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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 inhibition promotes EMT. (A) HCC827 cells were transfected with pCMV6-AC-IRES-GFP vector or pCMV6-PDK4-IRES-GFP (WT, 254A, or 257AA), then cultured in the presence of 1.5 μg/ml puromycin for 3 weeks. After that, the GFP-positive cells were enriched through FACS, cultured in the presence or absence of 2 ng/ml TGFβ for 10 days, and lysed for immunoblotting. (B) A549 and HCC827 cells were transfected with siNTC pool no. 2 or siPDK4 pool at 1 day and 3 days post-seeding. Two days after the second transfection, the cells were lysed for immunoblotting and qRT-PCR. VDAC and GAPDH are protein loading controls for mitochondrial lysate and whole cell lysate, respectively. In A and B, the red asterisk denotes the upper band that is specific for PDK4. (C, D) HCC827 (C) and HCC4006 (D) cells were transfected with siNTC pool no. 2 or siPDK4 pool at 20 nM siRNA at 1 day and 3 days post-seeding. Twenty-four hours after the second transfection, the media was replaced with growth media containing 2 μM erlotinib and cells were continuously cultured in such media with fresh media replenished every 3 to 4 days for 2 to 3 weeks. For the control plates without erlotinib treatment, the day before the second transfection, a fraction of the cells was re-plated at low density and subjected to a second transfection the next day. At the end of the experiment, cells were stained with crystal violet. The quantification of colony numbers represents four independent experiments, and for each experiment, the colony number in the siNTC plate is normalized to that in the siPDK4 plate. Paired t-test was performed for C and D. Data are plotted as mean ± SEM. *p < 0.05; **p < 0.01.
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Fig3: PDK4 inhibition promotes EMT. (A) HCC827 cells were transfected with pCMV6-AC-IRES-GFP vector or pCMV6-PDK4-IRES-GFP (WT, 254A, or 257AA), then cultured in the presence of 1.5 μg/ml puromycin for 3 weeks. After that, the GFP-positive cells were enriched through FACS, cultured in the presence or absence of 2 ng/ml TGFβ for 10 days, and lysed for immunoblotting. (B) A549 and HCC827 cells were transfected with siNTC pool no. 2 or siPDK4 pool at 1 day and 3 days post-seeding. Two days after the second transfection, the cells were lysed for immunoblotting and qRT-PCR. VDAC and GAPDH are protein loading controls for mitochondrial lysate and whole cell lysate, respectively. In A and B, the red asterisk denotes the upper band that is specific for PDK4. (C, D) HCC827 (C) and HCC4006 (D) cells were transfected with siNTC pool no. 2 or siPDK4 pool at 20 nM siRNA at 1 day and 3 days post-seeding. Twenty-four hours after the second transfection, the media was replaced with growth media containing 2 μM erlotinib and cells were continuously cultured in such media with fresh media replenished every 3 to 4 days for 2 to 3 weeks. For the control plates without erlotinib treatment, the day before the second transfection, a fraction of the cells was re-plated at low density and subjected to a second transfection the next day. At the end of the experiment, cells were stained with crystal violet. The quantification of colony numbers represents four independent experiments, and for each experiment, the colony number in the siNTC plate is normalized to that in the siPDK4 plate. Paired t-test was performed for C and D. Data are plotted as mean ± SEM. *p < 0.05; **p < 0.01.

Mentions: To determine whether PDK4 plays a functional role during EMT, we tested whether ectopic PDK4 expression would impede EMT. We generated HCC827 cells stably expressing wild-type or kinase-dead PDK4 (Figure 3A). Based on the structure of PDK4 [24], we generated two kinase-dead mutants by mutating the canonical ATP-binding sites—Glu254Ala, referred to as 254A, and Lys257Ala/Asp258Ala, referred to as 257AA. Although we still observed EMT following TGFβ treatment in cells expressing recombinant PDK4, importantly, wild-type PDK4 was indeed able to partially block the expression of the mesenchymal markers Zeb1 and Vimentin, whereas the kinase-dead mutants of PDK4 could not impede EMT. These findings suggest that the kinase activity of PDK4 is required for its role in EMT.Figure 3


