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. 2019 May 9;9(10):2999-3013.
doi: 10.7150/thno.31301. eCollection 2019.

"VSports在线直播" Metabolic enzyme PDK3 forms a positive feedback loop with transcription factor HSF1 to drive chemoresistance

Jinye Xu  1 , Qiqi Shi  1 , Wenxia Xu  1 , Qiyin Zhou  2 , Rongkai Shi  1 , Yanning Ma  3 , Dingwei Chen  4 , Liyuan Zhu  1 , Lifeng Feng  1 , Alfred Sze-Lok Cheng  5 , Helen Morrison  6 , Xian Wang  2 , Hongchuan Jin  1
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Metabolic enzyme PDK3 forms a positive feedback loop with transcription factor HSF1 to drive chemoresistance (VSports app下载)

Jinye Xu (VSports) et al. Theranostics. .

VSports注册入口 - Abstract

Background & Aims: Dysregulation of metabolism plays an important role in the development and progression of cancers, while the underlying mechanisms remain largely unknown. This study aims to explore the regulation and relevance of glycolysis in chemoresistance of gastric cancer. Methods: Biochemical differences between chemoresistant and chemosensitive cancer cells were determined by metabolism profiling, microarray gene expression, PCR or western blotting. Cancer cell growth in vitro or in vivo were analyzed by viability, apoptosis and nude mice assay. Immunoprecipation was used to explore the interaction of proteins with other proteins or DNAs. Results: By metabolic and gene expression profiling, we found that pyruvate dehydrogenase kinase 3 (PDK3) was highly expressed to promote glycolysis in chemoresistant cancer cells. Its genetic or chemical inhibition reverted chemoresistance in vitro and in vivo. It was transcriptionally regulated by transcription factor HSF1 (Heat shock factor 1). Interestingly, PDK3 can localize in the nucleus and interact with HSF1 to disrupt its phosphorylation by GSK3β VSports手机版. Since HSF1 was subjected to FBXW7-catalyzed polyubiquitination in a phosphorylation-dependent manner, PDK3 prevented HSF1 from proteasomal degradation. Thus, metabolic enzyme PDK3 and transcription factor HSF1 forms a positive feedback loop to promote glycolysis. As a result, inhibition of HSF1 impaired enhanced glycolysis and reverted chemoresistance both in vitro and in vivo. Conclusions: PDK3 forms a positive feedback loop with HSF1 to drive glycolysis in chemoresistance. Targeting this mitonuclear communication may represent a novel approach to overcome chemoresistance. .

