Glucose Metabolism Activates Hsf1via MTOR Signal Transduction Pathway In Hepatoma Cells

BackgroundHepatocyte carcinoma is the third highest incidence and the great mortality cancer in China. To figureout the molecular mechanism underlying the HCC will be significant for development of the therapeuticdrugs, early prevention, diagnosis and therapy.Tumor cells uptake a pound of glucose to keep their high–speed growth and metastasis. The tumorcells metabolize the glucose dominantly by the glycolysis to provides not only ATP, but also theintermediates and low concentration of superoxides, which are essential for synthesis of amino acids, riboseand tumor cell unlimited growth and metastasis. This phenomenon is known as Warburg effect. In addition,the tumor cells need to synthesize a lot of proteins for its unlimited proliferation, which usually activate thetumor cells’ protein quality control response and heat shock response to refold the neonatal proteinsstructure and function.However,it is unclear whether the glucose metabolism has any regulatory associationwith the heat shock response.Heat shock factor1-mediated heat shock response has been found to be activated in most of tumortissues, which are characterized by upregulating the expression of a number of Heat shock proteins Hsp90,Hsp70, Hsp40, Hsp60and small heat shock proteins(Hsp27,alpha B-crystallin).These heat shock proteinswork as molecular chaperone to refold the neonatal or denatured proteins and participate in many cellularfunctional regulations e.g. anti-apoptosis, signal transduction, cell cycles and cell differentiation.Knockdown Hsf1by siRNA was found to inhibit tumor cells’ growth in vitro. In mouse models knockdownhsf1gene can suppress the DEN-induced hepatocyte carcinoma, p53-mutation-induced lymphoma andErbB2-mutation-induced breast cancer. These results suggest activation of Hsf1transcription activity isessential for tumor cell initiation and development, and Hsf1could be considered as a novel tumortherapeutic target. However, the molecular mechanism that regulates Hsf1activation in tumor tissues is stillunclear. It is known that Hsf1can convert from non-active monomer to the active homotrimmercompanying with hyperphosphorylation, sumoylation and nuclear translation, where the active Hsf1bindsto the HSE elements in the promoter of targeting genes e.g. heat shock proteins. Map-kinases, GSK-3b, AMPK and mTOR have been reported to be involved in activating Hsf1by phosphorylation. AMPK andmTOR are two critical kinases that can sense the nutrient-deficiency and growth signal, and control themetabolism of glucose and protein synthesis. They are activated in most of tumor tissues. It is reported thatthe ways of activation of Hsf1are different from heat shock and tumor. mTOR is found to be an importantkinase that can mediate the tumor stress signal to activate Hsf1by phosphorylation of Serine326(ser326).On the other hand Hsf1is found to be involved in the regulation of insulin pathway. Knockdown Hsf1canupregulate the insulin receptor expression and reduce that glucose tolerance suggesting there is associationbetween Hsf1and glucose metabolism. According to these data we hypothesize that at the physiologicalcondition, glucose metabolism can activate the heat shock response, which then synergistically facilitatetumor cells unlimited growth and protect tumor cells from intrinsic stresses. mTOR, which is regulated bythe glucose, participates in the glucose-metabolism-mediated Hsf1activation.With HCC cell lines andmouse model our data demonstrate that glucose metabolism can activate Hsf1by increasing Hsf1/ser326phosphorylation and upregulate Hsf1-controlled expression of Hsp70and alpha B-crystallin. mTOR,whichis activated by Glucose, is responsive for the phosphorylation of Hsf1/ser326.AimsTo test our hypothesis that glucose metabolism can induce the Hsf1-mediated heat shock responseactivation by up-regulating mTOR pathway in HCC cells.Methods1. The hepatocyte carcinoma cells(SMMC-7721and plc/prf/5) and immortalized liver cell line(Changeliver) were pretreated in the glucose-free DMEM for24h and then recovered in the completemedium containing30mM glucose or2-deoxy-D-glucose for0h、1h、3h、6h、12h. The treated cells weresubjected to the rest of experiments. To determine whether mTOR is involved in glucose-mediated Hsf1activation, the cells that were pretreated with glucose-free media were recovered in the complete mediacontaining sham or mTOR inhibitor Rapamycin. The expression of Hsf1and heat shock proteins wereexaminated with immunoblotting and Realtime PCR. The Hsf1,ps326-hsf1, phospho-mTOR,phospho-p70s6K and phospho-AKT/473and actin were immunoblotted with their correspondingantibodies. 2. C57BL/6J mice were fast for24hours and then feed with water containing10%glucose for6hours.The ps326-Hsf1, Hsf1and Hsp70were immunoblotted.3. Luciferase assay: the Hsp70promoter-luciferase reporter were applied to test the activation of Hsf1in glucose treatment.Results1. The phosphorylation of Hsf1/ser326, which was reduced in the cells that were treated withglucose-free media, was upregulated when the glucose-free-medium-treated cells were refeeding withmedia containing high concentration of glucose in both SMMC-7721and plc/prf/5cells. The proteinexpression of Hsf1,Hsp70and Hsp90does not change. In contrast, the Hsp27and alpha B-crystallin,whichwere down regulated in the glucose-free media, were upregulated in the cell that were treated with recoverymedia (containing the30mM glucose). Replacement of glucose with2-deoxy-D-glucose in the recoverymedia could not induce these effects in plc/prf/5cells. Real-time PCR results indicate that the mRNAexpression of Hsf1and Hsp70was induced at6hours recovery treatment of glucose-medium but not at12hs in the plc/prf/5cells. The luciferase assay indicates that Hsp70promoter activity is significantupregulated in the cells treated with recovery media for12hs comparing to the24hs glucose-free mediumtreatment.However,in contrast to the SMMC-7721and plc/prf/5cells, The phosphorylation of Hsf1/S326and the protein expression of Hsf1, Hsp70,Hsp27and alpha B-crystallin are not affected by the glucoseconcentration in the immortalized human liver cells(Changeliver cells).2. The phosphorylation of mTOR/Ser2481and p70s6K/Thr389, which is downregulated in the cellstreated with glucose-free media, is upregulated in the cells treated with the glucose-recovery media. ThemTOR inhibitor Rapamycin can inhibit the glucose-induced the phosphorylation of mTOR and p70s6K andHsf1/S326, and downregulate the expression of alpha B-crystallin and Hsp70promoter activity.3. Fast and refeeding glucose to the mice can activate the expression of hsf1and Hsp70in mouse livercompared to the mice with normal feeding.Conclusions1. Glucose metabolism can activate Hsf1by inducing Hsf1/ser326phosphorylation leading toupregulation of the expression of alpha B-crystallin. 2. In plc/prf/5and SMMC-7721cells and normal mouse liver, through the activation of mTORpathway,glucose activate the phosphorylation of Hsf1/ser326, which is involved in the maintenance oftranscriptional activity of Hsf1in tumor cells.2-deoxy-D-glucose cannot activate the Hsf1. Presumably,glucose may activate mTOR and Hsf1through its metabolites.3. Glucose metabolism are involved in regulation of the heat shock response mediated by Hsf1.Presumably, the heat shock response is involved in the intracellular stability of HCC cells.