The role of YAP (Yes-associated protein 1) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G protein-coupled receptors) and RhoA, in the growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor-initiating cells derived from patient-derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using short hairpin RNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression, and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion, whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP 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, blocked cell invasion and migration, whereas knockdown of HBEGF (heparin-binding epidermal growth factor-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.
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We thank Jacqueline Sayyah for initial help with culture and analysis of 1321N1 cells, and Silvio Gutkind for assistance with interrogation and analysis of TCGA data
OMY completed much of the experimental work, data analysis, and figure preparation; JAB advised on PDX cell experiments and performed orthotopic xenograft injections; FBF contributed PDX cells and advice on data interpretation; SWP generated the CRISPR knockout cells; DR and AK carried out experiments examining TAZ and MRTF-B involvement; JS generated lentiviral shRNA constructs; KLG advised on manuscript preparation and YAP signaling; BD and JG assisted with RNA-seq and analysis under the direction of AR; OMC contributed studies on cell proliferation and finalized manuscript preparation. SM advised on design of the experiments and analysis of data and contributed to the manuscript preparation; JHB provided overall study supervision, data interpretation, and manuscript preparation.
This work was supported by NIH Grants GM036927, HL028143, HL085577. and CA170682 to JHB; HL097037 to SM; NS080939 to FBF and JAB; AI40127 and AI109842 to AR. OMY and SWP were supported by T32 GM007752; OMY also received support from T32 DK00754; BD is an HHMI postdoctoral fellow from the Jane Coffin Childs Fund; OMC is a PDE fellow from The Brazilian National Council for Scientific and Technological Development.
Conflict of interest
The authors declare that they have no conflict of interest.
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Yu, O.M., Benitez, J.A., Plouffe, S.W. et al. YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity. Oncogene 37, 5492–5507 (2018). https://doi.org/10.1038/s41388-018-0301-5
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