approved polymers available in the form of wafers
that are inserted into tumor activity to release the drug following the surgery. This wafers have
shown improve survival rates in the patients

CED, convection enhanced delivery system directly distributes the macromolecules to the brain
parenchyma cells. This system generates a pressure gradient that delivers drug to the targeted
tumor cells.

Monoclonal antibodies: Monoclonal antibodies provide high specificity, efficiency and affinity
for their target sites with no or minimal affecting the normal non-cancerous cells. Bevacizumab
is a monoclonal antibody that binds to its target site to VEGF (vascular endothelial growth
factor) thus prevents the synthesis of angiogenesis. Bevacizumab has been approved by FDA
after pre-clinical trials. (Hamza, 2014, p. 135-140; Gilbert, 2014, p. 699-708).However,
monoclonal antibodies show limitations in phase II or III clinical trials because of their poor
penetration attributed to their large size and inability to cross blood brain barrier

Anti-angiogenic Compounds: The hypothesis behind the development of anti-angiogenic

compounds is to cut the supply of blood of the tumor cells. Bevacizumab was developed on the
same principle, it halts tumor growth by working on vascular endothelial growth factor (VEGF-
A). Avastin is an anti-angiogenic agent that interferes with the development and synthesis of new
blood vessels critical for the survival of the cancer cells (Friedman, 2009, p. 4733-4740)

Tumor Treating Fields (TTFields): Tumor treating fields employ the low-intensity electrical
fields of 200 KHz frequency (Wenger, 2015, p. 7339-7357). These electrical fields are FDA
approved and selectively target the tumor cells and inhibit replication of cancer cell (Davies,
2013, p. 86-95). NovoTTF-100A (Optune®), Novocure patent, is the first FDA approved tumor
targeting field. TTF in combination with TMZ promises increased survival rate than the TMZ
therapy alone (Stylli, 2020)

Proton Beam Therapy (PBT): Bragg Peak Effect targets specifically to the tumor cells, it
reduces exposure of radiations to the normal healthy brain cells by limiting therapeutic volumes
to the small level. Gamma Knife Radio-surgery, a proton beam therapy provides high dose to the
tumor cells and in being used to provide treatment in recurrent glioblastoma (Larson, 2014, p.
142-148)
 30

T-cell therapy: T-cell therapy employs T-cells that are able to express chimeric antigen receptor
(CAR). Vaccine stimulate antigen specific effector T cell against specific antigen proteins that
are expressed in tumors but are absent in normal healthy cells. Examples include HSPPC-96 for
recurrent glioblastoma (Schuster, 2014, p. 274-279)

Viral-based therapy: viral based therapy includes the delivery of the oncolytic gene using a
viral vector. Oncolytic viruses produce their cytotoxic effects by replicating inside the tumor
cells and providing immunogenic effect. Its example includes DNX-2401, an adenovirus that
employs integrins to produce its anti-cancer effect. Integrins are produced specifically by tumor
cells (Lang, 2018, p. 1419)

Immunotherapy for GB: Cancer cells grow and invade into the neighboring tissues by
bypassing the immune system, they possess low immunogenicity, modulate antigen or suppress
immune system (Bhatia, 2011, p. 209-217 ; Sampson, 2010, p. 4722) Anticancer immunotherapy
is aimed at identifying and then destroying the cancer cells. Two therapeutic approaches in
immunotherapy are passive immunity and active immunity with the passive immunity
activating the body’s immune system and check points. The passive immunity providing agents
include anti-EGFR mAb (clinical trial of United States with trial No. NCT02540161) and anti-
EGFR-CD3 bsAb (clinical trial of United States with trial No. NCT02521090).

Anticancer active immunotherapy is tumor vaccination in which body is exposed to tumor

antigen to activate the response of body against this antigen. These two approaches either elevate
body’s already present immune response or activate a new response. (Reardon, 2014, p. 1441-
1458)

Dendritic cells vaccine:

Dendritic cells play an important role in body’s immune system by forming a link between
body’s innate immunity and its adaptive immunity. If a foreign particle invades body, they
recognize it, process it and present them on their cell surface for presentation to B and T
lymphocytes and thus maintaining a tolerance level for body’s own antigens (Batich, 2015, p.
79-94).The dendritic cells are the body’s most potent activator against foreign antigens. Till date,
sipuleucel-T, the only one cancer vaccine has been approved by United States (Kantoff, 2010, p.
411-422). But the main drawbacks of this therapy lies in optimized maturation of the dendritic
 31

cells, loading of tumor antigens and the ability of dendritic cells to vaccine draining lymph
nodes. Dendritic cells are generated by isolating patient’s CD14+ monocytes and then by
culturing with granulocyte macrophage colony-stimulating factor (GM-CSF). After several days,
they get matured into cytokines, Interleukin-1β, Interleukin-6 and tumor necrosis factor alpha
(Romani, 1994, p. 83-93). Dendtritic cell vaccination elicits an immune response against the
malignant cells of tumor in the body. For this purpose, they must have been inserted into the
body after their ex-vivo production. Tumor antigens can be categorized as Tumor associated
antigens or TAAs and Tumor specific antigens TSAs. TAAs can be employed as the target
molecules for dendritic cells as they are overly expressed in glioblastoma but their presence on
the normal healthy brain cells make them less suited. The other proteins which can be employed
as target sites include EphA2, HER2neu, telomerase and EGFR as these receptors have
immunological potential (Fenstermaker, 2014, p. 377-385).

Glioblastoma

Age < 65-70 years Age > 65-70 years

MGMT Methylated MGMT un-methylated or

unknown

TMZ/RT TMZ/RT or TMZ

RT

Diagnosis of recurrence

Repeat Surgery (If gross total Clinical Trial

resection is possible or if diagnostic
value (biological markers,
histology) Best Supportive care
Poor KPS, High age,
MGMT Unmethylated or
MGMT Methylated Contraindications for
unknown
alternative options

TMZ Nitrosourea BEV

Early or late progression upon TMZ Contraindication: Availability is restricted in EU
may be prognostic Preexisting hemorrhage, thromboembolism
Contraindication: Preexisting myelosppression,
myelosppression, hepatopathy hepatopathy
 32

Dendritic cells for eliciting body’s immune response can be loaded with macromolecules;
peptides (a single peptide or mixture of different peptides can be used), DNA, RNA or tumor
lysate. DCVax-L is a dendritic vaccine and is under phase III trial, it has shown 3 year survival
rate, improvement in the quality of life, increased survival in patients with recurrent glioblastoma
(Trail No. NCT00045968).

In vivo CD205 is a receptor that uptakes antigen and present these cells to the T cells for eliciting
immune response, this property has made CD205 a powerful candidate for DC (dendritic cell )
development. Recently, human CD205 antibody fused with tumor antigen NY-ESO-1, 45 was
trialed on 45 patients, most of the patients get stabilized or the disease regression was observed.
(U.S. clinical trial NCT00948961) (Dhodapkar, 2014).

Conclusion: This article focuses on the genetic mutations involved in the development of
Glioblastoma Multiforme. GBM is characterized by the in-activation of the suppressor genes
responsible to modulate cell cycle, amplification processes and hyper-activation of the pathways
involved in cell proliferation, cell survival, and division. Angiogenesis and specific cell receptors
that are present on the surface of tumor cells are the hallmarks of tumor genes. Metastatic
property of tumor cells makes them immortal thus contributes to the failure of the present active
treatment. So the advance methodologies of glioma cells treatment are under investigation in
research studies as well as clinical trials. These new techniques includes dendritic cells, proton
beam therapy and angiogenic components to arrest tumor cells in active form.
 33

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