Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts

doi:10.1016/0003-9861(91)90084-V

Archives of Biochemistry and Biophysics

Volume 286, Issue 2, 1 May 1991, Pages 586-590

https://doi.org/10.1016/0003-9861(91)90084-V Get rights and content

Abstract

Fructose 2,6-bisphosphate (F-2,6-P₂) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P₂ than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F₁) or inorganic phosphate (P_i) further stimulated lactate production in both cell lines. F-2,6-P₂ stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P₂. In the absence of F-2,6-P₂ the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mm, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mm ATP. F-2,6-P₂ reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 μm (in the presence of 2 mm ATP and 1 μm F-2,6-P₂) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 μm F-2,6-P₂) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P₂ was observed after transfection of rat-1 flbroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.

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Cancer has affected the lives of millions worldwide and is truly regarded as a devastating disease process. Despite advanced understanding of the genomic underpinning of cancer development and progression, therapeutic challenges are still persistent. Among all the human cancers, around 33% are attributed to mutations in RAS oncogene, a crucial component of the signaling pathways. With time, our understanding of RAS circuitry has improved and now the fact that it activates several downstream effectors, depending on the type and grades of cancer has been established. The circuitry is controlled via post-transcriptional mechanisms and frequent distortions in these mechanisms lead to important metabolic as well as immunological states that favor cancer cells' growth, survival, plasticity and metastasis. Therefore, understanding RAS circuitry can help researchers/clinicians to develop novel and potent therapeutics that, in turn, can save the lives of patients suffering from RAS-mutant cancers. There are many challenges presented by resistance and the potential strategies with a particular focus on novel combinations for overcoming these, that could move beyond transitory responses in the direction of treatment. Here in this review, we will look at how understanding the circuitry of RAS can be put to use in making strategies for developing therapeutics against RAS- driven malignancies.
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Citation Excerpt :
PFK1 exists in tetrameric form depending on tissue and cell types, where each contains three kinds of subunits: PFKL (liver and kidney), PFKM (muscle), and PFKP (platelets). A high cellular ATP/ADP ratio inhibits PFK1 activity [146]. PFK2 regulation has a major role in cancer metabolism. [147].
Cancer cells undergo metabolic alterations to meet the immense demand for energy, building blocks, and redox potential. Tumors show glucose-avid and lactate-secreting behavior even in the presence of oxygen, a process known as aerobic glycolysis. Glycolysis is the backbone of cancer cell metabolism, and cancer cells have evolved various mechanisms to enhance it. Glucose metabolism is intertwined with other metabolic pathways, making cancer metabolism diverse and heterogeneous, where glycolysis plays a central role. Oncogenic signaling accelerates the metabolic activities of glycolytic enzymes, mainly by enhancing their expression or by post-translational modifications. Aerobic glycolysis ferments glucose into lactate which supports tumor growth and metastasis by various mechanisms. Herein, we focused on tumor glycolysis, especially its interactions with the pentose phosphate pathway, glutamine metabolism, one-carbon metabolism, and mitochondrial oxidation. Further, we describe the role and regulation of key glycolytic enzymes in cancer. We summarize the role of lactate, an end product of glycolysis, in tumor growth, and the metabolic adaptations during metastasis. Lastly, we briefly discuss limitations and future directions to improve our understanding of glucose metabolism in cancer.
Tristetraprolin posttranscriptionally downregulates PFKFB3 in cancer cells
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Among the four isozymes, PFKFB3 is of particular interest because it is overexpressed in cancers [5–7]. In cancer cells, PFKFB3 expression is induced by stimuli such as oncogenic Ras signaling [8,9] and hypoxia [8]. Inhibition of PFKFB3 by small molecule antagonists [10], Ras inhibition [11], or knockdown of PFKFB3 [12] decreases glycolytic activity and suppresses tumor growth.
