| Objective:Gastric cancer is still one of the malignant tumors that threaten human health globally,and it will cause more than 1,000,000 new cases and approximately 783,000 deaths in 2020(equivalent to 1 death in every 12 deaths in the world).Especially in our country,the incidence of gastric cancer is also showing a significant upward trend.Although important progress has been made in the understanding and research of epidemiology,pathology,molecular mechanisms,and treatment options and strategies,the threat of gastric cancer to public health is still high.G-protein-coupled receptors(GPCRs)play a central role in signal transmission,thereby controlling many facets of cellular function.Increasing evidence suggests a role of GPCRs and their ligands in different aspects of tumor biology.G-protein-coupled receptor kinases(GRKs),a subfamily of AGC(protein kinase A/G/C-like)kinases originally identified as regulators of GPCR signaling,are emerging as potentially relevant onco-modulators.GRKs are a family of 7 serine/threonine protein kinases that specifically phosphorylate activated GPCRs.Mammalian GRKs consist of 3 subgroups based on sequence similarity,including the rhodopsin kinases(GRK1 [rhodopsin kinase]and GRK7 [cone opsin kinase]),the ?-ARK subgroup(GRK2 and GRK3),and the GRK4 subgroup(GRK4,GRK5,and GRK6).GRK3 is best known to phosphorylate the agonist-occupied form of the ?-adrenergic and related G-protein-coupled receptors,leading to broad regulation of receptor function.GRK3 is an isoform of GRKs that phosphorylates and desensitizes GPCRs.GRK3 shows varied expression levels and function in different tumor types.For example,down-modulation of GRK3 correlated with increased growth of glioblastoma cells and associated with worse outcomes in pancreatic ductal adenocarcinoma and hepatocellular carcinoma.In contrast,over-expression of GRK3 in prostate tumor cells and colon cancer promoted tumor growth and metastases through the induction of angiogenesis.However,the role of GRK3 in GAC remains unclear and needs investigation.Methods:We first performed PCR experiments on 23 pairs of gastric cancer and paired adjacent tissues from the Oncology Department of the First Affiliated Hospital of China Medical University and MD Anderson Cancer Center.At the same time,we conducted immunohistochemistry(IHC)experiments tissue micro-arrays(TMAs)which consisted393 pairs of gastric cancer tissues and paired adjacent non-cancerous tissues from the Department of Oncology,the First Affiliated Hospital of China Medical University.Analyze the correlationship between GRK3 expression and clinicopathological data or follow-up information.Verify through database analysis.Next,we used ascites tumor cells derived from gastric cancer patients to establish animal models to detect the expression of GRK3 in subcutaneous tumors in situ and mouse ascites tumor cells.Finally,GRK3 immunofluorescence staining was performed on the patient's ascites cells to evaluate its expression in peritoneal metastatic cells.Knockout and overexpression of GRK3,conduct MTS and colony formation experiments to explore the effect of GRK3 on the proliferation of gastric cancer cells.Use drug library to screen GRK3 inhibitor LD2,treat tumor cells in vitro,MTS,colony formation and transwell experiments were used to explore the effect of inhibitors on tumor proliferation and migration.In vivo,the mice were treated with inhibitors to study the effect of inhibiting GRK3 on tumor growth.In order to further explain the mechanism of GRK3 affecting the behavior of peritoneal metastasis,we used RPPA experiments to explore the changes in downstream targets after GRK3 inhibition.PCR was used to verify the correlation between GRK3 and YAP1 expression in gastric tumor tissues,adjacent non-cancerous tissues and ascites tumor cells.In the same gastric cancer TMAs,IHC experiments were performed on YAP1 to verify the correlation between GRK3 and YAP1.After GRK3 was knocked out and overexpressed,q RT-PCR and Western blot were used to verify whether the expression of YAP1 was affected by GRK3.After the cells were treated with LD2,q RT-PCR and Western blot were used to verify the expression of YAP1.Treated with LD2,the tumor sephere experiment and flow cytometry were applied on to analyze the influence of GRK3 on the stemness of tumor cells.LD2 was used to treat wild-type and YAP1knock-out clones,MTS assay was used to observe YAP1 dependence of LD2 in inhibiting tumor cell proliferation.Results:The results of the study found that GRK3 expression in gastric tumor tissues was higher than that in paired adjacent non-cancerous tissues.Immunohistochemistry on gastric cancer TMAs showed that the positive rate of GRK3 in gastric cancer tissue61.1%(240/393)was significantly higher than that in paired adjacent non-cancerous tissues 32.3%(127/393)(P<0.01).The high expression of GRK3 in primary gastric cancer tissue was significantly related to poor differentiation(P=0.044),Lauren's type(diffused type)(P=0.010),lymph node metastasis(P=0.001)and higher TNM staging(P=0.043).The survival time of patients with high GRK3 expression was significantly lower than that of patients with negative GRK3 expression(P=0.002).The database analyzed a cohort of 876 patients with gastric cancer,and the results showed that the high expression of GRK3 was also significantly associated with a shorter survival time and a higher risk of death(P<0.001,HR=1.74).In the PDX mouse model,it was found that the expression of GRK3 in ascites cells was significantly higher than its corresponding in subcutaneous tumor tissue.Immunofluorescence staining of the ascites cells of gastric cancer showed that 90% of the ascites tumor cells of gastric cancer patients showed strong GRK3 fluorescence signal.In gastric cell lines,it can be seen that GRK3 is almost not expressed in normal gastric mucosal epithelial cell lines,and positive expressions vary in strength in gastric tumor cell lines.In the four gastric cancer patients derived ascites tumor cells,GRK3 shows a strong expression level.We found that after knocking out GRK3,tumor cell proliferation was inhibited.After over-expression of GRK3,the proliferation ability of tumor cells is enhanced.Screening of the GRK3 specific inhibitor LD2 and can significantly inhibit the expression of GRK3 in gastric cancer cells,the proliferation and migration ability of tumor cells is also inhibited.In vivo,the mice were treated with LD2.Compared with the control group,the tumor volume and tumor weight of the LD2 treatment group were significantly reduced.The RPPA experiment found that after GRK3 was inhibited,the expression level of the transcription factor YAP1 in the Hippo/YAP1 pathway decreased.The results of PCR and immunohistochemistry experiments showed that the expression levels of GRK3 and YAP1 were positively correlated.After knocking out GRK3 in gastric cancer cells or treating the cells with LD2,it was found that the expression level of YAP1 was down-regulated.After overexpression of GRK3,YAP1 expression was up-regulated.Treatment of cells with LD2 significantly inhibited the characteristics of gastric cancer stem cells.The inhibitory effect of LD2 on wild-type cells was significantly stronger than that of YAP1 knockout cells.Conclusion: The expression of GRK3 in gastric cancer tissues is higher than that in the paired adjacent non cancerous tissues,and it is related to the poor differentiation,metastasis,advanced TNM stage of gastric cancer and poor postoperative prognosis of gastric cancer patients.GRK3 is highly expressed in gastric cancer cell lines and ascites tumor cells of gastric cancer patients.Inhibition of GRK3 reduces tumor proliferation;overexpression of GRK3 enhances tumor cell proliferation;LD2,a GRK3 inhibitor can significantly reduce GRK3 expression,cancer proliferation and migration.GRK3 regulates the stemness of tumor cells and the occurrence of peritoneal metastasis by regulating the transcription factor YAP1. |
PDF Full Text Request