The Role And Mechanism Of Mitochondrial Pyruvate Carrier 1 In The Progression Of Renal Carcinoma Cell

Renal cell carcinoma?RCC?represents 90%-95%of kidney cancer.Although the mortality rate of RCC has been decreasing due to the progression in nephrectomy,immunotherapy and target therapy,invasion and subsequent metastasis are still inevitable in25%-30%patients with RCC and finally lead to the death of patients.Therefore,clarifying the mechanisms on invasion of RCC is critical for the development of novel therapeutic strategies.Accumulating evidence reveals pivotal roles of aberrant mitochondrial metabolism in tumorigenesis,and key molecular associated with mitochondrial metabolism are tightly involved in the tumor progression.Mitochondrial pyruvate carrier?MPC?,including two members,MPC1 and MPC2,is originally described as gatekeeper responsible for pyruvate transportation and oxidation in mitochondrial,and cancer-related functions of MPC are also identified in several types of cancer.For example,depletion of MPC or blocking its activity by specific inhibitor results in aerobic glycolysis and aggressive growth of tumor cells.Decreased expression of MPC protein is correlated with poor survival of patients with various types of cancer,including colon cancer,prostate cancer and esophageal cancer.Reversely,overexpression of MPC in colorectal cancer cell lines depresses tumorigenicity of subcutaneously inoculated xenografts in mouse model.Moreover,in colon cancer and esophageal cancer,MPC suppresses the Warburg effect,which is one of the most important features in cancer cells.Interestingly,a recent work by Ohashi et al showed that MPC1 is an indicator for favorable prognosis in intrahepatic cholangiocarcinoma and inhibits invasion and distant metastasis.Taken together,these data indicate that MPC might function as tumor suppressor through the regulation on metabolism and epithelial-mesenchymal transition?EMT?.Despite the potential involvement of MPC in cancer invasion and metastasis,the clinical relevance and functional mechanism of MPC in RCC remain to be elucidated.In this study,therefore,we investigated the expression of MPC in human RCC and adjacent non-tumor tissues.Our data suggested that the expression of MPC1,but not MPC2,was downregulated in RCC tissues compared with paired non-tumor tissues,and negatively associated with tumor size,advanced lymph node metastasis,and overall survival of patients.Additionally,knockdown of MPC1 resulted in the increased migration and invasion of RCC cells,which was further confirmed with inhibitor targeting MPC.In contrast,overexpression of MPC1 inhibited the migration,invasion,and tumorigenesis of human RCC cells in xenograft models.Mechanistically,we observed that MPC1 expression impaired the mobility of RCC cells potentially through inhibiting Hypoxia Inducible Factor1 Alpha Subunit?HIF1??.Thus,our data implied that MPC1 might control the progression of RCC and acts as a novel prognostic indicator for human RCC.The main methods,results and conclusions of this study are showed as follows:1.MPC1 was identified as a poteintial target in RCC among mitochondrial biomarkers.We detected the gene expression of mitochondrial markers?n=40?on the kidney cancer tissue and normal tissue from GDS605 and selected four genes?MPC1,TOMM6,TOMM7 and CYCS?.qRT-PCR were used to detect the expression of these four genes in10 RCC and corrponding normal tissues.The results indicated that MPC1 may be involved in the regulation of progression of RCC cells.2.Clinical relavance of MPC1 expression in RCC.?1?MPC1 is lowly expressed in RCC tissue compared with corrponding normal tissue.The Western blotting were applied to detect the expression of MPC1 and MPC2 in 12paired RCC tissues.The result indicated that the expression of MPC1 was downregulated in RCC tissues.However,we did not find obvious difference of MPC2 expression in RCC tissues versus adjacent non-tumor tissues.IHC staining were also performed to detect the expression of MPC1 in 150 RCC tissue sample and 30 paired adjacent normal tissues.The data indicated that the proportion of MPC1^low cells in RCC tissues?60.67%,91/150?was markedly higher than that in corresponding para-cancer tissues?33.33%,10/30?.?2?The relationship between MPC1 level and clinical pathological parameters.The cutoff value of the IHC score was determined by relative risk analysis with the statistical software X-tile.These cut off value was used to divide patients with MPC1^low and with MPC1^high.Chi-square test showed that the expression level of MPC1 was negatively correlated with TNM stage?P=0.010?;age?P=0.001?;TNM stage?P=0.010?.However,the expression of MPC1 was not related with gender?P=0.254?;location?P=0.950?;lymph node metastasis?P=0.830?and histological grade?P=0.628?.?3?The relationship between MPC1 expression