Yes Associated Protein 1 Promotes Resistance To 5-Fu In Gastric Cancer By Regulating Glycometabolism Of TAMs

Background and purpose:Gastric cancer(GC)is the third most common malignancy and the third leading cause of cancer death worldwide.Many patients with gastric cancer are already in the advanced stage at the time of diagnosis,and chemotherapy has become the main treatment for most patients.However,the development of multidrug resistance usually results in the failure of chemotherapy,even after combination chemotherapy,which leads to tumor recurrence and further progression.In recent years,many studies have shown that the resistance to chemotherapy treatment is closely related to tumor microenvironment(TME).As one of the main components of TME,tumor associated macrophages(TAMs)are involved in regulating tumor resistance to chemotherapy treatment.Yes associated protein 1(YAP1)is a transcriptional coactivator and negative regulator of the Hippo pathway.It regulates diverse cellular processes,such as cancer development,contact inhibition and embryonic development,organ size,and cell differentiation.Our previous research shows that,YAP1,as an important downstream factor of HER4,promoted trastuzumab resistance in HER2 positive gastric cancer by inducing epithelial and mesenchymal transition.However,the exactly role of YAP1 in the resistance of gastric cancer to 5-Fu remains unknown.Our previous study reveals that YAP1 was overexpressed in GC tissues compared to paired normal gastric mucosa tissues.According to the clinical data,the overexpression of YAP 1 was associated with poor prognosis in patients with gastric GC.Mechanically,the secretory of YAP1 from GC cells could upregulate the expression of GLUT3,which stained in the membrane of M2 type macrophage.The overexpression of GLUT3 in macrophage stimulated the uptake of glucose and promoted the glucose metabolism.CCL8 was secreted more from macrophage after co-cultured with YAP1 overexpressed gastric cancer cells compared to vector groups.In turn,the secretory of CCL8 from GLUT3-dependent TAMs induced the resistant to 5-Fu treatment in gastric cancer through JAK3/STAT1 signal pathway.Methods:The expression of YAP 1,CD 163,GLUT3 and CCL8 were detected by IHC,IF,RT-qPCR and western blot.The proliferation of GC was detected by CCK8 assay and in vivo.The migration of GC was detected by cell scratch assay.Besides,cell activity of gastric cancer cells treated with 5-fu was detected by MTT assay and in vivo.Results:Part1.Overexpression of YAP1 is associated with 5-Fu resistance and poor prognosis of GCWe first analyzed the mRNA expression of YAP1 in 34 paired GC samples by RT-qPCR.Overexpression of YAP1 was observed 16 out of 32 GC tissue compared to paired normal gastric mucosa(p=0.046).We then carried on YAP1 IHC-P staining in 81 GC tissues.We divided all 81 GC samples into two groups,YAP1high and YAP1low group by the IHC-P score of YAP1.Survival analysis revealed that YAP1high group has lower overall 5-year DFS and OS survival rate than YAP1low group(p<0.005).In vitro,RT-qPCR was performed for the analysis of the mRNA expression of YAP 1 in most GC cells and the results showed that SGC7901 expressed highest and MKN45 expressed lowest of all.In order to rule out the interference of endogenous expression,we select the SGC7901 and MKN45 for the knocking down and overexpress model cells respectively.We designed the siRNA of YAP1 and YAP1 plasmid for the detailed research.RT-PCR and Western blot confirmed the effective of the siRNA of YAP1 and YAP 1 plasmid.To discuss the effect of YAP1 on 5-fu chemotherapy resistance of gastric cancer.The lentivirus-transfected cell lines with stably overexpression of YAP1 were established and the fluorescence showed transfected efficiency.In vivo,growth rate was increased in tumors of MKN45-YAP1 group,but the Ctrl group tumor became more sensitive to the treatment of 5-Fu,when compared.What confused us was that overexpression of YAP 1 in GC cells MKN45,there was no significant difference between MKN45-YAP1 and MKN45-Vector under the treatment of 5-Fu by MTT assay.Part2.GC cell secreted YAP1 stimulates resistance to 5-Fu via inducing M2 subtype macrophage glycometabolism reprogrammingAccumulating evidence suggests that tumor associated macrophages(TAMs)play a crucial role in tumor development,chemotherapy resistance and are potential therapeutic targets for