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YAP and MRTF-A, transcriptional co-activators of RhoA- mediated gene expression, are critical for glioblastoma tumorigenicity

ABSTRACT: The role of YAP (Yes associated protein 1 gene) and MRTF-A (Megakaryoblastic Leukemia gene, MLK1), two transcriptional co-activators regulated downstream of GPCRs (G-protein coupled receptor) and RhoA, in growth of glioblastoma cells and in vivo GBM tumor development was explored using human glioblastoma cell lines (1321N1) and tumor initiating cells derived from patient derived xenografts (PDX)PDX (GSC-23) cells. Knockdown of these co-activators in PDX cells using shRNA significantly attenuated in vitro proliferation and stemness assessed by limiting dilution and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors with lower morbidity than wild-type cells. In vitro studies of cellular responses to the GPCR agonist sphingosine 1-phosphate (S1P) demonstrated that YAP was required for glioblastoma cell invasion and migration, whereas MRTF-A, was required for cell adhesion. S1P- stimulated proliferation was abolished by knockout of either YAP or MRTF-A. Gene expression analysis by RNA-sequencing of S1P- treated 1321N1 cells in which MRTF-A and or YAP were deleted knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor; TF), a target gene regulated selectively through YAP, when knocked down blocked 1321N1 cell invasion and migration, whereas knockdown of HBEGF (Hheparin binding EGF-like growth factor) (HBEGF), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of several target genes dually regulated through both YAP and MRTF-A (CCN1 and MYC) or of those regulated by either factor. Expression of these same genes was decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in the in vivo GBM tumor environment and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE111571 | GEO | 2018/10/10

REPOSITORIES: GEO

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Project description:Rho-kinase signaling and YAP signaling are related to aortic dissection (AD) formation. However, as important biomechanical signaling pathways, their relationships with aortic smooth muscle cell (AoSMC) mechanics have not been investigated in AD formation. This study aims to explore the correlation between RhoA/ROCK1 and YAP, as well as their relationship with intrinsic AoSMC stiffness during AD formation. The expression of RhoA/ROCK1 and YAP as well as F-actin polymerization were analyzed in AoSMCs isolated from normal and AD human aortas. The intrinsic cell stiffness of normal and AD AoSMCs was measured using atomic force microscopy (AFM). The correlations among RhoA/ROCK1, YAP, and intrinsic AoSMC stiffness were explored by manipulating the activity of RhoA and ROCK1, and YAP expression. Finally, the role of RhoA/ROCK1/YAP/F-actin and AoSMC stiffness during AD formation was studied in pharmaceutically induced AD mouse models.Compared to normal human AoSMCs, the expression of RhoA and ROCK1 was downregulated, while the phosphorylation of YAP was increased in AD human AoSMCs, coupled with impaired F-actin polymerization and decreased intrinsic cell stiffness. Pharmaceutical inhibition of RhoA or ROCK1 activities and depletion of YAP all led to impaired F-actin polymerization and decreased intrinsic AoSMC stiffness. Moreover, abnormal collagen deposition and impaired cell-ECM interactions were found when RhoA/ROCK1/YAP/F-actin signaling was inhibited in normal human AoSMCs. We further analyzed the normal human AoSMC treated by Y27632 or not using RNA sequencing to investigate the impact of RhoA/ROCK1/YAP/F-actin on AoSMCs. GO analysis showed that differentially expressed genes (DEGs) related to cAMP-mediated signaling, cytoskeleton organization, cell-matrix adhesion, and extracellular matrix (ECM) organization were affected when ROCK1 activity was inhibited by Y27632 in normal AoSMCs. KEGG analysis showed differences in PI3K-Akt signaling, focal adhesion, MAPK, actin cytoskeleton, and ECM-receptor interaction. Consistently, cAMP, actin cytoskeleton organization, and ECM structural constituents were all downregulated in AoSMCs treated with Y27632 according to GSEA. We also analyzed the downregulated genes in AoSMCs treated with Y27632. Results demonstrated that the downregulated genes were mainly related to the cell-ECM unit, PI3K, MAPK, focal adhesion, and cAMP signaling pathways.

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YAP and MRTF-A, transcriptional co-activators of RhoA- mediated gene expression, are critical for glioblastoma tumorigenicity

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