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Metabolic and transcriptional profiling reveals pyruvate dehydrogenase kinase 4 as a mediator of epithelial-mesenchymal transition and drug resistance in tumor cells.

Sun Y, Daemen A, Hatzivassiliou G, Arnott D, Wilson C, Zhuang G, Gao M, Liu P, Boudreau A, Johnson L, Settleman J - Cancer Metab (2014)

Bottom Line: Such rewiring was at least partially mediated by the reduced expression of pyruvate dehydrogenase kinase 4 (PDK4), which serves as a gatekeeper of the TCA cycle by inactivating pyruvate dehydrogenase (PDH).We identified a novel interaction between PDK4 and apoptosis-inducing factor (AIF), an inner mitochondrial protein that appears to play a role in mediating this resistance.Together, these findings implicate PDK4 as a critical metabolic regulator of EMT and associated drug resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Discovery Oncology, Genentech Inc, 1 DNA Way, 94080 South San Francisco, CA USA.

ABSTRACT

Background: Accumulating preclinical and clinical evidence implicates epithelial-mesenchymal transition (EMT) in acquired resistance to anticancer drugs; however, mechanisms by which the mesenchymal state determines drug resistance remain unknown.

Results: To explore a potential role for altered cellular metabolism in EMT and associated drug resistance, we analyzed the metabolome and transcriptome of three lung cancer cell lines that were rendered drug resistant following experimental induction of EMT. This analysis revealed evidence of metabolic rewiring during EMT that diverts glucose to the TCA cycle. Such rewiring was at least partially mediated by the reduced expression of pyruvate dehydrogenase kinase 4 (PDK4), which serves as a gatekeeper of the TCA cycle by inactivating pyruvate dehydrogenase (PDH). Overexpression of PDK4 partially blocked TGFβ-induced EMT; conversely, PDK4 inhibition via RNAi-mediated knockdown was sufficient to drive EMT and promoted erlotinib resistance in EGFR mutant lung cancer cells. We identified a novel interaction between PDK4 and apoptosis-inducing factor (AIF), an inner mitochondrial protein that appears to play a role in mediating this resistance. In addition, analysis of human tumor samples revealed PDK4-low as a predictor of poor prognosis in lung cancer and that PDK4 expression is dramatically downregulated in most tumor types.

Conclusions: Together, these findings implicate PDK4 as a critical metabolic regulator of EMT and associated drug resistance.

No MeSH data available.