cancers.To validation whether TAMs functions as a potent tumor microenvironment(TME)factor that results in the resistance to the treatment of 5-Fu in GC,we firstly carried on the CD 163 IHC staining by tissue arrays.We compared the outcome of YAP1 and CD163 IHC staining and discovered that YAP1 high expression was correlated with more CD 163 positive macrophage infiltered.In addition,we stimulated the PMA-treated THP-1 cells with the supernatant of MKN45-YAP1 cells and MKN45-Vector cells,the positive correlation between the expression of YAP1 and CD 163 was verified by immunofluorescence staining.Then,in vitro,the supernatant of THP-1 cells co-cultured with MKN45-YAP1 cells was used for MTT assays.The result showed that the MKN45-YAP1 promote the resistance to 5-fu in GC through THP-1.Thus,we hypothesis that YAP1 promotes 5-Fu resistance by mediating tumor associated macrophage M2 polarization and reprogramming.We first demonstrate that YAP1 was secreted from GC cells by detecting the purified protein of serum.Western blot showed that,MKN45-YAP1 secreted more YAP1 proteins than MKN45-Ctrl groups.RT-qPCR confirmed that M1 subtype associated marker iNOS-1,CD80 expressed higher and M2 subtype marker IL10,IL13 and ARG1 were increased in PMA induced THP-1 after co-cultured with MKN45-YAP1 compared to Vector group.This result is due to the plasticity of macrophages.To elucidate the potential mechanism that YAP1 induces 5-Fu resistance by activating M2 subtype macrophage,we analyzed the metabolic difference after MKN45-YAP1 or MKN45-Vector treated THP-1.Surprisingly,we found that glycometabolism was activated including the overexpression of the key enzymes GPI,PGK1,GLUT1,LDHA,PFKFB3 and HK2 in MKN45-YAP1 treated THP1.Besides,the fatty acid metabolism and amino acid metabolism pathway changed no significant.Then,we verified the protein change of glycometabolism associated enzymes by western blot.Therefore,we think that secreted protein YAP 1 could induce the M2 polarization and activating the glycometabolism of macrophage.Part3.Secretary YAP1 activated GLUT3-dependant glycolysis by binding to GLUT3We have disclosed the phenomenon that secretary YAP1 could promote the glycometabolism of macrophages,but the mechanism remains unclear.Co-IP assays found that YAP1 could interact with the membrane expression glucose transporter 3(GLUT3),which could enhance the uptake of glucose from TME.After co-cultured with MKN45-YAP1 cells,THP1 expressed higher GLUT3 according to the IF staining and western blot.In addition,we analyzed the difference in phenotype and glycometabolism between THP-1 cells transfected with GLUT3 plasmid and vector by RT-qPCR.We found that M1 subtype associated marker iNOS-1,CD80,CD86 expressed higher and M2 subtype marker IL10 and CD206 were increased in THP-1 cells transfected with GLUT3 plasmid.Those results indicated that the YAP1 overexpressed GC cells secreted more protein YAP1.Secreted YAP1 could activate the M2 polarization and glycolysis reprogramming by binding to the membrane protein GLUT3 of THP1.The overexpression of GLUT3 increased the uptake of glucose by opening the glucose transporter door.Part4.Activated macrophage induced gastric cancer 5-Fu resistance by increasing CCL8 secretoryFirst,we found the expression of CCL8 in higher in THP-1 cells cultured with the supernatant of MKN45-YAP1 cells compared to vector groups by RT-qPCR.Then,the TIMER tool and RT-qPCR confirmed that GLUT3 expression was positively correlated with CCL8 expression in macrophages.We then use the CCL8 cytokine to treat gastric cancer cells.MTT assay illustrated that gastric cancer cell became more resistance to 5-Fu treatment after the stimulation of CCL8.In addition,we found that CCL8 promoted proliferation and invasion of GC cells in vitro through CCK8 and cell scratch experiment.In order to discuss how CCL8 can promote the resistance to 5-fu in GC cells,we used the GSEA and found that CCL8 may promote the resistance to 5-fu by activating the JAK/STAT signaling pathway.Then,we verified that CCL8 promoted the up-regulation of JAK1 and STAT3 phosphorylation by western blot.Thus,we concluded that YAP1 overexpressed GC upregulated glucose transporter GLUT3 in the membrane of macrophage and promote the M2 polarization and glycometabolism.so that the activated M2 macrophage secreted more CCL8 and induced the 5-Fu resistance by JAK1/STAT3 signal pathway of GC.