Molecular Mechanism For CD147-mediated Glucose Metabolic Reprogramming In Hepatocellular Carcinoma Cells

Cancer cells exhibit aberrant glucose metabolism (also named metabolic reprogramming),which plays important roles in tumorigenesis and progression. The molecular mechanismsunderlying metabolic reprogramming of cancer cells are complex and become a hot fieldin cancer rearch. However, most previous studies related to tumor metabolism havemainly focused on the intracellular signal transduction, payed no more attention on themembrane protein. In previous study, we have demonstrated that CD147, a typicalmembrane protein, is highly expressed on hepatocellular carcinoma (HCC), and palys acritical role in promoting glucose uptake and lactate export in HCC cells, suggesting that itmaybe a key molecule invovled in tumor metabolic reprogramming. Therefore, based onour previous understanding on the CD147and HCC,we aim to elucidate the biologicalfunctions of CD147in glycolysis, oxidative phosphorylation of HCC cells by usingCD147-knockout HCC cell and mouse model, thus to comprehensively explain themechanisms of CD147in the regulation of metabolic reprogramming in HCC cells. Thisproject has facililated the understanding on molecular pathology of HCC and provide newpotential targets for HCC treatment.Part1: CD147enhances the glycolysis in HCC cells To systematically investigate the role of CD147in the regulation of glycolysis in HCCcells, we used SMMC-7721cell lines differing only in their CD147status. To rule out thepossibility that our results are specific to SMMC-7721cells, HepG2/CD147-sh cell line,in which CD147expression was stably reduced by shRNA, was established in this study.In vitro assays showed that CD147-WT HCC cells exhibited a significantly higher glucoseuptake, lactate secretion and LDH activity when compared with CD147silencing cells. Asour expected, the pH value in culture medium of CD147-KO and CD147-sh cells wasobviously higher than that in culture medium of CD147-WT cells. In accordance with theregulatory function of CD147on glycolysis, knockout or knockdown of CD147expression was accompanied by a significant drop in intracellular concentration of glucoseand pyruvate. In comparision, silencing of CD147resulted in an increase of intracellularlactate concentration, consistent with a facilitory role of CD147in export of lactate.Moreover, an in vivo assay was also performed based on the nude mice modeltransplanted with CD147-WT, CD147-KO, CD147-R and CD147-sh cells. In line with ourin vitro studies, xenograft tissues developed by CD147-KO cells exhibited a significantlydecreased ability of18F-FDG uptake.Part2: CD147inhibits mitochondrial respiratory function in HCC CellsFour biggest microarray series of primary human HCC were downloaded from NCBIGEO database. we then compared the expression profile of CD147with that of1013mitochondrial nuclear-encoded genes (MNEGs) derived from the GEO database. Ofinterest, a notably strong bias was found for negative correlation of CD147with MNEGs.Furthermore, knockout or knockdown of CD147in HCC cells resulted in a significantincrease in mtDNA content. And also CD147silencing HCC cells have an obviousproliferation of mitochondria when compared with the control cells. Consistent with theresults from HCC cell lines, the level of HSP60(a mitochondrial marker protein) is alsosignificantly inversely correlated with CD147in HCC tissues. We then furtherdemonstrated that, At37degrees C the CD147silencing HCC cells exhibited significanthigher oxygen consumption rate than wildtype cells. Measurement of electron transport complex Ⅳ activity in HCC cells also revealed a significant difference between CD147wildtype and CD147silencing cells. Consistent with the function of CD147in theregulation of mitochondrial biogenesis, CD147extinction was accompanied by asignificant increase in citrate, succinate and fumatate. Taken together, these data illustratedthat overexrpession of CD147decreases the respiratory function of mitochondria byinhibiting mitochondrial biogenesis in HCC cells.Part3: the mechanism underlying the regulatory role of CD147in glucose metabolicreprogrammingTo gain further mechanistic insight into CD147-mediated reprogramming of glucosemetabolism, transcriptomic analysis was conducted using RNA isolated from CD147-WTand CD47-KO SMMC-7721cells. As our expected, of the nine gene sets mostsignificantly exchanged in CD147KO cells, three were independently defined as gene setsinvolvded in glucose metabolism. We then further demonstrated that, the reprogrammedglucose metabolism in HCC cells is triggered by loss of CD147expression and theconsequent dysregulated expression and location of MCT1, which leads to the inhibitionof PI3K/Akt/MDM2pathway by suppressing lactate export and decreased p53degradation by the proteasome. The resulting p53-dependent loss of Glut1and inhibitionof PFK are responsible for the reduction of glycolysis. On the contrary, the p53-dependentincrease of PGC-1α, TFAM and P53R2are responsible for the induction in mitochondrialmass in CD147-silencing hepatocellular carcinoma cells.