Multi-Omics Analysis of Glioblastoma and Glioblastoma Cell Line: Molecular Insights Into the Functional Role of GPR56 and TG2 in Mesenchymal Transition

Abstract

G protein-coupled receptor 56 (GPR56/ADGRG1) is an adhesion GPCR with an essential role in brain development and cancer. Elevated expression of GPR56 was observed in the clinical specimens of Glioblastoma (GBM), a highly invasive primary brain tumor. However, we found the expression to be variable across the specimens, presumably due to the intratumor heterogeneity of GBM. Therefore, we re-examined GPR56 expression in public domain spatial gene expression data and single-cell expression data for GBM, which revealed that GPR56 expression was high in cellular tumors, infiltrating tumor cells, and proliferating cells, low in microvascular proliferation and peri-necrotic areas of the tumor, especially in hypoxic mesenchymal-like cells. To gain a better understanding of the consequences of GPR56 downregulation in tumor cells and other molecular changes associated with it, we generated a sh-RNA-mediated GPR56 knockdown in the GBM cell line U373 and performed transcriptomics, proteomics, and phospho-proteomics analysis. Our analysis revealed enrichment of gene signatures, pathways, and phosphorylation of proteins potentially associated with mesenchymal (MES) transition in the tumor and concurrent increase in cell invasion and migration behavior of the GPR56 knockdown GBM cells. Interestingly, our analysis also showed elevated expression of Transglutaminase 2 (TG2) - a known interactor of GPR56, in the knockdown cells. The inverse expression of GPR56 and TG2 was also observed in intratumoral, spatial gene expression data for GBM and in GBM cell lines cultured in vitro under hypoxic conditions. Integrating all these observations, we propose a putative functional link between the inverse expression of the two proteins, the hypoxic niche and the mesenchymal status in the tumor. Hypoxia-induced downregulation of GPR56 and activation of TG2 may result in a network of molecular events that contribute to the mesenchymal transition of GBM cells, and we propose a putative model to explain this functional and regulatory relationship of the two proteins.

GPR56 signaling pathway network and its dynamics in the mesenchymal transition of glioblastoma

“…In our earlier study (Ganesh et al 2022), we generated transcriptome, proteome, and phosphoproteome data for GPR56 knockdown U373 GBM cells. Our analysis showed 1010 transcripts, 47 proteins, and 70 phosphoproteins that were differentially expressed (Ganesh et al 2022).…”

“…Interestingly TG2 itself is a multifunctional protein present intra and extracellularly and interacts even with other cell surface receptors (Nurminskaya and Belkin 2012). Further, the two proteins are also expressed reciprocally in spatial regions of GBM (Ganesh et al 2022). So, the interaction between GPR56 and TG2 ligand may involve a complex network of molecular cascades connected not only to GPR56 activity but also to the independent activity of TG2, representing an extraordinary regulatory pathway system with implications in the process of mesenchymal transition.…”

“…This included molecules involved in one or more of the top enriched pathways such as Inflammation by Chemokine and cytokine signaling, Wnt, Integrin, Ca signaling, and angiogenesis. These pathways and their pathway axes are known to be associated with the process of mesenchymal transition in general, some of them were also found to be hub entities in the interaction network (Ganesh et al 2022) (Supplementary Tables 1A and 2A).…”

“…GBM is a highly aggressive brain tumor characterized by its complex molecular and genetic heterogeneity, which has led to the identification of distinct molecular subtypes-(neural (N), proneural (PN), classical (CL), and mesenchymal (MES)) (Patel et al 2014;Verhaak et al 2010;Wang et al 2017). In our earlier publication, we have shown that the GPR56 expression in GBM is heterogeneous-high in PN and low in the MES subtype, and its expression is negatively associated with TG2 in PN and MES cells (Ganesh et al 2022). The inverse relationship between GPR56 and TG2 was also supported by spatial expression data (microvascular proliferation and pseudopalisiding cells around necrosis (PAN) of GBM tissues and also in GBM cells cultured under hypoxic conditions (Ganesh et al 2022).…”

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G protein-coupled receptor 56 (GPR56/ADGRG1) is a multifunctional adhesion GPCR involved in diverse biological processes ranging from development to cancer. In our earlier study, we reported that GPR56 is expressed heterogeneously in glioblastoma (GBM) and is involved in the mesenchymal transition, making it a promising therapeutic target (Ganesh et al, 2022). Despite its important role in cancer, its mechanism of action or signaling is not completely understood.

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