PO-197 A possible link between the mitochondrial gatekeeper pyruvate dehydrogenase enzyme complex and EMT

Abstract

Introduction In recent years, mitochondria have attracted much attention in metastatic cancer research. The first finding of the importance of mitochondria in cancer was reported by Otto Warburg in 1920. In the following years,it was found that cancer is closely related to mitochondrial characteristics such as glucose metabolism and chemoresistance. In this context,epithelial mesenchymal transition (EMT), due to its potential of being a molecular marker for tumour metastasis, clarification of this process is essential. Although increments have been made in metastatic cancer research,there are no studies showing the relationship between pyruvate dehydrogenase enzyme complex (PDH) and EMT. Material and methods To inhibit PDH activity, pharmacological (PDH inhibitor, Cpi-613) and genomic approaches (stable cell lines were established by lentivirus) were used in A549 cells. The antiproliferative effect of Cpi-613 was investigated by xCELLigence System, SRB and ATP viability assays. After determining anti-proliferative doses of Cpi-613 and/or getting monoclonal cell lines (shCtrl and shPDHA1); wound healing, invasion and drug sensitivity tests were also applied. To demonstrate the EMT phenotype in cells, EMT-related protein expressions were analysed via western blotting in cell fractionations. Genes that regulate the activity of PDH, were also checked by qRt-PCR. Cell cycle specifity of Cpi-613 was also investigated. Glycolysis and/or survival-related proteins and cancer stem cell characteristics were also determined.SB431542 (TGF-βRI inhibitor) was also used to determine Cpi-613 induced EMT depends on TGF-β signalling. Results and discussions Inhibition of PDH by Cpi-613 and PDHA1 knockdown induced morphological changes which are characteristics of EMT. A more rapid wound healing, increased invasive potential and cemoresistance were also shown. The effect of Cpi-613 was non-specific for cell cycle. The possible inactivating kinase of PDH was found to be PDK4. SB431542 treatment also reversed the EMT phenotype. In shPDHA1 cells the expressions of GLUT1; MDR1 and MRP1; AKT, PI3K, MAPK; and OCT-4 proteins found to be increased. The proportion of CD133 + cells were also higher in shPDHA1 cells than those of shCtrl cells. Conclusion Knockdown of PDHA1 expression or inhibition of PDH activity induced the EMT phenotype and more importantly resulted in resistance to anticancer drugs. The induction of PDH enzyme activity could be a novel approach for the development of anti-metastatic cancer drugs.