4. Discussion
High-grade gliomas are particularly challenging tumours to treat due to
their invasive nature which requires substantial understanding of
molecular mechanism behind their aggressive behaviour compared to low
grade gliomas. Since LGGs are less common and exhibit milder symptoms
than HGG delayed diagnosis or misdiagnosis are frequent. LGG are also
slow-growing and less invasive compared to high-grade gliomas and in
some cases do not require immediate surgical intervention. Because of
these reasons it is challenging to get the low-grade glioma tissue
samples compared to HGG. The current study is the first LC-MS/MS based
proteomics study of high-grade and low-grade tumor tissues investigating
the protein level differences and identifying the key molecular pathways
differentially regulated between the two cohorts. We first performed the
label free quantification of glioma samples to identify the
differentially regulated proteins in HGGs. Proteins like Fibronectin,
Vitronectin Fibrinogens (FGA, FGB, FGG), Collagens (COL1A1, COL1A2,
COL4A1, COL4A2, COL6A1, COL6A2, COL6A3), Plasminogen and Transforming
growth factor-β came over-expressed in the HGGs, while other proteins
like Tenascin R, Contactin-1, Neurofascin, Ankyrin-2 and Neurogranin
were found downregulated in HGG.
Fibronectin and vitronectin both are glycoproteins found in the
extracellular matrix (ECM), they play a crucial role in cancer cell
invasion and metastasis. Fibronectin provides a substrate for cancer
cells to adhere to and migrate from the primary tumor site and invade
surrounding tissues (41). Fibronectin and vitronectin both are known to
interact with integrin receptors on the cancer cell surface, promoting
cell adhesion and initiating signaling pathways that regulate migration.
Cancer cells secrete enzymes called matrix metalloproteinases (MMPs),
which degrade the ECM components facilitating tumor cell invasion (40,
41).
Another class of proteins found overexpressed in HGG were fibrinogens
such as FGA, FGB and FGG. In high-grade glioma, the disrupted
blood-brain barrier allows fibrinogen from the blood to penetrate the
tumor microenvironment where it is converted into insoluble fibrin by
the enzyme thrombin, resulting in the formation of fibrin deposits
within the tumor. Fibrin deposits in the tumor microenvironment act as a
provisional matrix further promoting the migration and invasion of
glioma cells (7).
Our data also showed high expression of collagens in HGG, like
fibronectin and fibrinogens, collagens are also key components of the
extracellular matrix (ECM). Upregulated collagens, particularly collagen
types I and IV, play a role in ECM remodeling within HGG. These
collagens are deposited around tumor cells and contribute to the
formation of a denser and stiffer ECM. This altered ECM composition and
structure can facilitate tumor cell invasion and migration through the
surrounding brain tissue (43). In HGG, upregulated collagens also
contribute to angiogenesis, the formation of new blood vessels that
supply nutrients and oxygen to the tumor. They promote endothelial cell
adhesion, migration, and tube formation, facilitating the development of
an extensive vascular network within the tumor (5).
Gene set enrichment analysis of identified proteins revealed epithelial
mesenchymal transition (EMT) pathway to be highly enriched in HGGs
followed by enriched focal adhesion and actin cytoskeleton pathway. EMT
and the focal adhesion pathway are interconnected and play significant
role in the progression of cancer. EMT is a process through which
epithelial cells acquire mesenchymal-like characteristics, including
enhanced migratory and invasive properties (44). In high-grade gliomas,
EMT has been observed and is associated with tumor progression and
metastasis. EMT enables glioma cells to detach from the primary tumor
mass, invade surrounding brain tissue, and migrate to distant sites.
Glioma cells undergoing EMT show phenotypic changes, such as loss of
cell-cell adhesion molecules (e.g., E-cadherin) and gain of mesenchymal
markers (e.g., N-cadherin, vimentin, fibronectin), facilitating their
invasive behavior (5,44). Transforming growth factor-beta-induced
protein (TGF-β), a strong inducer of EMT transcription factors, drives
the EMT in both SMAD and non-SMAD signalling pathways (45). We have
observed high expression of TGF-β in high grade gliomas indicating the
over expression of EMT pathway in HGG. EMT has also been linked to
resistance to chemotherapy and radiation therapy in gliomas. The altered
molecular profile and increased migratory properties conferred by EMT
can contribute to therapy resistance and tumor recurrence (44). It has
been associated with the acquisition of stem cell-like properties in
glioma cells. Cells undergoing EMT exhibit enhanced self-renewal
capabilities, increased resistance to apoptosis, and a capacity for
multilineage differentiation (44). These stem-like properties contribute
to tumor heterogeneity and therapeutic resistance, which is well known
phenomenon in HGG.
Another enriched pathway observed in HGG was focal adhesion pathway
which is critical for cell adhesion, migration, and
mechano-transduction. In HGG, the focal adhesion pathway is upregulated
and contributes to invasive and migratory behaviors. Focal adhesion
proteins also participate in ECM remodeling by mediating the interaction
between cells and the surrounding matrix (46). Dysregulated focal
adhesions in gliomas contribute to ECM degradation, facilitating tumor
invasion and angiogenesis. Focal adhesion proteins, including FAK (focal
adhesion kinase), activate downstream signaling pathways involved in
tumor progression, such as MAPK and PI3K/AKT pathway (46) . These
signaling cascades regulate cell survival, proliferation, and migration,
promoting glioma progression and resistance to therapy. Understanding
the interplay between EMT and the focal adhesion pathway in high-grade
glioma progression is crucial for developing targeted therapies.
