| BackgroundGastric cancer is one of the most common malignant tumors of digestive system in the world at present.The clinical efficacy and survival prognosis of patients with gastric cancer are poor.According to the latest epidemiological data published by GLOBOCAN,in 2018,there were about 1.033 million new cases of gastric cancer and about 783,000 cases of death in the world.The data showed that the incidence and mortality of gastric cancer were in the fifth and second place of all kinds of cancer respectively.China is a country with a high incidence of gastric cancer.According to the data of the National Cancer Registry,the number of gastric cancer cases and deaths in China accounts for about half of the world.Clinical data showed that the main cause of death in 60%of patients with advanced gastric cancer was metastasis.Compared with other types of cancer,gastric cancer was more likely to develop peritoneal implants metastasis,in which the incidence of omental metastasis was about 50%,and the 5-year survival rate was less than 10%.Therefore,omental metastasis is an important cause of poor survival and prognosis in patients with gastric cancer.At present,the underlying mechanism of omental metastasis is still unclear,and most of the basic researches focus on the invasion phenotype.Therefore,it is important to study the mechanism of omental metastasis for improving the survival and prognosis of gastric cancer patients.Fatty acid oxidation is one of the most important energy sources for tumor cells.In order to maintain its own proliferation,tumor cells promote fatty acid oxidation by increasing lipid uptake and fatty acid synthesis.Fatty acids as a substrate for energy metabolism have two main forms including exogenous uptake and endogenous synthesis,such as the way in which human normal cells obtain fatty acids mainly through exogenous intake of food.Cancer cells,on the other hand,tend to use the endogenous form of denovo synthesis to obtain fatty acids.Previous studies have shown that adipocytes in tumor microenvironment can play a key role in promoting the reprogramming of lipid metabolism of cancer cells and improving the proliferation and metastasis of tumor cells.It has been reported that gastric cancer cells will detach from the primary extracellular matrix before forming distant metastasis.In the process of metastasis,gastric cancer cells need to resist anoikis in order to colonize in the omentum finally.The results show that tumor microenvironment is an important factor affecting the metastasis of cancer cells,which is mainly composed of different tumor cells and many kinds of stromal cells,cytokines and chemokines.The omentum contains a large number of adipocytes,nerves and blood vessels.Previous studies have proved that adipocytes in the omentxun activate fatty acid oxidation pathway by supplying fatty acids to tumor cells.Furthermore,adipocytes promote the reprogramming of lipid metabolism of tumor cells and finally induce colonization of tumor cells to form a new metastasis of omentum.Phosphatidylinositol transfer protein,cytoplasmic 1(PITPNC1)is a member of the class II PITP family that promotes the transfer of phosphatidylinositol and phosphatidylinositol in the endocytic system,which is related to lipid metabolism.There are few reports about PITPNC1 in the field of cancer research.Previous studies have found that PITPNC1 can be located in Golgi complex and recruit RAB1B to promote the malignant secretion of metastasis factors,and it is highly expressed in metastatic breast cancer,melanoma,colon cancer and other different types of cancer.Therefore,we speculate that PITPNC1 may mediate adipocytes to promote lipid metabolism reprogramming of gastric cancer cells and become a new key molecular target for promoting omental metastasis of gastric cancer.Targeting PITPNC1 may provide a new strategy for clinical treatment of gastric cancer omental metastasis.Research contents1.Expression of PITPNC1 in primary gastric cancer and omental metastasis as well as its relationship with clinical efficacyWe collected pathological specimens of 126 cases of gastric cancer patients with omental metastasis including primary gastric tumors,paracancerous tissues and omental metastases from multiple clinical centers(Nanfang Hospital,Zhujiang Hospital,Nanjing General Hospital,Canton Medical Second Hospital,People's Hospital of Nanhai).The expression of PITPNC1 in primary and omental metastases of gastric cancer was analyzed by immunohistochemistry,and the correlation between the incidence of omental metastasis and the expression of PITPNC1 was analyzed.At the same time,we collected 6 fresh gastric cancer specimens,and further verified the difference of PITPNC1 expression in paracancerous tissues,primary tumors and omental metastases by Western blot assay.We collected blood samples from patients with advanced gastric cancer in Department of Oncology from Nanfang Hospital before and after treatment,and detected the difference of PITPNC1 expression before and after treatment by Real-Time PCR assay.We provide written consent for all patients and inform them of the details about the study,and this study was reviewed by the Nanfang Hospital Ethics Committee(Guangzhou).At last,the role of PITPNC1 in promoting omental metastasis was further verified by constructing a nude mouse model of omental metastasis in vivo.2.PITPNC1 promote fatty acid ?-oxidation pathway to mediate the adhesion of gastric cancer cells and omental metastasisFirstly,AGS and BGC823 stably transformed cell lines with overexpression and silencing of PITPNC1 were constructed,and the effects of overexpression and silencing of PITPNC1 on adhesion and oxidative phosphorylation(OXPHOS)of gastric cancer cells were detected.The changes of E-Cadherin and apoptotic index of gastric cancer cell after overexpression and silencing of PITPNC1 were determined by Western blot.Then,the fatty acid oxidation inhibitor(Etomoxir,ETX)was used to treat gastric cancer cells in control group and overexpression PITPNC1 group to observe whether Etomoxir could reverse the increase of cell adhesion induced by overexpression of PITPNC1,and it was determined whether PITPNC1 could increase the cell adhesion ability of the gastric cancer by the fatty acid oxidation pathway.Finally,the omental metastasis model of gastric cancer in nude mice was constructed by over-expression of PITPNC1,and the number of omental metastasis in nude mice was observed by Etomoxir treatment.3.Adipocytes enhance adhesion ability of gastric cancer cells through fatty acid?