Related in: MedlinePlus

PDK4 downregulation is observed in mesenchymal cells, and mediates metabolic rewiring. (A) A schematic representation of the metabolic rewiring observed in TGFβ-induced mesenchymal cells. Red highlight indicates increased flux from glucose to the TCA cycle and then to glutamate in the mesenchymal cells. (B) Heatmap based on microarray gene expression data showing the mRNA levels of PDK1-PDK4 in the parental cells (P) and mesenchymal derivatives (M). (C) Quantitative RT-PCR showing PDK4 mRNA levels in parental and mesenchymal cells. In B and C, data shown is the average of three separate biological samples generated at the same time. (D) Western blots with the mitochondria showing the PDK4 protein levels in parental and mesenchymal derivatives. VDAC was used as the protein loading control. The red asterisk denotes the upper band that specifically corresponds to PDK4. (E) Immunoblotting for PDK4 and EMT markers in A549 parental (Par) and resistant cells. The resistant clones were generated by culturing A549 cells in the presence of 0.5 μM GDC-0973 and 0.2 μM GDC-0941 for 2 months. Data from two independent clones is shown. (F, G) HCC827 parental and mesenchymal cells were infected with lentivirus expressing RFP or PDK4 with a GFP reporter. The GFP-positive cells were selected by FACS, cultured in growth media containing 13C-U-glucose overnight, and then subjected to LC-MS analysis. (F) Glucose to TCA cycle/glutamate contributions were plotted based on the percentages of (M + 2) citrate, (M + 2) malate and (M + 2) glutamate in each metabolite’s respective total pool. (G) Pyruvate to glutamate contribution was plotted based on the ratio of glucose → glutamate (the percentage of (M + 2) glutamate in total glutamate pool) over glucose → pyruvate (the percentage of (M + 3) pyruvate in total pyruvate pool). Pyruvate to citrate or malate contribution was similarly plotted. Data are plotted as mean ± SEM. **p < 0.01.
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Fig2: PDK4 downregulation is observed in mesenchymal cells, and mediates metabolic rewiring. (A) A schematic representation of the metabolic rewiring observed in TGFβ-induced mesenchymal cells. Red highlight indicates increased flux from glucose to the TCA cycle and then to glutamate in the mesenchymal cells. (B) Heatmap based on microarray gene expression data showing the mRNA levels of PDK1-PDK4 in the parental cells (P) and mesenchymal derivatives (M). (C) Quantitative RT-PCR showing PDK4 mRNA levels in parental and mesenchymal cells. In B and C, data shown is the average of three separate biological samples generated at the same time. (D) Western blots with the mitochondria showing the PDK4 protein levels in parental and mesenchymal derivatives. VDAC was used as the protein loading control. The red asterisk denotes the upper band that specifically corresponds to PDK4. (E) Immunoblotting for PDK4 and EMT markers in A549 parental (Par) and resistant cells. The resistant clones were generated by culturing A549 cells in the presence of 0.5 μM GDC-0973 and 0.2 μM GDC-0941 for 2 months. Data from two independent clones is shown. (F, G) HCC827 parental and mesenchymal cells were infected with lentivirus expressing RFP or PDK4 with a GFP reporter. The GFP-positive cells were selected by FACS, cultured in growth media containing 13C-U-glucose overnight, and then subjected to LC-MS analysis. (F) Glucose to TCA cycle/glutamate contributions were plotted based on the percentages of (M + 2) citrate, (M + 2) malate and (M + 2) glutamate in each metabolite’s respective total pool. (G) Pyruvate to glutamate contribution was plotted based on the ratio of glucose → glutamate (the percentage of (M + 2) glutamate in total glutamate pool) over glucose → pyruvate (the percentage of (M + 3) pyruvate in total pyruvate pool). Pyruvate to citrate or malate contribution was similarly plotted. Data are plotted as mean ± SEM. **p < 0.01.

Mentions: Next, we addressed the mechanism underlying the observed glutamate accumulation in the mesenchymal cells. The likely potential carbon sources are either glutamine or glucose. Glutamine can be converted directly to glutamate via deamination. Glucose, in contrast, can be converted to glutamate through a multi-step process [10]. During the early steps of glycolysis, glucose is first converted to pyruvate and then to acetyl-CoA, which directly enters the TCA cycle. Alpha-ketoglutarate, a critical intermediate metabolite in the TCA cycle, serves as the backbone for the synthesis of many amino acids, including glutamate. To assess the glucose and glutamine contributions to glutamate and glutathione, we performed mass isotopologue distribution analysis (MIDA) [19]. We cultured the epithelial and mesenchymal cells with C-13 uniformly labeled glucose or glutamine, and quantified the incorporation of C-13 label into glutamate using liquid column-mass spectrometry (LC/MS). Although glutamine was still a major source of the glutamate pool in both the parental and mesenchymal cells, we found significantly decreased percentages of glutamine-derived glutamate (Additional file 2: Figure S1E) and corresponding increased percentages of glucose-derived glutamate (Figure 1H) in all three mesenchymal models compared to their corresponding parental cells. Importantly, we also observed significantly increased percentages of glucose-derived TCA cycle metabolites, including citrate (the first metabolite in the TCA cycle), α-ketoglutarate (αKG, a critical precursor for amino acid synthesis), and malate (Figure 1I), consistent with the notion that the TCA cycle contributes to the observed glutamate accumulation during EMT. Collectively, these data demonstrate a diversion of glucose to the TCA cycle → glutamate axis in the mesenchymal cells (Figure 2A).Figure 2


Metabolic and transcriptional profiling reveals pyruvate dehydrogenase kinase 4 as a mediator of epithelial-mesenchymal transition and drug resistance in tumor cells.

