The Role And Mechanism Of MPC1 In The Development Of Lung Adenocarcinoma

With the increasing incidence and mortality of lung cancer in our country,Lung cancer is the largest threaten to people’s health and life.Lung adenocarcinoma is one of the most common pathological types of lung cancer,accounting for about 40%of lung cancer patients.Although the diagnosis and treatment of lung adenocarcinoma have been improved,The survival rates of lung cancer do not change significantly.The main reason is the maintenance of tumor stemness,invasion and metastasis.Therefore,it is necessary for us to explore the molecular mechanism of lung cancer progression for early diagnosis and effective treatment.As we all known,malignant progression of tumors is a complex process which is regulated by multi-molecular and multi-signaling networks.Mitochondrial Pyruvate Carrier1（MPC1）is involved in pyruvate transportation from cytoplasm to mitochondria.Recent Studies have showed that MPC1 is lowly expressed in a variety of tumors,but its relationship with lung adenocarcinoma remains unclear.Our previous studies have indicated that MPC1 expression is low in lung adenocarcinoma,and MPC1 deletion can maintain the stemness,strongly promotes invasion and migration of lung cancer cells.Meanwhile,our preliminary results suggested that MPC1 can form a complex with STAT3,indicating that MPC1 plays a part in the malignant biological behavior of lung adenocarcinoma by regulating the STAT3 signal pathway.Based on this,we speculate that MPC1 and STAT3 co-regulate the occurrence and development of lung adenocarcinoma.Herein,we first examined the expression of M PC1in lung adenocarcinoma tissues and cells;the effect of MPC1 on the invasion and the migration ability of the lung adenocarcinoma cell line is detected by transwell assay.Furthermore,MPC1 is found to inhibit the dry of the lung adenocarcinoma cell;Further study revealed that MPC1 could interact with mitochondrial signal transducer and activator of transcription 3（mito-STAT3）,disrupting the distribution of STAT3 and reducing cytoplasmic signal transducer and activator of transcription 3（cyto-STAT3）as well as its phosphorylation,while the activation of cyto-STAT3 by IL-6 reversed the attenuated malignant progression in MPC1-overexpression LAC cells.main results and conclusions are as follows:1.MPC1 can be used as a good prognostic indicator for patients with lung adenocarcinoma:（1）Abnormal expression of MPC1 in lung adenocarcinoma tissues and cell lines.The mRNA and protein of MPC1 protein in lung adenocarcinoma was significantly lower than that in adjacent mucosa.Furthermore,The expression levels of MPC1 protein and mRNA in lung adenocarcinoma cell lines were remarkable lower than those in normal human bronchial epithelial cells.（2）the overall survivals（OS）of LAC patients with MPC1^low showed shorter than that with MPC1^high in our clinical cohort and GEO database Univariate and multivariate analyses further indicated that MPC1 was an independent prognostic indicator for OS of LAC patients.Therefore,MPC1 might act as an indicator of favorable prognosis for patients with LAC.2.MPC1 is involved in the"stemness"maintenance of lung adenocarcinoma:（1）The sphere formation assay shows that the diameter and number of tumor sphere after silencing MPC1 or using MPC1 inhibitor UK5099 were greatly higher than those in control group.（2）The expression levels of stem cell markers NANOG,OCT4 and SOX2 in lung adenocarcinoma cells were significantly down-regulated after overexpression of MPC1 whearas the expression levels of the above cell stem markers were significantly up-regulated after MPC1 silencing or using MPC1 inhibitor.3.MPC1 deletion can enhance the migration and invasion of LAC cells:（1）Deletion of MPC1 promotes invasion and migration of lung adenocarcinoma cells.Compared with the control group,over-expression of MPC1 can inhibit the invasion and migration of lung adenocarcinoma cells A549 and H1299;while knockdown the MPC1 of H1299 cells or treatment of it with UK5099 can promote the invasion and migration.（2）Immunofluorescence pseudopod formation assay showed that invadopodia positive cells were reduced after MPC1 over-expression,while invadopodia positive cells were increased after MPC1 silencing.Western-blot results showed that the expression of MMP7,MMP3and MMP2 proteins in MPC1 over-expressed cells were down-regulated compared with the control group.After the silencing of MPC1,the expression levels of MMP7,MMP3 and MMP2 proteins were up-regulated compared with the control group.4.MPC1 is involved in the regulation of STAT3 phosphorylation and mitochondrial translocation:（1）Mass spectrometry showed that MPC1 could interact with STAT3.We further use Co-IP experiments to confirm the interaction between them.It was found that MPC1 can interact with mito-STAT3 via mitochondrial separation experiment.（2）overexpression of MPC1 down regulated p-STAT3（Y705）,whereas knockdown of MPC1 up regulated p-STAT3（Y705）,but without affecting p-STAT3（S727）and total STAT3 expression Since cyto-STAT3 was recruited into mitochondrial inner membrane by some chaperone and MPC1 was located in the inner membrane of mitochondrial,we proposed that the binding of MPC1 with STAT3 in mitochondria prevents the process that cyto-STAT3 was transported into mitochondria.Western blotting shows that overexpression of MPC1increased the level of STAT3 anchored on mitochondrion,but decreased the level of STAT3and p-STAT3（Y705）in cytoplasm Cell fractionation analysis demonstrated that overexpression of MPC1 reduced p-STAT3（Y705）in cytoplasm and transport into the nuclei..The axis of MPC1/STAT3 regulates the malignant biological behavior of lung adenocarcinoma cells:（1）IL-6 is an activator of phosphorylated STAT3.We found that the expression of phosphorylated STAT3 was up-regulated with the increase of IL-6 concentration.IL-6treatment could reverse the inhibition of over-expression of MPC1 on the ability of globulation,invasion and migration of lung adenocarcinoma cells.（2）IL-6 could reverse the expression of SOX2 and MMP2 proteins in lung adenocarcinoma cells by over-expression of MPC1.6.In vivo experiments indicated that high expression of MPC1 inhibited progression of lung adenocarcinoma:It was found that over-expression of MPC1 could significantly inhibit the tumorigenicity of the cells,and the volume and weight of the tumor decreased significantly.Immunohistochemical staining showed that the expression of phosphorylated STAT3,SOX2 and MMP2 was significantly down-regulated in lung adenocarcinoma xenografts with over-expression of MPC1.In summary,MPC1 is significantly low expressed in lung adenocarcinoma tissues and cells.Over-expression of MPC1 can inhibit the ability of lung adenocarcinoma to invasive,migration and tumor sphere formation assay.Interaction between MPC1 and STAT3 inhibits STAT3 activation and nuclear translocation in cytoplasm,promote mitochondrial translocation of STAT3.In addition,over-expression of MPC1 can also inhibit the tumorigenic ability of lung adenocarcinoma in vivo.