and prognosis of RCC.Kaplan-Meier survival analysis using Cohort-150 showed significantly shorter overall survival?OS?for the patients with MPC1^low RCC than those with MPC1^high RCC?P=0.0016?.Moreover,survival analysis on 164 RCC patients from the TCGA-database consistently indicated that low MPC1 expression was associated with poor OS?P=0.0128?.Univariate and multivariate analyses further indicated that low MPC1 expression was an independent prognostic indicator?P=0.046;P=0.014?for OS of RCC patients.3.MPC1 deficiency promotes tumor progression in RCC.?1?Knockdown or overexpression of MPC1 markedly changed the invasiveness and mobility of RCC cells.We established MPC1-depleted and overexpressed 786-O cells.Transwell assay with or without Matrigel showed that loss of MPC1 increased the migration and invasion of RCC cells.In addition,treatment with MPC inhibitor,UK5099 enhanced cell migration and invasion compared with vehicle treatment.In contrast,overexpression of MPC1significantly impaired the migration and invasion of RCC cells.?2?Matrix metalloprotein,but not EMT,is affected by MPC1 in RCC cells.We first examined the two EMT markers,Vimentin and E-Cadherin in RCC cells.However,neither of the two genes were obviously altered under MPC1 depletion,MPC inhibitor treatment or MPC1 overexpression.we also evaluated the expression of MMPs.We observed that the mRNA and protein of MMP7 and MMP9 were significantly upregulated in 786-O cells transfected with si MPC1 versus siCtrl.Consistently,MPC inhibitor,UK5099 treatment also induced the expression of MMP7 and MMP9 in RCC cells.Reversely,RCC cells with forced expression of MPC1 suppressed the expression of MMP7 and MMP9.?3?MPC1 inhibits tumor growth by preventing cell in G2 stage.CCK-8 assay was applied to evaluate the growth in MPC1-depleted and overexpressed786-O cells.We found that abrrant MPC1 decreased tumor cell growth.Furthermore,the percentage of G2 stage of 786-O cell with abrrant MPC1 was higher than that of786-O-Mock cell,suggesting MPC1 prevents tumor growth via regulating cell cycle.4.MPC1 is involved in tumor intitation and progression in vivo.To further examine the effect of MPC1 on RCC cells in vivo,RCC cell line?786-O?with overexpression or knockdown of MPC1 were subcutaneously transplanted into mice which were sacrificed 30 days after implantation.The average weight and volume of tumors formed by RCC cells with MPC1 knockdown were larger than those formed by RCC cells with MPC1 overexpression.Moreover,IHC staining showed that the expression of MMP7 decreased in tumors derived from overexpressed-MPC1 RCC cells compared with those derived from Mock cells.Therefore,the in vivo data further demonstrated that MPC1 expression is negatively associated with the growth of RCC cells5.MPC1 is involved in hypoxia/HIF1?axis in RCC cells.1.Hypoxia is frequently detected in RCC and leaded to increased migration and invasion of RCC cells.In addition,the proteins in mitochondrial are highly sensitive to hypoxia.Accordingly,we speculated that MPC1 might be responsive to hypoxia.As expected,CoCl₂ treatment or culture under 1%O₂,which are two ways to mimic hypoxia condition reduced the expression of MPC1.We found MPC1 is lowly expressed in normal condition as compared with that in hypoxia,suggesting that MPC1 may be involved in hypoxia-related pathway,which mediated RCC progression.2.It has been well-known that HIF1?is a crucial regulator in hypoxia and is linked to the development and progression of RCC.Therefore,we also investigated the relationship between MPC1 and HIF1?.Interestingly,we observed that suppression of MPC1expression leaded to increase of HIF1?expression.Overexpression of MPC1 leaded to the decrease of HIF1?.Moreover,IHC staining were used to evaluate MPC1 expression and HIF1?expression from Cohort-150.We found that the expression of HIF1?in MPC1^lowow RCC cells was higher than that in MPC1^high RCC cells,which were further confirmed using TCGA database.Altogether,MPC1 might be a novel upstream regulator for HIF1?in RCC cells.Conclusions1.MPC1 is lowly expressed in tumor tissue comared with that in corresponding normal tissue among the mitochondrial biomarker set.2.Low expression of MPC1 is asscociated with tumor progression and poor prognosis.The expression of MPC1 is negatively related with tumor invasion,TNM staging of RCC and OS of the patients.MPC1 is an independent prognostic factor of RCC.3.MPC1 deficiency promotes tumor progression in vivo and in vitro.MPC1 was not only decreased the mobility of RCC by MMP pathway,but also decrease the proliferation of the cell by preventing cell in G2 stage.4.MPC1/HIF1?axis was of importance in the progression of RCC.Overexpression of MPC1 decreased HIF1?expression,while knockdown or treateing with MPC inhibitor increased HIF1?expression in RCC cell.