Sun Y, Daemen A, Hatzivassiliou G, Arnott D, Wilson C, Zhuang G, Gao M, Liu P, Boudreau A, Johnson L, Settleman J - Cancer Metab (2014)

PDK4 downregulation is observed in mesenchymal cells, and mediates metabolic rewiring. (A) A schematic representation of the metabolic rewiring observed in TGFβ-induced mesenchymal cells. Red highlight indicates increased flux from glucose to the TCA cycle and then to glutamate in the mesenchymal cells. (B) Heatmap based on microarray gene expression data showing the mRNA levels of PDK1-PDK4 in the parental cells (P) and mesenchymal derivatives (M). (C) Quantitative RT-PCR showing PDK4 mRNA levels in parental and mesenchymal cells. In B and C, data shown is the average of three separate biological samples generated at the same time. (D) Western blots with the mitochondria showing the PDK4 protein levels in parental and mesenchymal derivatives. VDAC was used as the protein loading control. The red asterisk denotes the upper band that specifically corresponds to PDK4. (E) Immunoblotting for PDK4 and EMT markers in A549 parental (Par) and resistant cells. The resistant clones were generated by culturing A549 cells in the presence of 0.5 μM GDC-0973 and 0.2 μM GDC-0941 for 2 months. Data from two independent clones is shown. (F, G) HCC827 parental and mesenchymal cells were infected with lentivirus expressing RFP or PDK4 with a GFP reporter. The GFP-positive cells were selected by FACS, cultured in growth media containing 13C-U-glucose overnight, and then subjected to LC-MS analysis. (F) Glucose to TCA cycle/glutamate contributions were plotted based on the percentages of (M + 2) citrate, (M + 2) malate and (M + 2) glutamate in each metabolite’s respective total pool. (G) Pyruvate to glutamate contribution was plotted based on the ratio of glucose → glutamate (the percentage of (M + 2) glutamate in total glutamate pool) over glucose → pyruvate (the percentage of (M + 3) pyruvate in total pyruvate pool). Pyruvate to citrate or malate contribution was similarly plotted. Data are plotted as mean ± SEM. **p < 0.01.
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Fig2: PDK4 downregulation is observed in mesenchymal cells, and mediates metabolic rewiring. (A) A schematic representation of the metabolic rewiring observed in TGFβ-induced mesenchymal cells. Red highlight indicates increased flux from glucose to the TCA cycle and then to glutamate in the mesenchymal cells. (B) Heatmap based on microarray gene expression data showing the mRNA levels of PDK1-PDK4 in the parental cells (P) and mesenchymal derivatives (M). (C) Quantitative RT-PCR showing PDK4 mRNA levels in parental and mesenchymal cells. In B and C, data shown is the average of three separate biological samples generated at the same time. (D) Western blots with the mitochondria showing the PDK4 protein levels in parental and mesenchymal derivatives. VDAC was used as the protein loading control. The red asterisk denotes the upper band that specifically corresponds to PDK4. (E) Immunoblotting for PDK4 and EMT markers in A549 parental (Par) and resistant cells. The resistant clones were generated by culturing A549 cells in the presence of 0.5 μM GDC-0973 and 0.2 μM GDC-0941 for 2 months. Data from two independent clones is shown. (F, G) HCC827 parental and mesenchymal cells were infected with lentivirus expressing RFP or PDK4 with a GFP reporter. The GFP-positive cells were selected by FACS, cultured in growth media containing 13C-U-glucose overnight, and then subjected to LC-MS analysis. (F) Glucose to TCA cycle/glutamate contributions were plotted based on the percentages of (M + 2) citrate, (M + 2) malate and (M + 2) glutamate in each metabolite’s respective total pool. (G) Pyruvate to glutamate contribution was plotted based on the ratio of glucose → glutamate (the percentage of (M + 2) glutamate in total glutamate pool) over glucose → pyruvate (the percentage of (M + 3) pyruvate in total pyruvate pool). Pyruvate to citrate or malate contribution was similarly plotted. Data are plotted as mean ± SEM. **p < 0.01.
Mentions: Next, we addressed the mechanism underlying the observed glutamate accumulation in the mesenchymal cells. The likely potential carbon sources are either glutamine or glucose. Glutamine can be converted directly to glutamate via deamination. Glucose, in contrast, can be converted to glutamate through a multi-step process [10]. During the early steps of glycolysis, glucose is first converted to pyruvate and then to acetyl-CoA, which directly enters the TCA cycle. Alpha-ketoglutarate, a critical intermediate metabolite in the TCA cycle, serves as the backbone for the synthesis of many amino acids, including glutamate. To assess the glucose and glutamine contributions to glutamate and glutathione, we performed mass isotopologue distribution analysis (MIDA) [19]. We cultured the epithelial and mesenchymal cells with C-13 uniformly labeled glucose or glutamine, and quantified the incorporation of C-13 label into glutamate using liquid column-mass spectrometry (LC/MS). Although glutamine was still a major source of the glutamate pool in both the parental and mesenchymal cells, we found significantly decreased percentages of glutamine-derived glutamate (Additional file 2: Figure S1E) and corresponding increased percentages of glucose-derived glutamate (Figure 1H) in all three mesenchymal models compared to their corresponding parental cells. Importantly, we also observed significantly increased percentages of glucose-derived TCA cycle metabolites, including citrate (the first metabolite in the TCA cycle), α-ketoglutarate (αKG, a critical precursor for amino acid synthesis), and malate (Figure 1I), consistent with the notion that the TCA cycle contributes to the observed glutamate accumulation during EMT. Collectively, these data demonstrate a diversion of glucose to the TCA cycle → glutamate axis in the mesenchymal cells (Figure 2A).Figure 2

