Glycolytic Enzyme PDK1 Promotes Breast Cancer Stem Cell Properties Under Hypoxia

1.BackgroundBreast cancer is the highest incidence of cancer in women.In recent years,as the basic research of breast cancer translation to clinical therapy,the effect of breast cancer treatment has been greatly improved,but drug-resistance,recurrence and metastasis during breast cancer treatment are increasingly serious.In many tumors,including breast cancer,a subpopulation of self-renewal and multi-directional differentiation potential tumor cells,which could initiate or reconstructs the phenotype of tumor tissue,are called as cancer stem cells.Numerous studies have shown that cancer stem cells participate in the process of tumor metastasis,recurrence and chemoradiotherapy.Therefore,the therapeutic strategy of targeting cancer stem cells will bring new orientation to the treatment of cancer.However,as cancer stem cells possessed different subgroups and displayed high plasticity and dynamics,there is still no effective target and intervention mechanism for the treatment of cancer stem cells.PDK1(pyruvate dehydrogenase kinase 1)can phosphorylate the pyruvate dehydrogenase(PDH)E1 alpha subunit and inactivates the PDH enzyme complex,which converts the pyruvate into acetyl coenzyme A,leading to the inhibition of pyruvate oxidation via tricarboxylic acid cycle.Recent studies have shown that PDK1 are highly expressed in a variety of malignancies,including breast cancer.As an important glycolytic enzyme,PDK1 is widely involved in the cell metabolism and plays an important role in tumor progression and reprogramming of stem cells.Previous studies showed that PDK1is associated with tumor cell proliferation,metastasis,radio-and chemo-resistance and poor prognosis.However,the mechanisms of PDK1 in regulating tumor progression,especially modulating cancer stem cell maintenance remain exclusive,which need to be further explored.2.Methods(1)(1)The key glycolytic enzymes in the glucose metabolic pathway were compared between the CD44~+/CD24~-and CD44~-/CD24~+subpopulations from genome mRNA expression profiling data(GSE15192).(2)Cancer stem cell(CSC)populations in MDA-MB-231 cells were enriched by sphere formation assay and ALDH~+cell sorting,and compared the mRNA levels of PDK1 and other glycolytic enzymes in CSC with non-CSC by RT-qPCR;the protein level of PDK1 of breast tumor tissues and their adjacent tissues was detected by Western blotting.(3)The ratio of ALDH~+cell population was detected by flow cytometry,after transfection of siRNA or plasmid to knock down or overexpress PDK1;the knockdown or overexpression PDK1 stable cell lines were established using lentiviral infection,and sphere formation ability was analyzed by silencing or overexpressing PDK1;the volume of xenografted tumors in PDK1 knockdown cells were detected.(4)Cell proliferation,cell cycle and cell viability were determined by BrdU staining,cell cycle analysis and CCK8 kit in knockdown or overexpression PDK1 cells.(2)(1)The location of HIF-1αand PDK1 in tumor cells were detected by immunofluorescence,and tissue morphology was observed by HE staining.(2)Stemness-related factors were examined by immunofluorescence in peripheral and central regions tumor cells;the tumor tissues isolated from peripheral and central regions were digested to perform primary culture,and the mRNA level of PDK1 was detected by RT-qPCR;in vivo limiting dilution assays performed by plating decreasing numbers of primary xenografted tumor cells into immunodeficient mice calculated with extreme limiting dilution assay analysis to detect the ability of tumorigenesis in peripheral and central region tumor cells;and sphere formation assay was performed in peripheral and central region tumor cells.(3)Glucose uptake,lactate production,ATP levels and PDH activity were detected by kit in peripheral and central region tumor cells.