The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 3 (PFKFB3) catalyzes the first committed rate-limiting step of glycolysis and is upregulated in cancer cells. The mechanism of PFKFB3 expression upregulation in cancer cells has not been fully elucidated. The PFKFB3 3′-UTR is reported to contain AU-rich elements (AREs) that are important for regulating PFKFB3 mRNA stability. However, the mechanisms by which PFKFB3 mRNA stability is determined by its 3′-UTR are not well known. We demonstrated that tristetraprolin (TTP), an ARE-binding protein, has a critical function regulating PFKFB3 mRNA stability. Our results showed that PFKFB3 mRNA contains three AREs in the 3′-UTR. TTP bound to the 3rd ARE and enhanced the decay of PFKFB3 mRNA. Overexpression of TTP decreased PFKFB3 expression and ATP levels but increased GSH level in cancer cells. Overexpression of PFKFB3 cDNA without the 3′-UTR rescued ATP level and GSH level in TTP-overexpressing cells. Our results suggested that TTP post-transcriptionally downregulated PFKFB3 expression and that overexpression of TTP may contribute to suppression of glycolysis and energy production of cancer cells in part by downregulating PFKFB3 expression.
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This change occurred via the stimulation of the cytosolic part of the glutamate-aspartate shuttle [202], which is normally used to reoxidize cytosolic NADH [220,221]. It was also shown that KRAS upregulates aspartate transaminase (GOT) and represses the expression of glutamate dehydrogenase 1 (GLUD1) [222,223]. Thus, KRAS rewires metabolic fluxes through a novel pathway whereby glutamine-derived aspartate produced by the mitochondrial aspartate aminotransferase (GOT2) is converted to oxaloacetate by the cytosolic aspartate aminotransferase (GOT1).
The RAS pathway is a highly conserved cascade of protein-protein interactions and phosphorylation that is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Recent findings indicate that the RAS pathway plays a role in the regulation of energy metabolism via the control of mitochondrial form and function but little is known on the participation of this effect in RAS-related rare human genetic diseases. Germline mutations that hyperactivate the RAS pathway have been discovered and linked to human developmental disorders that are known as RASopathies. Individuals with RASopathies, which are estimated to affect approximately 1/1000 human birth, share many overlapping characteristics, including cardiac malformations, short stature, neurocognitive impairment, craniofacial dysmorphy, cutaneous, musculoskeletal, and ocular abnormalities, hypotonia and a predisposition to developing cancer. Since the identification of the first RASopathy, type 1 neurofibromatosis (NF1), which is caused by the inactivation of neurofibromin 1, several other syndromes have been associated with mutations in the core components of the RAS-MAPK pathway. These syndromes include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), which was formerly called LEOPARD syndrome, Costello syndrome (CS), cardio-facio-cutaneous syndrome (CFC), Legius syndrome (LS) and capillary malformation–arteriovenous malformation syndrome (CM-AVM). Here, we review current knowledge about the bioenergetics of the RASopathies and discuss the molecular control of energy homeostasis and mitochondrial physiology by the RAS pathway.
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Identification of a blood-based biomarker that can detect early cognitive decline presents a significant healthcare challenge. We prepared peripheral blood mononuclear cells (PBMCs) from individuals who had a poorer than predicted performance in their delayed recall performance on the Logical Memory II Subtest of the Wechsler Memory Scale (WMS) relative to their IQ estimated by the National Adult Reading Test (NART); we described these individuals as IQ-discrepant, compared with IQ-consistent, individuals. Stimulation with Aβ + LPS increased production of TNFα to a greater extent in cells from IQ-discrepant, compared with IQ-consistent, individuals. This was associated with a shift towards glycolysis and the evidence indicates that 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3 plays a role in driving glycolysis. A similar shift towards glycolysis was observed in MDMs prepared from IQ-discrepant, compared with IQ-consistent, individuals. The important finding here is that we have established an increased sensitivity to Aβ + LPS stimulation in PBMCs from individuals that under-perform on a memory task, relative to their estimated premorbid IQ, which may be an indicator of early cognitive decline. This may be a useful tool in determining the presence of early cognitive dysfunction.

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^∗: Present address: Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.

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Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts

Abstract

FEBS Lett

Biochem. Biophys. Res. Commun

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Cell

Cell

Nature