The Role Of Geranylgeranylation Signaling In Cancer Cell Proliferation And Migration

Background:Geranylgeranylation(GGylation)is an important protein modification in cells.Many intracellular signaling molecules need to be modified by GGylation to play their biological functions in cell proliferation,differentiation,migration and survival.GGylation-mediated protein modification process has been proved to play an important role in cancer progression,but the mechanism and signaling pathway of its tumor-promoting effect are still unclear.Objective:This research focused on gastric cancer and triple negative breast cancer as the research system,emphatically discussed the regulation of the GGylation signal on the tumor-promoting protein YAP/TAZ,to determine the promotion effect of GGylation-YAP/TAZ signaling pathway on tumor growth,metastasis and invasion,to reveal the internal mechanism of GGylation-YAP/TAZ signaling pathway to activate tumor cell proliferation,migration and invasion.At the same time,new molecular targets for the diagnosis and treatment of gastric cancer and triple negative breast cancer were identified,which provided theoretical basis for the clinical development of new targeted therapeutic drugs and prognosis prediction for tumor patients.Methods:1.The cells were treated with atorvastatin(AT)and geranylgeranyl-transferase I inhibitor(GGTI-298)to inhibit the GGylation.Geranylgeraniol(GGOH)was used to rescue the AT-caused defect in GGylation.2.The lentiviral vector mammalian shRNA expression system was used to construct the YAP1,TAZ,CYR61,E2F1 knockdown cell lines in the gastric cancer AGS or the triple negative breast cancer(TNBC)MDA-MB-231 cells.3.Cell proliferation was determined by the cell counting assay;cell migration was determined by the wound healing assay and the transwell migration assay;cell invasion was detected by the transwell invasion assay;the subcellular localization of proteins in cells was observed and quantified by immunofluorescence staining;expression of related genes was detected by the conventional RT-PCR or the quantitative real-time PCR(qRT-PCR);and expression of proteins in cells was detected by Western blotting.4.The microtubule inhibitor Nocodazole was used for synchronization of cell cycle.Cytokinesis was analyzed by the Nocodazole release assay.Nucleus or mitosis was visualized by fluorescent staining with DAPI or Hoechst.5.Expression of the YAP/TAZ target gene product CYR61 in both tumor and adjacent normal tissue from 214 cases of gastric cardia adenocarcinoma(GCA)was detected by the tissue microarray assay with immunohistochemical staining.The correlation between CYR61 expression and patient survival,TNM stage,tumor invasion(T category),lymph node metastasis(N category),tumor size,or differentiation in GCA were statistically analyzed.Results:1.Inhibition of GGylation significantly reduced proliferation,migration and invasion of the gastric cancer AGS cells or the proliferation of TNBC MDA-MB-231 and MDA-MB-453 cells,indicating that proliferation,migration and invasion of cancer cells were dependent on the GGylation signaling.2.Inhibition of GGylation down-regulated expression of the YAP/TAZ target genes CYR61 and CTGF in AGS cells.Furthermore,knockdown of YAP1 or TAZ inhibited proliferation,migration and invasion of AGS cells or MDA-MB-231 cells.These results suggest that YAP and TAZ are the downstream effectors of the GGylation signaling.3.Inhibition of GGylation impaired translocation of YAP1 and TAZ to nucleus in AGS cells or MDA-MB-231 cells,indicating that GGylation signaling activates YAP/TAZ.4.Inhibition of GGylation or knockdown of YAP1 inhibited mitosis of MDA-MB-231 cells,suggesting that the GGylation/YAP signaling promotes the progression of cancer cell cycle.5.Inhibition of GGylation or knockdown of YAP1 down-regulated expression of the centromere proteins and the transcription factor E2F1 in MDA-MB-231 cells,while knockdown of TAZ had an insignificant effect on expression of centromere protein.In addition,knockdown of E2F1 inhibited proliferation of MDA-MB-231 cells and expression of centromere genes.These results suggest that the GGylation/YAP signaling promotes cancer cell proliferation through up-regulating E2F1 thus enhancing expression of centromere proteins.6.Overexpression of the YAP/TAZ target gene CYR61 was observed in gastric cardia adenocarcinoma(GCA).Statistical analysis indicated that overexpression of CYR61 is positively correlated with TNM stage and N category of GCA,and negatively correlated with the cumulative survival of the GCA patients.These results suggest that CYR61 may be a driving protein for metastasis of GCA and a biomarker for poor prognosis.7.Knockdown of CYR61 inhibited migration but not proliferation of AGS cells,suggesting that CYR61 may mediate the effect of the GGylation/YAP/TAZ signaling on cancer cell migration,but not proliferation.Conclusions:The GGylation signaling activates the YAP/TAZ pathway to promote proliferation,migration and invasion in gastric and TNBC cells,and metastasis of GCA.The mechanism by which the GGylation signaling promotes cancer cell proliferation is activating YAP1 to up-regulate expression of E2F1 that enhances transcription of centromere genes,thus facilitating cancer cell cycle progression.The mechanism by which the GGylation signaling promotes cancer cell migration and invasion is activating YAP/TAZ to enhance expression of CYR61,thus facilitating cancer cell migration and invasion.