Metabolic Enzyme PDK3 Forms A Positive Feedback Loop With Transcription Factor HSF1 To Drive Chemoresistance In Gastric Cancer

Background:Chemotherapy remains the major treatment for most perioperative and advanced cancer patients,especially for cancers with limited targeted therapy options such as gastric cancer.To date,chemoresistance is still the main cause of the treatment failure and unsatisfied overall survival.Dysregulation of glucose metabolism plays an important role in drug resistant cancer cells.Our previous study has shown that glycolysis was enhanced in gastric chemoresistant cancer cells.However,the underlying mechanisms still remain largely unknown.Pyruvate dehydrogenase kinase(PDK)is the key enzyme in mitochondria that determines the fate of pyruvate.It can stimulate glycolysis in cancer cells by inhibiting pyruvate into the TCA cycle via negatively regulating the pyruvate dehydrogenase complex.Our early work showed that Pyruvate dehydrogenase kinase 3(PDK3)is highly expressed in drug resistant cancer cells when gene expression was profiled.However,the regulation of PDK3 gene expression and the role of PDK3 in the chemoresistant cells of gastric cancer hasn’t been reported yet.Objective:The goal of this study is to clarify the role of PDK3 in stimulating glycolysis and the effect on chemoresistance in the drug resistant cells of gastric cancer.We plan to explore the underlying mechanism behind the regulation of PDK3 in the chemoresistantcells.Method:GC-TOF/MS was employed to detect the alternation of glucose metabolism profile in SGC7901,SGC-R,PDK3 and HSF1(Heat shock factor 1)high expression SGC7901 cells,which were confirmed with colorimetry.To identify the association of glycolysis with chemoresistant cells,global gene expression profiles in BGC-R and SGC-R cells as well as their parental chemosensitive cells,BGC823 and SGC7901 was analyzed with Gene Set Enrichment Analysis(GSEA).Changes in chemosensitivity of gastric cancer cells in vitro or in vivo were analyzed by viability,flow cytometry and western blot and nude mice assay after genetic alternation or treated with chemical inhibition.Luciferase reporter assay and ChIP(Chromatin Immunoprecipitation)were applied to identify the transcriptional regulation of HSF1 on PDK3.IP(Immunoprecipitation),confocal microscopy and GST-pull down were performed to explore the underlying mechanism behind the regulation of PDK3 on HSF1.Results:As a result,chemoresistant cells displayed a distinguished glycolysis metabolism profile from the parental SGC7901 cells.GSEA analysis showed that the genes involved in glycolysis were significantly enriched in chemoresistant cells.Among these deregulated glycolysis-associated genes,PDK3 was the most upregulated gene in chemoresistant cells.Knockdown of PDK3 or its chemical inhibition could impaire glycolysis to reverse chemoresistance in vitro and in vivo.In contrast,PDK3 overexpression drives glycolysis to promote chemoresistance in sensitive cells.In chemoresistant cancer cells,both mRNA and protein level of PDK3 were increased.Therefore,we proposed that transcription regulation of PDK3 expression is dominant in chemoresistance.Herein,for the first time we found that PDK3 is a direct target of transcription factor HSF1.Indeed,HSF1 expression was increased in chemoresistant cells and PDH activity was reduced by HSF1 knockdown in chemoresistant cells,which was rescued by PDK3 overexpression.Interestingly,PDK3 can not only stimulate glycolysis by inhibiting the activity of PDH in the mitochondria,but also localize in the nucleus and directly interact with HSF1.The 226-315 region of HSF1 in the regulatory domain is critical to this interaction with PDK3.GSK3β and ERK1 could catalyze the phosphorylation of serine 303 and 307 sites in this region of HSF1,respectively,to promote its interaction with FBXW7.By interacting with this region of HSF1,PDK3 competed with GSK3β to attenuate serine phosphorylation and subsequent FBXW7-dependent polyubiquitination of HSF1.Moreover,the glucose metabolism in HSF1 over-expressing cells displayed a clear distinction from parental cells and resembled SGC-R and PDK3 over-expressing cells.Consistent with previous results in PDK3 over-expressing cells,the high expression of HSF1 can enhance glycolysis to confer chemoresistance.Its genetic or chemical inhibition reverted chemoresistance in vitro and in vivo.Conclusion:PDK3 was increased to promote chemoresistance as a result of HSF1-driven transcription.It can not only stimulate glycolysis,but also localize in the nucleus and directly interact with HSF1 to protect it from FBXW7-dependent polyubiquitination and degradation.Thus,the metabolic enzyme PDK3 and transcription factor HSF1 forms a positive feedback loop to promote glycolysis and chemoresistance.Genetic or chemical inhibition of this feedback loop to impair enhanced glycolysis may represent a novel approach to overcome chemoresistance.