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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

AIF interacts with PDK4 and plays a role in EMT. (A) HEK293T cells were transfected with the pCMV6-Entry empty vector or pCMV6-FLAG-Myc-PDK4. Forty-eight hours post-transfection, the cells were lysed and FLAG IP was performed. Mass spectrometry was performed to analyze the immunoprecipitates, and the top five proteins identified in the immunoprecipitates are shown. (B) Immunoblotting was performed with samples prepared as in A to confirm the PDK4-AIF interaction. (C) A549 cells and HCC827 cells were transfected with siNTC pool no. 2, siAIF pool, or siPDK4 pool at 1 day and 3 days post-seeding, then lysed for immunoblotting. (D) HCC827 cells were transfected with siNTC pool no. 2, siAIF pool, and siPDK4 pool, treated with erlotinib (erl), and stained with crystal violet as in Figure 3C. (E) HCC827 cells were transfected as in D. Two days after the second transfection, the cells were stained with CM-H2DCFDA and ROS levels were analyzed using flow cytometry.
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Fig4: AIF interacts with PDK4 and plays a role in EMT. (A) HEK293T cells were transfected with the pCMV6-Entry empty vector or pCMV6-FLAG-Myc-PDK4. Forty-eight hours post-transfection, the cells were lysed and FLAG IP was performed. Mass spectrometry was performed to analyze the immunoprecipitates, and the top five proteins identified in the immunoprecipitates are shown. (B) Immunoblotting was performed with samples prepared as in A to confirm the PDK4-AIF interaction. (C) A549 cells and HCC827 cells were transfected with siNTC pool no. 2, siAIF pool, or siPDK4 pool at 1 day and 3 days post-seeding, then lysed for immunoblotting. (D) HCC827 cells were transfected with siNTC pool no. 2, siAIF pool, and siPDK4 pool, treated with erlotinib (erl), and stained with crystal violet as in Figure 3C. (E) HCC827 cells were transfected as in D. Two days after the second transfection, the cells were stained with CM-H2DCFDA and ROS levels were analyzed using flow cytometry.

Mentions: To gain additional insight into PDK4’s role in EMT, we sought to identify proteins that interact with PDK4. Considering the very limited information regarding PDK4-interacting proteins, we performed an unbiased immunoprecipitation (IP) mass spectrometry experiment to identify novel PDK4-binding proteins. We transiently overexpressed FLAG-tagged PDK4 in HEK293T cells and examined the proteins that specifically bound to PDK4 using IP, followed by mass spectrometry (Additional file 8: Table S2). Interestingly, AIF was one of the proteins that showed a prominent interaction with PDK4 (Figure 4A and Additional file 8: Table S2), which we confirmed by IP-Western blot analysis (Figure 4B).Figure 4


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)

AIF interacts with PDK4 and plays a role in EMT. (A) HEK293T cells were transfected with the pCMV6-Entry empty vector or pCMV6-FLAG-Myc-PDK4. Forty-eight hours post-transfection, the cells were lysed and FLAG IP was performed. Mass spectrometry was performed to analyze the immunoprecipitates, and the top five proteins identified in the immunoprecipitates are shown. (B) Immunoblotting was performed with samples prepared as in A to confirm the PDK4-AIF interaction. (C) A549 cells and HCC827 cells were transfected with siNTC pool no. 2, siAIF pool, or siPDK4 pool at 1 day and 3 days post-seeding, then lysed for immunoblotting. (D) HCC827 cells were transfected with siNTC pool no. 2, siAIF pool, and siPDK4 pool, treated with erlotinib (erl), and stained with crystal violet as in Figure 3C. (E) HCC827 cells were transfected as in D. Two days after the second transfection, the cells were stained with CM-H2DCFDA and ROS levels were analyzed using flow cytometry.
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Related In: Results  -  Collection

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Fig4: AIF interacts with PDK4 and plays a role in EMT. (A) HEK293T cells were transfected with the pCMV6-Entry empty vector or pCMV6-FLAG-Myc-PDK4. Forty-eight hours post-transfection, the cells were lysed and FLAG IP was performed. Mass spectrometry was performed to analyze the immunoprecipitates, and the top five proteins identified in the immunoprecipitates are shown. (B) Immunoblotting was performed with samples prepared as in A to confirm the PDK4-AIF interaction. (C) A549 cells and HCC827 cells were transfected with siNTC pool no. 2, siAIF pool, or siPDK4 pool at 1 day and 3 days post-seeding, then lysed for immunoblotting. (D) HCC827 cells were transfected with siNTC pool no. 2, siAIF pool, and siPDK4 pool, treated with erlotinib (erl), and stained with crystal violet as in Figure 3C. (E) HCC827 cells were transfected as in D. Two days after the second transfection, the cells were stained with CM-H2DCFDA and ROS levels were analyzed using flow cytometry.
Mentions: To gain additional insight into PDK4’s role in EMT, we sought to identify proteins that interact with PDK4. Considering the very limited information regarding PDK4-interacting proteins, we performed an unbiased immunoprecipitation (IP) mass spectrometry experiment to identify novel PDK4-binding proteins. We transiently overexpressed FLAG-tagged PDK4 in HEK293T cells and examined the proteins that specifically bound to PDK4 using IP, followed by mass spectrometry (Additional file 8: Table S2). Interestingly, AIF was one of the proteins that showed a prominent interaction with PDK4 (Figure 4A and Additional file 8: Table S2), which we confirmed by IP-Western blot analysis (Figure 4B).Figure 4

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