PEPCK-M recoups tumor cell anabolic potential in a PKC-ζ-dependent manner

Abstract

Background Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4-dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated. Methods [U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo. Results PEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS. In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo. Conclusions All in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress.

From OCR and ECAR to energy: Perspectives on the design and interpretation of bioenergetics studies

“…For example, measurement of the electrochemical potential across the inner mitochondrial membrane (IMM) is proportional to the energetic state of the ETS and can be measured potentiometrically or with fluorescent organic cations ( e.g. , tetramethyl rhodamine methyl ester) ( 31 , 32 ). The redox potential of the reduced pyridine nucleotide pool (NAD(P)H) within specific subcellular compartments can be proportional to the energetic state of specific pathways and can be monitored via autofluorescence (ideally fluorescence lifetime) ( 33 , 34 ).…”

“…It is directly proportional to the power transferred through the phosphate potential ( Box 1 ), which if measured over the duration of a cellular process is proportional to the total work required by the process itself ( 74 ). Directly measuring J ATP is complex and limited by technical challenges ( 75 ), but estimating J ATP from EFA data is straightforward and has been recently described in detail ( 9 , 10 , 31 ). Importantly, there is a major caveat to this approach: i.e.…”

Phosphoenolpyruvate carboxykinase in cell metabolism: Roles and mechanisms beyond gluconeogenesis

“…PCK diverts TCA intermediate metabolites into cytosol, thus probably participating in anaplerosis and cataplerosis involving amino acids. PCK2-overexpressing cells exhibit high anabolic activity with higher amino acid consumption from culture media, especially for glycine and proline [ 26 ]. After breaking down and entering TCA, amino acid products are further converted into PEP catalyzed by PCK and fed into the serine biosynthetic pathway [ 27 ].…”

“…Thereafter, PEP is utilized in anabolism to produce amino acids required for growth, especially serine, glycine, and proline. Silencing or inhibiting PCK2 shuts down the TCA cycle and increases ROS, leading to impaired proteostasis and limited growth of cancer cells [ 26 ]. Vincent et al.…”

Multifaceted Role of AMPK in Viral Infections

“…Activated AMPK promotes catabolic pathways with respect to glucose metabolism, such as glucose uptake and glycolysis, and inhibits gluconeogenesis activation ( Figure 3 ). AMPK regulates glucose uptake by translocation of glucose transporter 4 (GLUT4) and glucose transporter 1 (GLUT1) by phosphorylating and inhibiting RAB GTPase-activating protein (GAP), TBC1 domain family member 1 (TBC1D1) [ 42 ] and thioredoxin-interacting protein (TXNIP) [ 43 ], respectively. Uptake of glucose is also promoted by AMPK phosphorylation of phosphoinositide phosphate kinase, phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) and phospholipase D1 (PLD1), which regulates GLUT4 translocation, thus indirectly increasing glucose uptake [ 44 , 45 ].…”