Bottom Line: Such rewiring was at least partially mediated by the reduced expression of pyruvate dehydrogenase kinase 4 (PDK4), which serves as a gatekeeper of the TCA cycle by inactivating pyruvate dehydrogenase (PDH).We identified a novel interaction between PDK4 and apoptosis-inducing factor (AIF), an inner mitochondrial protein that appears to play a role in mediating this resistance.Together, these findings implicate PDK4 as a critical metabolic regulator of EMT and associated drug resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Discovery Oncology, Genentech Inc, 1 DNA Way, 94080 South San Francisco, CA USA.

ABSTRACT

Background: Accumulating preclinical and clinical evidence implicates epithelial-mesenchymal transition (EMT) in acquired resistance to anticancer drugs; however, mechanisms by which the mesenchymal state determines drug resistance remain unknown.

Results: To explore a potential role for altered cellular metabolism in EMT and associated drug resistance, we analyzed the metabolome and transcriptome of three lung cancer cell lines that were rendered drug resistant following experimental induction of EMT. This analysis revealed evidence of metabolic rewiring during EMT that diverts glucose to the TCA cycle. Such rewiring was at least partially mediated by the reduced expression of pyruvate dehydrogenase kinase 4 (PDK4), which serves as a gatekeeper of the TCA cycle by inactivating pyruvate dehydrogenase (PDH). Overexpression of PDK4 partially blocked TGFβ-induced EMT; conversely, PDK4 inhibition via RNAi-mediated knockdown was sufficient to drive EMT and promoted erlotinib resistance in EGFR mutant lung cancer cells. We identified a novel interaction between PDK4 and apoptosis-inducing factor (AIF), an inner mitochondrial protein that appears to play a role in mediating this resistance. In addition, analysis of human tumor samples revealed PDK4-low as a predictor of poor prognosis in lung cancer and that PDK4 expression is dramatically downregulated in most tumor types.

Conclusions: Together, these findings implicate PDK4 as a critical metabolic regulator of EMT and associated drug resistance.

No MeSH data available.


Related in: MedlinePlus