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 inhibition promotes EMT. (A) HCC827 cells were transfected with pCMV6-AC-IRES-GFP vector or pCMV6-PDK4-IRES-GFP (WT, 254A, or 257AA), then cultured in the presence of 1.5 μg/ml puromycin for 3 weeks. After that, the GFP-positive cells were enriched through FACS, cultured in the presence or absence of 2 ng/ml TGFβ for 10 days, and lysed for immunoblotting. (B) A549 and HCC827 cells were transfected with siNTC pool no. 2 or siPDK4 pool at 1 day and 3 days post-seeding. Two days after the second transfection, the cells were lysed for immunoblotting and qRT-PCR. VDAC and GAPDH are protein loading controls for mitochondrial lysate and whole cell lysate, respectively. In A and B, the red asterisk denotes the upper band that is specific for PDK4. (C, D) HCC827 (C) and HCC4006 (D) cells were transfected with siNTC pool no. 2 or siPDK4 pool at 20 nM siRNA at 1 day and 3 days post-seeding. Twenty-four hours after the second transfection, the media was replaced with growth media containing 2 μM erlotinib and cells were continuously cultured in such media with fresh media replenished every 3 to 4 days for 2 to 3 weeks. For the control plates without erlotinib treatment, the day before the second transfection, a fraction of the cells was re-plated at low density and subjected to a second transfection the next day. At the end of the experiment, cells were stained with crystal violet. The quantification of colony numbers represents four independent experiments, and for each experiment, the colony number in the siNTC plate is normalized to that in the siPDK4 plate. Paired t-test was performed for C and D. Data are plotted as mean ± SEM. *p < 0.05; **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: PDK4 inhibition promotes EMT. (A) HCC827 cells were transfected with pCMV6-AC-IRES-GFP vector or pCMV6-PDK4-IRES-GFP (WT, 254A, or 257AA), then cultured in the presence of 1.5 μg/ml puromycin for 3 weeks. After that, the GFP-positive cells were enriched through FACS, cultured in the presence or absence of 2 ng/ml TGFβ for 10 days, and lysed for immunoblotting. (B) A549 and HCC827 cells were transfected with siNTC pool no. 2 or siPDK4 pool at 1 day and 3 days post-seeding. Two days after the second transfection, the cells were lysed for immunoblotting and qRT-PCR. VDAC and GAPDH are protein loading controls for mitochondrial lysate and whole cell lysate, respectively. In A and B, the red asterisk denotes the upper band that is specific for PDK4. (C, D) HCC827 (C) and HCC4006 (D) cells were transfected with siNTC pool no. 2 or siPDK4 pool at 20 nM siRNA at 1 day and 3 days post-seeding. Twenty-four hours after the second transfection, the media was replaced with growth media containing 2 μM erlotinib and cells were continuously cultured in such media with fresh media replenished every 3 to 4 days for 2 to 3 weeks. For the control plates without erlotinib treatment, the day before the second transfection, a fraction of the cells was re-plated at low density and subjected to a second transfection the next day. At the end of the experiment, cells were stained with crystal violet. The quantification of colony numbers represents four independent experiments, and for each experiment, the colony number in the siNTC plate is normalized to that in the siPDK4 plate. Paired t-test was performed for C and D. Data are plotted as mean ± SEM. *p < 0.05; **p < 0.01.
Mentions: To determine whether PDK4 plays a functional role during EMT, we tested whether ectopic PDK4 expression would impede EMT. We generated HCC827 cells stably expressing wild-type or kinase-dead PDK4 (Figure 3A). Based on the structure of PDK4 [24], we generated two kinase-dead mutants by mutating the canonical ATP-binding sites—Glu254Ala, referred to as 254A, and Lys257Ala/Asp258Ala, referred to as 257AA. Although we still observed EMT following TGFβ treatment in cells expressing recombinant PDK4, importantly, wild-type PDK4 was indeed able to partially block the expression of the mesenchymal markers Zeb1 and Vimentin, whereas the kinase-dead mutants of PDK4 could not impede EMT. These findings suggest that the kinase activity of PDK4 is required for its role in EMT.Figure 3

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