Keywords: HSF1; PDK3; chemoresistance; glycolysis; metabolism. V体育安卓版.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
PDK3 is upregulated in chemoresistant cancer cells. A, Principal component analysis (PCA) sore plots based on Glycosis&TCA metabolite profiles obtained from GC-TOF/MS analysis of SGC-R and SGC7901 cells. B, Metabolites changes in the glycolytic pathway in SGC-R and SGC7901 cells were detected by GC-TOF/MS. C, Gene expression profile of chemoresistant cells and their parental cells was analyzed by GSEA enrichment. D, Heatmap analysis of expression levels of genes in glycolytic pathway. E, The relative expression of PDKs mRNA levels in SGC-R, BGC-R and their parental chemosensitive cell lines (SGC7901 and BGC823). F, Immunoblot analysis of the protein expression of PDK3 in chemoresistant GC cells and their parental cells. G, PDH activitie was detected by PDH assay in chemoresistant cells relative to their parental cells. H and I, Kaplan-Meier analysis of the overall survival (OS) and progression-free survival (PFS) in GC cohorts according to PDK3 mRNA expression were achieved by using an online tool (http://www.kmplot.com) and the best cut-point was employed as the cutoff. Log-rank p-values and hazard ratios (HRs; 95% confidence interval in parentheses) were shown.
Figure 2
Figure 2
PDK3 drives glycolysis to promote chemoresistance. A, Principal component analysis (PCA) sore plots based on Glycosis&TCA metabolite profiles obtained from GC-TOF/MS analysis of SGC-R cells, stable PDK3 overexpression SGC7901 cells and the control SGC7901 cells. B, Metabolites changes in the glycolytic pathway induced by PDK3 overexpression were detected by GC-TOF/MS. C, PDH activity in SGC7901 cells with stable PDK3 expression was measured as in Figure 1G. D, SGC7901 cells with stable PDK3 expression were treated with various doses of DDP for 24 h and MTS was performed to detect their viability. E, Effect of PDK3 overexpression on c-PARP1 and c-caspase3 levels in SGC7901 cells treated with or without DDP were determined by immunoblot analysis. F, Effect of PDK3 overexpression on DDP-induced apoptosis of SGC7901 cells was determined by Flow cytometry analysis after PI and Annexin-V staining. G, PDH activity in chemoresistant cells with PDK3 knock-down was measured as in 2C. H, The effect of PDK3 knock-down on DDP-induced viability inhibition of chemoresistant cells were analyzed by MTS assay. I, Effect of PDK3 knock-down on DDP-induced cleavage of PARP1 and caspase3 were determined by immunoblot analysis. J, Effect of PDK3 knock-down on DDP-induced apoptosis of chemoresistant cells was determined by Flow cytometry analysis as in 2F.
Figure 3
Figure 3
Chemical inhibition of PDK3 impaired glycolysis to reverse chemoresistance. A, PDH activity in chemoresistant cells with or without DCA treatment were determined as in 1G. B-D, Glucose consumption (B) and production of pyruvic acid (C) or lactic acid (D) in chemoresistant cells were measured after exposure to 10 mM DCA for 6 h. E, Viability of various cells treated with various doses of DDP and DCA were evaluated by MTS assay. F and G, Apoptosis of SGC-R, BGC-R and MFC cells after treatment with DCA and/or DDP (6 mg/ml) were analyzed by immunoblot (F) and flowcytometry analysis (G). H-J, MFC cells were used for in vivo tumorigenicity analysis. Tumour sizes were measured every 3 days using caliper to plot growth curve (H) and tumor mass were weighted at the end of experiment (I and J). K and L, Cleaved PARP1 or caspase-3 in tumor tissues were evaluated by immunoblot analysis (K) or immunohistochemistry staining (L), respectively.
Figure 4
Figure 4
HSF1 stimulates PDK3 transcription. A, Schematic diagram of putative HSF1-binding sequence within a region 2000bp upstream to the TSS (transcription start site) of human PDK3. B, Immunoblot analysis of the protein expression of HSF1 in chemoresistant GC cells and their parental cells. C, The binding of HSF1 to PDK3 promoter was assessed by ChIP assay in SGC-R cells. D, PDK3 promoter-driven activity in the presence of wide-type (WT) or DNA-binding deficiency mutant (MT) HSF1 were analyzed in HEK293 cells by Luciferase reporter assay. E and F, The effect of WT or MT HSF1 on PDK3 expression in chemosensitive cells were analyzed by RT-PCR (E) and immunoblot (F) analysis. G-I, The effect of HSF1 knock-down on PDK3 mRNA. (G), PDK3 protein (H) and PDH activity (I) were determined by as previously described.
Figure 5
Figure 5
PDK3 upregulates HSF1 protein expression by preventing its degradation. A, Immunoblot analysis of HSF1 levels in chemosensitive GC cells with or without PDK3 overexpression. B, The effect of PDK3 overexpression on HSF1 reporter activity in HEK293 cells was analyzed by luciferase reporter assay. C and D, The effect of PDK3 knockdown on HSF1 expression (C) and activity (D) were evaluated as in A and B, respectively. E, The half-life of HSF1 protein in SGC-R cells pretreated with cycloheximide (50 μg/mL) were determined by immunoblot analysis. F, HSF1 proteins in SGC-R cells before and after PDK3 knock-down were immunoprecipitated by anti-HSF1 antibody and blotted with anti-ubiquitin antibody. G, HSF1 protein expression in SGC-R and BGC-R cells with or without PDK3 and FBXW7 knock-down were detected by immunoblot analysis. H, The interaction of HSF1 with FBXW7 in SGC7901 cells with or without PDK3 overexpression was analyzed by co-imunoprecipitation. I, HSF1 ubiquitination before and after PDK3 overexpression was analyzed by uiquitination assay.
Figure 6
Figure 6
PDK3 interacts with HSF1 to disrupt its phoshorylation by GSK3β. A, Immunofluorescence staining assay of intracellular distribution of exogenous PDK3 in SGC7901 cells. myc-PDK3: green; the mitochondrial marker MitoTracker: red; the nuclear marker DAPI: blue. B and C, Immunoblot analysis of the nuclear or cytoplasm distribution of exogenous (B) and endogenous (C) PDK3 in chemoresistant cells. D and E, The interaction of PDK3 with HSF1 was analyzed by co-immunoprecipitation. (D, HEK293 cells with exogenous PDK3 and HSF1 expression; E, SGC-R cells with endogenous PDK3 and HSF1 expression). F, Co-localization of PDK3 with HSF1 in chemoresistant cells were analyzed under confocal microscopy. PDK3: red; HSF1: green; DAPI: blue. G and H: The interaction region responsible for the interaction of PDK3 with HSF1 was defined by GST-pull down assay. I, Serine phosphorylation of HSF1 in SGC7901 cells before and after PDK3 overexpression were detected by immunoblot analysis. p-HSF1 (S303+S307): 303/307 serine phosphorylation specific antibody; p-serine: serine phsohorylation specific anitbody. J, the interaction of HSF1 with ERK1/2 or GSK3β in SGC7901 cells before and after PDK3 expression was analyzed by immunoprecipitation.
Figure 7
Figure 7
HSF1 stimulates glycolysis to confer chemoresistance. A, Principal component analysis (PCA) sore plots based on Glycosis&TCA metabolite profiles obtained from GC-TOF/MS analysis of various cells as indicated. B, The effect of HSF1 overexpression on glycolysis metabolites in SGC7901 cells was detected by GC-TOF/MS. C, The effect of HSF1 overexpression on PDH activity was analyzed as 1G. D-F, The effect of HSF1 overexpression on DDP-induced viability inhibition (D, MTS assay), and apoptosis activation (E, PARP1 and caspase-3 cleavage; F, PI and Annexin-V staining). G-I, The effect of HSF1 knock-down on DDP-induced viability inhibition (G, MTS assay), and apoptosis activation (H, PARP1 and caspase-3 cleavage; I, PI and Annexin-V staining).
Figure 8
Figure 8
Chemical inhibition of HSF1 reverses chemoresistance. A, Viability of various cells treated with or without DDP and KNK437 were evaluated by MTS assay. B and C, Apoptosis of various cells treated with or without DDP and KNK437 were evaluated by immunoblot (B, PARP1 and caspase-3 cleavage), and flowcytometry analysis (C, PI and Annexin-V staining). D-F, Nude mice treated as indicated were applied to evaluate the effect of KNK437 and DDP on tumorigenicity of MFC cells as in 3H-J. G and H, Cleaved PARP1 or caspase-3 in tumor tissues were evaluated by immunoblot analysis (G) or immunohistochemistry staining (H), respectively. I, working model: PDK3 was increased as a result of HSF1-driven transcription. It can not only stimulate glycolysis by inhibiting the activity of PDH in the mitochondria, but also directly interacted 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.
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