Abstract
Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism.
Highlights
The integrated stress response (ISR) is necessary to help the tumor adapt to its microenvironment
Glutamine represents the quantitatively most important TCA cycle fuel in cancer cells; we investigated whether PCK2 mRNA is regulated by withdrawal of these amino acids. qRT-PCR analysis confirmed that PCK2 mRNA content increased at least 2-fold after glutamine deprivation in all cell lines tested (Fig. 1D)
An amino acid response element (AARE) Consensus Site at the PCK2 Promoter Recruits ATF4 to Activate Transcription under amino acid response (AAR) or ER Stress—Transcriptional up-regulation was responsible for increased PEPCK-M protein levels observed; we evaluated whether a downstream transcriptional effector of ER or amino acid stress response directly mediated this induction
Summary
The integrated stress response (ISR) is necessary to help the tumor adapt to its microenvironment. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Eukaryotes have evolved complex mechanisms to allow cells to confront and adapt to variable conditions such as nutrient limitations One such process, known as integrated stress response (ISR), collectively groups several signaling pathways. 2 Recipient of a fellowship from the Ministerio de Economia y Competitividad (FPI). In the present work we describe the selective expression of PCK2 in several human tumors and all cancer cell lines studied, suggesting that PEPCK-M activity might support specific purposes in the context of tumor metabolic adaptations. The mechanism of PCK2 gene regulation in cancer cells under ISR requires recruiting ATF4 to a consensus amino acid response element (AARE) sequence located in the PEPCK-M proximal promoter. PEPCK-M up-regulation is a novel metabolic adaptation in cancer
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