Strategies aimed at inhibiting EMT, disrupting focal adhesion signaling,
or targeting key molecules within these pathways hold promise for
limiting glioma invasiveness and improving treatment outcomes.
Hypoxia is the other pathway which was found enriched in HGG,
characterized by low oxygen levels. Tumors often develop regions of
inadequate oxygen supply due to their rapid growth and aberrant
vasculature. Hypoxia triggers the release of various pro-angiogenic
factors, such as vascular endothelial growth factor (VEGF), to stimulate
the formation of new blood vessels (47) . This process, known as
angiogenesis, helps supply oxygen and nutrients to the tumor, promoting
its growth and survival. Increased angiogenesis also contributes to
tumor aggressiveness and metastasis. Hypoxic conditions force cancer
cells to adapt their metabolism to survive and grow. They undergo
metabolic reprogramming, shifting towards anaerobic glycolysis, known as
the Warburg effect, which allows them to generate energy even in the
absence of oxygen. This metabolic switch not only provides energy for
cancer cell growth but also leads to the accumulation of metabolic
byproducts that can promote tumor progression (47). Hypoxia can also
induce genetic and epigenetic changes in cancer cells that promote their
growth and survival. Hypoxia-inducible factors (HIFs) are key
transcription factors that are stabilized under low oxygen conditions
and regulate the expression of various genes involved in angiogenesis,
metabolism, cell survival, and invasion. HIFs can promote the expression
of pro-growth and pro-survival genes while inhibiting genes involved in
cell death (47) . Hypoxia has been implicated in promoting EMT.
Understanding the role of hypoxia in cancer cell growth is crucial for
developing effective therapeutic strategies. Targeting hypoxia-related
pathways, such as angiogenesis and HIF signaling has emerged as a
promising approach to inhibit tumor growth and improve treatment
outcomes in cancer.
The results of GSEA analysis were also in concurrence of protein-protein
interaction analysis which resulted focal adhesion, ECM receptor
interaction as top pathways. Two additional pathways identified were
complement coagulation cascade and N-glycan biosynthesis. Fibrinogens
(FGA, FGB, FGG), plasminogen, kininogen and antithrombin-III were mapped
to complement and coagulation pathways. Three subunits of
oligosaccharyltransferase enzyme RPN1, RPN2 and DAD1 were mapped to
N-glycan biosynthesis pathway. Whereas Multiple reaction monitoring
based targeted proteomics experiment identified another subunit DDOST to
be upregulated in HGGs.
Oligosaccharyltransferase (OST) is an enzyme complex with 11 subunits,
it is responsible for protein glycosylation. RPN1 and RPN2 subunits are
considered as receptors of membrane bound ribosomes hence called
ribophorins (48). DAD1 helps in maintaining the structural integrity of
the OST complex by forming a heterotetrameric complex with RPN1, RPN2
and DDOST while OST48 or DDOST keeps the OST complex in the ER with its
cytosolic domain (49). Higher OST expression is corelated with the
aberrant glycoprotein expression. Abnormal expression of glycoproteins,
can impact the adhesive properties of cancer cells, affecting cell-cell
and cell-ECM interactions. These alterations may contribute to increased
invasiveness and metastatic potential of high-grade glioma cells.Figure 6 summarizes the changes in the extra cellular matrix
during the epithelial to mesenchymal transition and role of OST in
cancer cell migration. In several tumour cells, higher expression of
glycosyltransferase has been linked to enhanced aggressiveness. In a
study by Wu et al. (50), immunohistochemistry identified
N-Acetylgalactosaminyltransferase-14 as a potential biomarker for breast
cancer. Further, higher expression of sialyltransferases and
fucosyltransferases is associated with enhanced malignant behaviour
(51). Similar findings have been observed in various other tumour cells,
including liver, ovary, and melanomas, demonstrating the significance of
glycosylation in tumour aggressiveness. Similarly, OST can also be
explored as potential biomarker for HGGs and it can act as a potential
therapeutic target to control mesenchymal behaviour of HGGs.
We have also performed in-silico molecular docking to identify
OST inhibitors which can suppress its expression. Molecular docking
results identified Irinotecan and Entrectinib to be potential inhibitors
of catalytic subunits of OST. Both Irinotecan and Entrectinib are FDA
approved therefore safe for human use and showed good binding affinity
towards OST isomers STT3A and STT3B. These inhibitors can be explored
further for their anti-cancer efficacy in cell culture or animal model.
High-grade gliomas, including glioblastoma, are highly heterogeneous
tumors. This heterogeneity poses challenges in identifying consistent
and reliable biomarkers that accurately represent the complex molecular
alterations within gliomas. In this study we have performed
comprehensive proteomic investigation of high-grade and low-grade
gliomas with the aim to identify potential clinical marker/therapeutic
targets. We have found oligosaccharyltransferase (OST) complex to be
highly upregulated in high grade gliomas. Four subunits of OST complex;
RPN1, RPN2, DAD1 and DDOST have shown higher expression in HGG compared
to LGG. While the specific role of oligosaccharyltransferase in
high-grade glioma metastasis remains to be fully elucidated, it is
likely that alterations in glycoprotein expression, potentially
influenced by oligosaccharyltransferase activity, can impact various
aspects of metastasis, including cell adhesion, invasion, immune
interactions, and ECM remodeling. Further research is needed to uncover
the specific mechanisms and implications of glycosylation in glioma
metastasis and the potential involvement of oligosaccharyltransferase in
this process.