Regulation of Glioblastoma Stem Cell Properties and Tumor Invasion by G⍺12 Signaling

Abstract

Glioblastoma multiforme (GBM), the most common malignant brain tumor, is associated with upregulation of a number of G‐protein coupled receptors. TCGA analysis of glioblastoma reveals that Gα12 mRNA levels are elevated in 28% of GBM patient tumors, the highest rate for all tumor types surveyed. There are concomitant alterations in expression of a subset of GPCRs that couple to Gα12. Gα12 signals through activation of RhoA and its downstream effectors. Our earlier studies revealed that gene programs induced through the RhoA‐regulated transcriptional co‐activators YAP and MRTF‐A contribute to glioma stem cell properties and their in vivo growth as orthotopic (brain) tumors (Yu et al., Oncogene 2018). To determine if Gα12 signaling pathways transduce the effects of known and orphan GPCRs activated in the tumor environment we knocked down Gα12 using shRNA in two human GBM cancer stem cell lines, GSC‐23 and HK‐281. Gα12 mRNA levels decreased by 60‐80% without compensatory upregulation of mRNA for the homologous Gα13. Loss of Gα12 significantly attenuated the stem cell properties of GSC‐23 cells as determined by limiting dilution assays and also reduced mRNA levels for multiple canonical stem cells genes (CCND1, NANONG, OCT4, SOX2 and NESTIN) by 35% to 60%. Tumor growth of GSC‐23 control and Gα12 knockdown (KD) cells was examined following orthotopic injection into mouse brain. Surprisingly, Gα12 deletion had no evident effect on tumor size nor did it prolong mouse survival. On the other hand, histopathological analyses revealed striking differences at the tumor border, indicative of diminished invasive properties and this was confirmed by immunohistochemical staining for the human nucleoli antigen. RNA sequencing and DESeq2 analysis in WT and KD tumors identified a number of genes encoding extra and intracellular proteins related to cell adhesion and migration dynamics which were differentially expressed in Gα12 KDs. Thrombospondin‐1 (Thbs‐1) was one of the most repressed genes in the Gα12 knockdown tumors and it was previously identified as a YAP‐dependent gene in these cells. Expression of Thbs‐1 and Gα12 is highly correlated in the more aggressive mesenchymal‐types of GBM by Gliovis and IvyGAP tools, and clinical reports show elevated Thbs‐1 as a marker of poor prognosis in GBM patients. To assess the effect of Gα12 and Thbs‐1 on glioblastoma stem cell migration we performed in vitro Transwell‐based assays. These studies demonstrated that both Gα12 and its transcriptional target Thbs‐1 are required for agonist‐stimulated GSC‐23 cell migration and invasion. We also demonstrated through chemogenetic activation of Gα12 by DREADD expression that Gα12 signaling in GSC‐23 cells is sufficient to drive Thbs‐1 expression and migration. Our findings indicate that Gα12 is a focal point for GPCR control of transcriptional programs for stemness and invasiveness in glioma stem cells derived tumors and suggest targeting this site as a therapeutic intervention to disrupt tumor cell invasion.