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.