GPER1 Regulates NADK1 To Promote Anoikis Resistance And Peritoneal Metastasis Of Gastric Cancer Cells

BackgroundGastric cancer(GC)is associated with high morbidity and mortality worldwide.The symptoms of early gastric cancer are not enough to attract people’s attention,and most patients are often in the advanced stage of gastric cancer when they are diagnosed.The commonly used treatment methods for advanced gastric cancer include radiotherapy,chemotherapy and targeted therapy,but the effective rate of these treatments is not more than 30%,and the clinical treatment effect of advanced gastric cancer is significantly lower than that of early gastric cancer.In the cases of gastric cancer metastasis,peritoneal metastasis occurs in 45%of patients with gastric cancer.Peritoneum is divided into mural peritoneum and visceral peritoneum,which is composed of mesothelium and connective tissue outside.The connective tissue on the peritoneum contains a large number of fat cells.Adipose tissue is not only the largest energy reservoir in the human body,but also the largest endocrine tissue in the human body,which can secrete a large number of hormones,growth factors,enzymes,cytokines and matrix proteins.Adipose tissue expresses aromatase(CYP19),which converts androgens into estrogen.Estrogen not only acts on the reproductive organs,but also has effects on other organs,such as the musculoskeletal,cardiovascular and gastrointestinal systems.In addition to its physiological functions,estrogen also affects the occurrence and development of many diseases such as breast cancer and colon cancer.It has been reported that estrogen interacts with related receptors to remove reactive oxygen species(ROS)and inhibit the development of breast cancer.However,whether or not estrogen receptor signaling plays a role in peritoneal metastasis of gastric cancer and its mechanism remains unclear..Anoikis is a process of apoptosis(cell death)that cells undergo after losing contact with the extracellular matrix or neighboring cells.It can block the metastasis of cancer cells.Due to the activation of redox signaling pathways,cancer cells can acquire anoikis resistance.Whether estrogen induced redox signaling affects peritoneal metastasis of gastric cancer is still unclear.PurposeEstrogen receptor signaling affects the occurrence and development of gastric cancer,but the specific molecular mechanism is still unclear.Studying the mechanism of estrogen receptor signaling in the resistance of gastric cancer to anoikis apoptosis can provide targets for inhibiting the metastasis of gastric cancer.The purpose of this study is,firstly,to determine the estrogen receptor molecules that affect the growth and metastasis of gastric cancer;secondly to explore the mechanism of estrogen receptor promoting gastric cancer cells to resist lost-nest apoptosis.Method1.The expression of estrogen receptors(ERα,ERβ and GPER1)in gastric cancer cell lines was detected by RT-qPCR,Western blot(WB)and immunofluorescence.2.In suspension culture,CCK-8 was used to detect the viability of SGC7901 and BGC823 in addition to E2,GPER1 agonist(G1),inhibitor(G15),or overexpression and knockout of GPER1.3.The GPER1 overexpression and control cells,GPER1 knockout and control cells were injected into nude mice by intraperitoneal injection to construct an intraperitoneal tumor model in nude mice.4.Gastric cancer cells were cultured in suspension,and the intracellular NADPH/NADP+and GSH/GSSG ratios,as well as ROS levels were detected.Cell death and proliferation were detected by Calcein AM/EthD-1 staining and soft AGAR clonal formation assay.5.WB was used to detect some of the key enzymes involved in NADPH anabolism.The mRNA level and ubiquitination level of NADK1 were detected by q-PCR,WB and Co-IP.6.WB and Co-IP verified that GPER1 regulates PKC to activate NADK1.7.NADK1 knockdown gastric cancer cell lines were constructed,and the intracellular NADPH/NADP+ and GSH/GSSG ratios and ROS levels were detected.Meanwhile,Calcein AM/EthD-1 double staining was performed to detect cell death.8.Gastric cancer cells with NADK1 knockdown(BGC823,SGC7901)were injected intrabitoneally into nude mice.After tumor formation,proliferation and apoptosis were detected