Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth

Abstract Background Pyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit....

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Autores principales:	Nancy P. Echeverri Ruiz, Vijay Mohan, Jinghai Wu, Sabina Scott, McKenzie Kreamer, Martin Benej, Tereza Golias, Ioanna Papandreou, Nicholas C. Denko
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Hypoxia Glucose metabolism Orthotopic pancreatic tumors Pyruvate dehydrogenase Mitochondria Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
Acceso en línea:	https://doaj.org/article/d422a91c2933419ab609c3d888089031
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spelling	oai:doaj.org-article:d422a91c2933419ab609c3d8880890312021-11-14T12:37:03ZDynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth10.1186/s40170-021-00275-42049-3002https://doaj.org/article/d422a91c2933419ab609c3d8880890312021-11-01T00:00:00Zhttps://doi.org/10.1186/s40170-021-00275-4https://doaj.org/toc/2049-3002Abstract Background Pyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1–4) and two reactivating phosphatases (PDP1, 2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth. Methods To isolate the function of the serine-dependent regulation of PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293, and 300 or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice. Results In this family of cells with non-phosphorylatable PDHA1, we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice. Conclusions We found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had a very modest reduction of growth in vitro, but failed to grow as tumors indicating that dynamic PDC adaptation to microenvironmental conditions is necessary to support pancreatic cancer growth in vivo.Nancy P. Echeverri RuizVijay MohanJinghai WuSabina ScottMcKenzie KreamerMartin BenejTereza GoliasIoanna PapandreouNicholas C. DenkoBMCarticleHypoxiaGlucose metabolismOrthotopic pancreatic tumorsPyruvate dehydrogenaseMitochondriaNeoplasms. Tumors. Oncology. Including cancer and carcinogensRC254-282ENCancer & Metabolism, Vol 9, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Hypoxia Glucose metabolism Orthotopic pancreatic tumors Pyruvate dehydrogenase Mitochondria Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
spellingShingle	Hypoxia Glucose metabolism Orthotopic pancreatic tumors Pyruvate dehydrogenase Mitochondria Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282 Nancy P. Echeverri Ruiz Vijay Mohan Jinghai Wu Sabina Scott McKenzie Kreamer Martin Benej Tereza Golias Ioanna Papandreou Nicholas C. Denko Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth
description	Abstract Background Pyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1–4) and two reactivating phosphatases (PDP1, 2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth. Methods To isolate the function of the serine-dependent regulation of PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293, and 300 or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice. Results In this family of cells with non-phosphorylatable PDHA1, we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice. Conclusions We found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had a very modest reduction of growth in vitro, but failed to grow as tumors indicating that dynamic PDC adaptation to microenvironmental conditions is necessary to support pancreatic cancer growth in vivo.
format	article
author	Nancy P. Echeverri Ruiz Vijay Mohan Jinghai Wu Sabina Scott McKenzie Kreamer Martin Benej Tereza Golias Ioanna Papandreou Nicholas C. Denko
author_facet	Nancy P. Echeverri Ruiz Vijay Mohan Jinghai Wu Sabina Scott McKenzie Kreamer Martin Benej Tereza Golias Ioanna Papandreou Nicholas C. Denko
author_sort	Nancy P. Echeverri Ruiz
title	Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth
title_short	Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth
title_full	Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth
title_fullStr	Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth
title_full_unstemmed	Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth
title_sort	dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth
publisher	BMC
publishDate	2021
url	https://doaj.org/article/d422a91c2933419ab609c3d888089031
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Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth

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