Abstract A19: Regulation of glioblastoma tumor growth and stem cell properties through Gα12 and tissue factor, upstream and downstream players in YAP signaling

Glioblastoma multiforme (GBM), the most common and aggressive form of brain tumor, has been associated with the dysregulation of G-protein coupled receptors signaling. Data from The Cancer Genome Atlas demonstrated remarkable upregulation of mRNA for the Gα12 protein, which is elevated in 26% of GBM patient tumors (the highest rate of tumor samples surveyed). The Gα12 protein couples GPCRs to guanine nucleotide exchange factors (GEFs) for RhoA and was originally isolated as an oncogene. Furthermore, the subset of GPCRs that couples to Gα12 to activate RhoA includes S1P, LPA, and thrombin. These are generated, respectively, through sphingosine kinase, autotaxin, and tissue factor, all of which are elevated in glioblastoma tumors or cell lines. Signaling to RhoA leads to activation of the transcriptional co-activator YAP (Yes-associated protein 1). The role of YAP in growth of glioblastoma cells and in vivo GBM tumor development was explored in our previous studies (Yu et al., Oncogene 2018) using human glioblastoma cell lines and GBM stem cells (GSCs) derived from patient-derived xenografts (PDX). We showed that shRNA mediated knockdown of YAP in GSC-23 PDX cells significantly attenuated in vitro self-renewal capability assessed by limiting dilution, neurosphere formation, and stem cell gene expression. Orthotopic xenografts of the YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. Our current work examines the effects of Gα12 since it transduces effects of multiple GPCRs upstream to RhoA that could be activated in the tumor environment. Targeting Gα12 using shRNA in two GBM cancer stem cell lines, GSC-23 and HK-281, decreased Gα12 mRNA levels by 80% without compensatory upregulation in the expression of the homologous Gα13 protein. Loss of Gα12 attenuated GSC-23 stemness as determined by limiting dilution assay and reduced mRNA expression levels of canonical stem cells genes (CCND1, NANONG, OCT4, SOX2, and NESTIN by approximately 50%). We are currently examining tumor growth of GSC-23 control and Gα12 knockdown cells as orthotopic xenografts. We are also examining the role of specific YAP-regulated target genes identified by RNA-sequencing of S1P-treated control and YAP knockout glioblastoma cells. In particular, one of the genes highly dependent on YAP is F3, which encodes the protein for tissue factor (TF) which is involved not only in coagulation but also in generation of thrombin, a ligand for PAR1, a GPCR that is upregulated in GBM and regulates RhoA signaling. Expression of TF was also selectively decreased in tumors from PDX cells lacking YAP, indicating that this transcriptional pathway is regulated in preclinical GBM models. Thus, Gα12/RhoA-mediated YAP signaling pathway appears to play a pivotal role in GBM tumor growth and progression. Citation Format: Olga M. Chaim, Jinhui Ma, Jacqueline Lara, Frank Furnari, Shigeki Myamoto, Joan H. Brown. Regulation of glioblastoma tumor growth and stem cell properties through Gα12 and tissue factor, upstream and downstream players in YAP signaling [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A19.