-oxidation pathwayTo determine how adipocytes affect tumor cells,we selected three pairs of primary gastric cancer and corresponding omental metastasis for RNA sequencing,and used Gene Set Enrichment Analysis to determine the relevant signaling pathways.Two kinds of gastric cancer cell lines,AGS and BGC823,were co-cultured with adipocytes by Transwell system,and then the suspended gastric cancer cells were inoculated on 96 well plates coated with Matrigel.The adhesion of gastric cancer cells was observed by crystal violet staining and MTT assay.At the same time,the expression of adhesion molecule E-Cadherin and fatty acid oxidation key enzyme CPTIB in gastric cancer cells was detected by Western blot assay after co-culture with adipocytes.The effect of coculture of adipocytes on basal respiration rate and maximal respiration rate of gastric cancer cells was determined by oxygen consumption rate.And then,AGS and BGC823 gastric cancer cells were treated with fatty acid P-oxidation inhibitor Etomoxir,and the adhesion of gastric cancer cells was observed by crystal violet staining and MTT method.Results1.PITPNC1 promotes omental metastasis of gastric cancerA total of 126 paraffin specimens of gastric cancer patients from various clinical centers were collected,and all 126 patients had matched specimens of omental metastases.The results of IHC of 126 specimens showed that the expression of PITPNC1 in omental metastases was higher in 71.4%of specimens than in primary tissues.The expression of PITPNC1 in omental metastases was similar to that in primary tissues in 30 cases(23.8%).The expression of PITPNC1 in primary tumors was lower than that in omental metastases in 4.8%of all specimens.The above results suggested that the patients with high expression of PITPNC1 in primary gastric tumor had a higher probability of omental metastasis.In addition,fresh specimens of primary tissues,paracancerous tissues and omental metastases were collected from 6 patients with gastric cancer.Western blot results showed that the expression of PITPNC1 was significantly higher in primary tumor tissues than in paracancerous tissues.The expression of PITPNC1 in omental metastases was significantly higher than that in primary tumors,which was consistent with the immunohistochemical results.Finally,we collected blood samples from 12 patients with advanced gastric cancer and used Real-Time PCR assay to verify the difference of PITPNC1 expression before and after treatment.The results showed that the expression of PITPNC1 in gastric cancer patients decreased significantly after chemotherapy.By constructing a nude mouse model of omental metastasis in vivo,it was found that the tumors in the overexpressed PITPNC1 group had stronger fluorescence.In conclusion,we found that the high expression of PITPNC1 in gastric carcinoma may be a relative index to predict the therapeutic effect of chemotherapy for gastric cancer,and the high expression of PITPNC1 may be related to the increase of omental metastasis of gastric cancer.2.PITPNC1 promote fatty acid P-oxidation pathway to mediate the adhesion of gastric cancer cells.Etomoxir can reduce the adhesion ability of gastric cancer cells,but overexpression of PITPNC1 can reverse this situation.Overexpression of PITPNC1 can make gastric cancer cells have higher basic respiration rate and maximum respiration rate,and increase the level of ATP in gastric cancer cells.The inhibitory effect of Etomoxir on oxidative phosphorylated was partially reversed.Western blot results showed that silencing PITPNC1 could decrease the expression of E-Cadherin and promote cell apoptosis in gastric cancer cells,but the reverse result was obtained after overexpression of PITPNC1.After adding Etomoxir,the fatty acid ?-oxidation was inhibited,while the expression of E-Cadherin decreased and apoptosis increased.Finally,by silencing PITPNC1,we found that the adhesion ability of gastric cancer cells was decreased,but the phenomenon could be partially reversed after co-culture with adipocytes.Finally,the use of Etomoxir to treat omental metastasis in nude mice showed that Etomoxir significantly reduced the number of tumor nodules in the PITPNC1 overexpression group.3.Adipocytes enhance Adhesion ability of gastric Cancer cells through Fatty Acid p-Oxidation Pathway.Gene set enrichment analysis identified two signaling pathways associated with TNF-? and IL-6.Western blot and qPCR results indicated Both TNF-? and IL-6 up-regulated the expression of PITPNC1 in gastric cancer cells.Gastric cancer cells AGS and BGC823 were co-cultured with adipocytes.The suspended gastric cancer cells were inoculated on the Matrigel coated plate.The adhesion of gastric cancer cells was observed by crystal violet staining and MTT method.The results showed that the adhesion ability of gastric cancer cells was significantly enhanced after co-culture.Western blot results showed that the expression of E-Cadherin and CPT1B in AGS and BGC823 gastric cancer cells increased after co-culture with adipocytes.The results of oxygen consumption test showed that adipocytes could promote the basic respiration rate and maximal respiration rate of gastric cancer cells.It was found that Etomoxir could inhibit the adhesion ability of gastric cancer cells treated with Etomoxir by fatty acid ?-oxidation,but the phenomenon could be reversed by co-culture with adipocytes.These results suggest that adipocytes can promote the adhesion of gastric cancer cells through fatty acid ?-oxidation pathway.Conclusion1.PITPNC1 can promote omental metastasis of gastric cancer and can be used as a predictor of clinical efficacy.2.PITPNC1 promotes cell adhesion and omental metastasis by activating fatty acid?-oxidation in gastric cancer cells.3.Adipocytes activate AKT by secreting TNF-? and IL-6,and promote the up-regulation of PITPNC1 to mediate the adhesion of gastric cancer cells. |
PDF Full Text Request