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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

Metabolic changes in three NSCLC cell lines upon TGFβ-induced EMT. A549, HCC827, and NCI-H358 lung cancer cells were cultured in the presence of 2 ng/ml TGFβ for 2 to 5 weeks to induce EMT. The following aspects of both the parental (P) and mesenchymal (M) cells were characterized. (A) Morphological changes of cells. (B) The expression of the epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, Vimentin, Zeb1, and Snail). (C) Erlotinib sensitivity of HCC827 and GDC-0973 sensitivity of A549 parental and mesenchymal cells. The cells were treated with the EGFR kinase inhibitor erlotinib or MEK inhibitor GDC-0973 for 3 days, and viability was measured using a CellTiter-Glo assay. (D) Glycolysis/OXPHOS ratio, defined by PPR/OCR and measured using the Seahorse metabolic analyzer. Average results from three to four independent experiments are shown. (E, F) Cellular glutamine (E) and glutamate (F) concentrations as measured by mass spectrometry. Each data point is from five separate biological samples generated at the same time. The boxes represent 10–90 percentile. (G) Glutamate secretion per cell during 24 h. Average of data from six wells in one experiment, which is representative of three independent experiments, is shown. (H, I) Cells were incubated with growth media containing 13C-U-glucose overnight, and then subjected to LC-MS analysis. (H) Glucose to glutamate contribution was plotted based on the percentage of (M + 2) glutamate in the total glutamate pool. (I) Glucose to TCA cycle contribution was plotted based on the percentages of (M + 2) citrate, (M + 2) α-ketoglutarate and (M + 2) malate in each individual metabolite’s total pool. For all panels, data are plotted as mean ± SEM. *p < 0.05; **p < 0.01, unless otherwise specified.
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Fig1: Metabolic changes in three NSCLC cell lines upon TGFβ-induced EMT. A549, HCC827, and NCI-H358 lung cancer cells were cultured in the presence of 2 ng/ml TGFβ for 2 to 5 weeks to induce EMT. The following aspects of both the parental (P) and mesenchymal (M) cells were characterized. (A) Morphological changes of cells. (B) The expression of the epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, Vimentin, Zeb1, and Snail). (C) Erlotinib sensitivity of HCC827 and GDC-0973 sensitivity of A549 parental and mesenchymal cells. The cells were treated with the EGFR kinase inhibitor erlotinib or MEK inhibitor GDC-0973 for 3 days, and viability was measured using a CellTiter-Glo assay. (D) Glycolysis/OXPHOS ratio, defined by PPR/OCR and measured using the Seahorse metabolic analyzer. Average results from three to four independent experiments are shown. (E, F) Cellular glutamine (E) and glutamate (F) concentrations as measured by mass spectrometry. Each data point is from five separate biological samples generated at the same time. The boxes represent 10–90 percentile. (G) Glutamate secretion per cell during 24 h. Average of data from six wells in one experiment, which is representative of three independent experiments, is shown. (H, I) Cells were incubated with growth media containing 13C-U-glucose overnight, and then subjected to LC-MS analysis. (H) Glucose to glutamate contribution was plotted based on the percentage of (M + 2) glutamate in the total glutamate pool. (I) Glucose to TCA cycle contribution was plotted based on the percentages of (M + 2) citrate, (M + 2) α-ketoglutarate and (M + 2) malate in each individual metabolite’s total pool. For all panels, data are plotted as mean ± SEM. *p < 0.05; **p < 0.01, unless otherwise specified.

Mentions: Human cancer cell lines provide essential models for dissecting fundamental mechanisms in tumor biology. We modeled EMT in cultured cancer cells using TGFβ treatment since TGFβ robustly induces EMT in many epithelial cell line models, and physiologically, hyperactivation of TGFβ signaling has been shown to be associated with the mesenchymal phenotype and cancer drug resistance [13, 14]. To identify EMT-associated changes in cancer cell biology that are not restricted to one specific genetic background, we examined three different human non-small cell lung cancer (NSCLC) cell lines—A549 (KRASG12S-driven), HCC827 (EGFRΔE746-A750-driven), and NCI-H358 (KRASG12C-driven). We cultured cells with continuous exposure to TGFβ for 3 weeks and observed dramatic morphological transformation—the cells changed from displaying a compact epithelial morphology with obvious cell-cell contacts to a fibroblast-like morphology with scattered spindle shapes (Figure 1A). Consistent with the morphological changes observed in the TGFβ-treated cells, we detected decreased expression of the epithelial marker E-cadherin as well as increased expression of the mesenchymal markers N-cadherin and Vimentin and the EMT-associated transcription factors Zeb1 and Snail (Figure 1B).Figure 1