(4)NTC and shPDK1 MDA-MB-231 cells were injected subcutaneously in nude mice,and the tumors isolated from peripheral and central regions were digested to conduct primary culture;the protein level of PDK1,PDH activity,glycolysis level and sphere formation ability were measured.(3)(1)Using RT-qPCR assay,nine hypoxia-related lncRNAs expression were measured between tumor central cells and peripheral cells,normoxic cells and hypoxic cells,mammosphere cells and monolayer cells.(2)Stable silencing H19 cells(MDA-MB-231 and MCF-7)were selected by shRNA lentivirus infection.Interfering efficiencies in these cells were identified by RT-qPCR assay.(3)Glucose uptake,lactate production and ATP levels were evaluated by kit between shH19 and NTC cells under normoxia and hypoxia.(4)Breast cancer cells(MDA-MB-231 and MCF-7)were transfected with siH19,RT-qPCR assay was performed to detect interfering efficiencies;the expression of stemness-related factors was measured by Western blotting in si H19cells;sphere formation assay was conducted in stable H19 silencing cells.(4)(1)RT-qPCR and Western blotting were used to examine PDK1 mRNA and protein levels in H19 ablation cells under hypoxia;PDH activity was assessed in H19knockdown cells.(2)Overexpression of H19 in PDK1 depletion cells,PDK1 protein level was detected using Western blotting assay;glucose uptake,lactate production and ATP levels were evaluated by kit;sphere formation ability was performed,xenografted tumor growth was monitored.(3)Twenty breast cancer patient tumor samples were collected,RT-qPCR was conducted to detect H19 and PDK1 level in these samples;χ~2test was used to analyze the relationship between H19 level and PDK1 level.(5)(1)To identify the mRNA and protein levels of HIF1A in H19 knockdown breast cancer cells,we perform RT-qPCR and Western blotting assay.(2)Nucleus and cytoplasmic RNA were extracted by kit,RT-qPCR was conducted to measure H19 level in normoxia and hypoxia;HIF1A 3’UTR full-length sequence(FL)including the putative miRNA(let-7)response element(MRE)were cloned into the psiCHECK2 vector,and the MRE was mutated in psiCHECK2-Mut vector,let-7 mimic significantly inhibited the relative luciferase activity of psi-HIF1A-FL and MRE,while displayed no effects on psi-HIF1A-Mut;RT-qPCR and Western blotting assay were performed to detect HIF1A mRNA and protein expression transfected with let-7 mimic or inhibitor under hypoxia.(3)The relative luciferase activity of psi-HIF1A-MRE was evaluated in cancer cells transfected with increasing amounts of wide-type H19;RT-qPCR and Western blotting assay were conducted to measure HIF-1αprotein expression transfected with siH19or/and let-7 inhibitor under hypoxia.(4)Stable HIF1A knock-down cells(MDA-MB-231 and MCF-7)were selected by shRNA lentivirus infection.Interfering efficiencies and PDK1 mRNA level in these cells were measured by RT-qPCR assay;PDK1 and HIF-1αprotein expression were detected by Western blotting in shHIF1A cells transfected with wide-type H19.(6)(1)H19 mRNA level was measured by RT-q PCR under treatment with aspirin in time-and dose-dependent manner;Western blotting assay was conducted to detect PDK1 protein level with aspirin treatment under hypoxia;PDH activity was measured under aspirin treatment by kit.(2)Glucose uptake,lactate production and ATP levels were examined with aspirin treatment by kit.(3)The expression of stemness-related factors were detected with aspirin treatment by Western blotting assay;sphere formation assay was performed under aspirin treatment;xenograft assay was addressed to evaluate tumor growth and volumes under aspirin treatment.3.Results(1)(1)By analyzing the expression profile GSE15192 from GEO Database,the expression of PDK1 in CD44~+/CD24~-subpopulations was higher than CD44~-/CD24~+.(2)The mRNA and protein level of PDK1 and stemness-related factors in cancer stem cells(CSCs)or tumor tissues were higher than non-CSC or normal tissues;Kaplan-Meier analysis showed that high PDK1 expression level of breast cancer indicated poor overall survival(OS),p<0.05.