by staining.Result1.Gastric cancer cells(HGC27,AGS,NCIN87,MKN7,MKN74,BGC823 and SGC7901)express three kinds of estrogen receptors,among which GPER1 mRNA level is relatively high.In five GC cell lines,SGC7901 and BGC823 expressed relatively higher levels of GPER1 protein than GES-1.The expression level of GPER1 protein in BGC823-M was higher than that in BGC823 cell line.In SGC7901 and BGC823 cells,GPER1 was mainly expressed on the plasma membrane of the cell,and a small amount of ERα and ERβ were expressed on the nucleus.2.The viability of SGC7901 and BGC823 cell lines was detected under suspension conditions.It was found that E2 slightly increased the viability of SGC7901 and BGC823 cells,and significantly increased the viability of cells in a dose-dependent response to G1(p<0.01).However,G15 significantly inhibited cell viability.In BGC823-M cells,G1 was found to increase cell viability more significantly than E2.3.We determined the role of GPER1 in promoting gastric cancer metastasis in vivo by establishing a mouse abdominal metastasis model.When GPER1 was knocked out,the metastatic nodules of enteromesenteric wall were significantly reduced in nude mice.Paraffin sections of tumors and peritoneum were obtained from nude mice in the GPER1 knockout group,and IHC staining showed a decrease in Ki-67 expression and an increase in the percentage of TUNEL-positive cells.However,overexpression of GPER1 significantly enhanced tumor peritoneal metastasis with increased Ki-67 expression and reduced percentage of TUNEL-positive cells.4.In suspension culture,the NADPH/NADP+ and GSH/GSSG ratios of GPER1 overexpressed GC cells were significantly higher than those of control group(p<0.01).In the GPER1 knockout group,the NADPH/NADP+and GSH/GSSG ratios of GC cells were significantly lower than those of the control group.In addition,ROS levels decreased and cell death decreased in GC cells overexpressing GPER1.After GPER1 knockout,intracellular ROS levels increased and cell death increased too.In addition,knockout of GPER1 in BGC823 and SGC7901 cells significantly inhibited communities in soft AGAR culture.5.The results of WB showed that among the enzymes related to NADPH production,NADK1 was significantly regulated by GPER1.Further results showed that GPER1 inhibited ubiquitination of NADK1 and increased its protein level expression.It has been reported that PKC signaling can phosphorylate NADK1 and then exert physiological functions,so we hypothesized that GPER1 might regulate NADK1 in a similar manner.WB results showed that PKC phosphorylation increased in response to GPER1 expression.Since no antibody against phosphorylated NADK1 has been detected so far,we performed immunoprecipitation on NADK1 and then used PKC substrate antibody to detect NADK1 phosphorylation status.The results showed that GPER1 could promote the expression of phosphorylated NADK1,and the expression of phosphorylated NADK1 was also promoted or inhibited by the addition of PKC agonist PMA or inhibitor Sotrastaurin.These results suggest that GPER1 promotes the phosphorylation of NADK1 and regulates its expression through PKC.6.Transfection of BGC823 and SGC7901 was confirmed by WB after lentivirus construction of GC cell lines with low NADK1 expression.After knockdown of NADK1,the ratio of NADPH/NADP+and GSH/GSSG in GC cells were significantly lower than those in the control group(p<0.01),while the level of ROS was significantly higher than that in the control group.Calcein AM/EthD-1 staining showed that decreased NADK1 expression increased GC cell death compared with the control group.7.In vivo experiments,the NADK1 knockdown nude mice had more mesenteric nodules,lower Ki67 expression and more TUNEL-positive cells than the control group.In vitro and in vivo experiments showed that decreased NADK1 expression inhibited peritoneal metastasis of GC cells.Conclusion1、In vitro suspension culture,GPER1 can promote GC cells to resist anoikis.2、In vivo,GPER1 promotes the growth of peritoneal metastases of GC cells.3、GPER1 promotes anoikis resistance of GC cells by inducing NADPH production4、GPER1 regulates the phosphorylation of NADK1 by PKC.5、Knockdown of NADK1 inhibits the peritoneal metastasis of gastric cancer.