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)

Metabolic changes in three NSCLC cell lines upon TGFβ-induced EMT. A549, HCC827, and NCI-H358 lung cancer cells were cultured in the presence of 2 ng/ml TGFβ for 2 to 5 weeks to induce EMT. The following aspects of both the parental (P) and mesenchymal (M) cells were characterized. (A) Morphological changes of cells. (B) The expression of the epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, Vimentin, Zeb1, and Snail). (C) Erlotinib sensitivity of HCC827 and GDC-0973 sensitivity of A549 parental and mesenchymal cells. The cells were treated with the EGFR kinase inhibitor erlotinib or MEK inhibitor GDC-0973 for 3 days, and viability was measured using a CellTiter-Glo assay. (D) Glycolysis/OXPHOS ratio, defined by PPR/OCR and measured using the Seahorse metabolic analyzer. Average results from three to four independent experiments are shown. (E, F) Cellular glutamine (E) and glutamate (F) concentrations as measured by mass spectrometry. Each data point is from five separate biological samples generated at the same time. The boxes represent 10–90 percentile. (G) Glutamate secretion per cell during 24 h. Average of data from six wells in one experiment, which is representative of three independent experiments, is shown. (H, I) Cells were incubated with growth media containing 13C-U-glucose overnight, and then subjected to LC-MS analysis. (H) Glucose to glutamate contribution was plotted based on the percentage of (M + 2) glutamate in the total glutamate pool. (I) Glucose to TCA cycle contribution was plotted based on the percentages of (M + 2) citrate, (M + 2) α-ketoglutarate and (M + 2) malate in each individual metabolite’s total pool. For all panels, data are plotted as mean ± SEM. *p < 0.05; **p < 0.01, unless otherwise specified.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Metabolic changes in three NSCLC cell lines upon TGFβ-induced EMT. A549, HCC827, and NCI-H358 lung cancer cells were cultured in the presence of 2 ng/ml TGFβ for 2 to 5 weeks to induce EMT. The following aspects of both the parental (P) and mesenchymal (M) cells were characterized. (A) Morphological changes of cells. (B) The expression of the epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, Vimentin, Zeb1, and Snail). (C) Erlotinib sensitivity of HCC827 and GDC-0973 sensitivity of A549 parental and mesenchymal cells. The cells were treated with the EGFR kinase inhibitor erlotinib or MEK inhibitor GDC-0973 for 3 days, and viability was measured using a CellTiter-Glo assay. (D) Glycolysis/OXPHOS ratio, defined by PPR/OCR and measured using the Seahorse metabolic analyzer. Average results from three to four independent experiments are shown. (E, F) Cellular glutamine (E) and glutamate (F) concentrations as measured by mass spectrometry. Each data point is from five separate biological samples generated at the same time. The boxes represent 10–90 percentile. (G) Glutamate secretion per cell during 24 h. Average of data from six wells in one experiment, which is representative of three independent experiments, is shown. (H, I) Cells were incubated with growth media containing 13C-U-glucose overnight, and then subjected to LC-MS analysis. (H) Glucose to glutamate contribution was plotted based on the percentage of (M + 2) glutamate in the total glutamate pool. (I) Glucose to TCA cycle contribution was plotted based on the percentages of (M + 2) citrate, (M + 2) α-ketoglutarate and (M + 2) malate in each individual metabolite’s total pool. For all panels, data are plotted as mean ± SEM. *p < 0.05; **p < 0.01, unless otherwise specified.
Mentions: Human cancer cell lines provide essential models for dissecting fundamental mechanisms in tumor biology. We modeled EMT in cultured cancer cells using TGFβ treatment since TGFβ robustly induces EMT in many epithelial cell line models, and physiologically, hyperactivation of TGFβ signaling has been shown to be associated with the mesenchymal phenotype and cancer drug resistance [13, 14]. To identify EMT-associated changes in cancer cell biology that are not restricted to one specific genetic background, we examined three different human non-small cell lung cancer (NSCLC) cell lines—A549 (KRASG12S-driven), HCC827 (EGFRΔE746-A750-driven), and NCI-H358 (KRASG12C-driven). We cultured cells with continuous exposure to TGFβ for 3 weeks and observed dramatic morphological transformation—the cells changed from displaying a compact epithelial morphology with obvious cell-cell contacts to a fibroblast-like morphology with scattered spindle shapes (Figure 1A). Consistent with the morphological changes observed in the TGFβ-treated cells, we detected decreased expression of the epithelial marker E-cadherin as well as increased expression of the mesenchymal markers N-cadherin and Vimentin and the EMT-associated transcription factors Zeb1 and Snail (Figure 1B).Figure 1

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