(3)The ratio of ALDH~+cell population,the protein level of PDK1and stemness-related factors and mammosphere formation diameters and numbers were decreased in PDK1 knockdown cells and were increased in PDK1 overexpression cells;the volumes of xenografted tumor was smaller in PDK1 depletion cells.(4)Knockdown and overexpression of PDK1 displayed no effect on cell proliferation,cell cycle and cell viability of breast cancer cells.(2)(1)The protein level of PDK1 was enriched in tumor central hypoxic regions than tumor peripheral regions by immunofluorescence and H&E staining.(2)The stemness-related factors were highly expressed in tumor central regions,and tumor central region primary cells exhibited high level of PDK1 protein,promoted sphere formation ability and xenografted tumorigenesis.(3)Glucose uptake,lactate production and ATP levels in central region cells were higher than peripheral region cells while PDH activity was opposite.(4)Compared to NTC tumor cells,there were no significant differences in PDK1 levels between peripheral and central cells isolated from shPDK1tumors;there were also no significant variations in glucose uptake,lactate production,ATP levels,PDH activity and sphere formation ability between central cells and peripheral cells in shPDK1 tumors.(3)(1)The expression level of H19 in central region cells,hypoxic cancer cells and mammosphere cells was much higher than peripheric region cells,normoxic tumor cells and monolayer cell,and H19 was also the highest differentially expressed lncRNA compared to others.(2)The expression level of H19 was knocked down in sh H19 cells;H19 knockdown significantly decreased glucose uptake,lactate production and ATP levels under hypoxia.(3)Silencing of H19 had no effect on glycolysis level under normoxia.(4)Depletion of H19 remarkably decreased the stemness-related factor expression and attenuated sphere formation ability under hypoxia.(4)(1)H19 depletion dramatically decreased PDK1 mRNA and protein expression under hypoxia,and elevated PDH activity.(2)PDK1 knockdown reversed H19-elevated cellular glucose uptake,lactate production and ATP levels.(3)PDK1 knockdown reversed H19-increased mammosphere diameters and numbers;ablation of PDK1restricted H19-enhanced xenografted tumor volume.(4)Linear regression analysis showed a positive relationship between H19 and PDK1(R~2=0.7262,P<0.001,n=15).(5)(1)Knockdown H19 markedly decreased HIF-1aprotein level,but had no significant changes in mRNA levels of HIF-1aunder hypoxia;(2)H19 was highly induced in the cytoplasm but not in the nucleus under hypoxia;let-7 mimic significantly inhibited the relative luciferase activity of reporter psi-HIF1A-FL and MRE,while exhibited no effects on psi-HIF1A-Mut;let-7 mimic decreased but let-7 inhibitor increased HIF-1aprotein levels,while both of them had no significant effect on the mRNA levels.(3)The relative luciferase activity of psi-HIF1A-MRE was upregulated in response to wild-type H19,but not by H19 with mutated let-7 binding site in a dose-dependent manner;let-7 released by H19 silencing decreased HIF-1aprotein expression,which could be rescued by ilet-7.(4)Ablation of HIF1A decreased PDK1 mRNA level and overexpression H19 increased PDK1 expression,knockdown HIF1A could reverse H19-elevated PDK1 expression.(6)(1)Aspirin decreased H19 level in time-and dose-dependent manner,reduced PDK1 protein expression under hypoxia and elevated PDH activity.(2)Aspirin significantly inhibited glycolysis by diminishing cellular glucose uptake,lactate production and ATP levels.(3)Aspirin remarkably decreased stemness-related factors expression,reduced mammosphere diameters and numbers and restricted xenografted tumor volumes.4.Conclusions(1)PDK1 is required for breast cancer stem-like traits.(2)PDK1 activates glycolysis to enhance stemness in hypoxia.(3)Hypoxia-induced H19 contributes to glycolysis and stemness in breast cancer.(4)Depletion of PDK1 antagonizes H19-mediated glycolysis and stemness.(5)H19 enhances PDK1 expression in a HIF-1αdependent manner.(6)Aspirin suppresses glycolysis and stemness maintenance through inhibiting H19 and PDK1.