Cancer cells exhibit a dynamic metabolic landscape and require a sufficient supply of nucleotides and other macromolecules to grow and proliferate. To meet the metabolic requirements for cell growth, cancer cells must stimulate de novo nucleotide synthesis to obtain adequate nucleotide pools to support nucleic acid and protein synthesis along with energy preservation, signaling activity, glycosylation mechanisms, and cytoskeletal function. Both oncogenes and tumor suppressors have recently been identified as key molecular determinants for de novo nucleotide synthesis that contribute to the maintenance of homeostasis and the proliferation of cancer cells. Inactivation of tumor suppressors such as TP53 and LKB1 and hyperactivation of the mTOR pathway and of oncogenes such as MYC, RAS, and AKT have been shown to fuel nucleotide synthesis in tumor cells. The molecular mechanisms by which these signaling hubs influence metabolism, especially the metabolic pathways for nucleotide synthesis, continue to emerge. Here, we focus on the current understanding of the molecular mechanisms by which oncogenes and tumor suppressors modulate nucleotide synthesis in cancer cells and, based on these insights, discuss potential strategies to target cancer cell proliferation.

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Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) ؉ glutamate (YNB ؉ Glu), or YNB ؉ aspartate (YNB ؉ Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB ؉ Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB ؉ Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB ؉ Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1. We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris. In addition to GDH2, enzymes such as aspartate aminotransferase (AAT), 2 malate dehydrogenase (MDH), and glutamine synthetase (GLN1) also play key roles in the metabolism of amino acids (4). In S. cerevisiae, AAT1 and AAT2 encode AAT localized in mitochondria (mAAT) and cytoplasm (cAAT). cAAT catalyzes the reversible conversion of glutamate and oxaloacetate into ␣-ketoglutarate and aspartate (4). The oxaloacetate thus generated is converted to malate by MDH present in the cytoplasm (cMDH) encoded by MDH2. Malate enters mitochondria via ␣-ketoglutarate-malate exchanger protein, which also transports ␣-ketoglutarate in the opposite direction. Malate is oxidized to oxaloacetate by the mitochondrial MDH (mMDH) encoded by MDH1, resulting in the formation of NADH, which enters the electron transport chain and generates energy (4). The mitochondrial oxaloacetate is converted to aspartate by mAAT, which is transported to cytoplasm via the aspartate-glutamate exchanger. Glutamate present in the cytoplasm is converted to glutamine by GLN1 (4). Saccharomyces cerevisiaeP. pastoris, a methylotrophic yeast, is extensively used for the production of recombinant proteins. Being a respiratory yeast, P. pastoris completely oxidizes sugars, avoiding formation of ethanol, and this results in efficient utilization of carbon sources yielding high biomass. During high cell density fermentation of P. pastoris, ammonium...

The ability of yeast cells to grow in the presence of diverse carbon sources offers a unique opportunity to study various metabolic pathways, which is not always feasible in higher eukaryotic systems. In addition to glucose, yeast cells can utilize acetate, ethanol, glycerol, or fatty acids as the sole source of carbon, and the study of their metabolism and regulation has been one of the fascinating areas of biochemistry. The regulation of metabolic pathways of respiratory yeasts such as Pichia pastoris has not been as well studied as that of Saccharomyces cerevisiae despite the extensive use of the former for the commercial production of recombinant proteins. P. pastoris, a methylotrophic yeast, can metabolize a number of compounds, such as glycerol, methanol, acetate, and oleic acid, in addition to glucose. However, very little information is available on the transcriptional regulation of metabolic pathways other than the methanol utilization (mut) 2 pathway in this yeast species. The expression of genes of the mut pathway is regulated by at least three zinc finger proteins (1-6). Of these, methanol expression regulator 1 (Mxr1p) activates the expression of genes of the mut pathway by binding to Mxr1p response elements (MXREs) in their promoters (2, 3). Rop1p has the same DNA binding specificity as Mxr1p and functions as a repressor of genes of the mut pathway in P. pastoris cultured in nutrientrich medium containing yeast extract, peptone, and methanol (YPM) but not minimal medium containing a yeast nitrogen base, ammonium sulfate, and methanol (YNBM) (4, 5). Trm1p is also essential for the expression of genes of the mut pathway (6). However, its mechanism of action remains unknown. The differential regulation of methanol metabolism in YNBM and YPM by Mxr1p and Rop1p led us to investigate the transcriptional regulation of other metabolic pathways in cells cultured in minimal and nutrient-rich media. In this study, we demonstrate that Mxr1p regulates acetate metabolism only in cells cultured in nutrient-rich medium containing yeast extract, peptone, and acetate (YPA) but not minimal medium containing yeast nitrogen base, ammonium sulfate and acetate (YNBA). Experimental ProceduresYeast and Bacterial Strains-P. pastoris (GS115, his Ϫ ) was cultured in either nutrient-rich medium (1.0% yeast extract and 2.0% peptone) containing 2.0% glucose (YPD) or 2.0% acetate (YPA) or minimal yeast nitrogen base medium (0.17% yeast nitrogen base without amino acids and 0.5% ammonium sulfate) supplemented with 2.0% glucose (YNBD) or 2.0% sodium acetate (YNBA). Casamino acids (CAAs) and glutamate were added to YNBA medium to final concentrations of 1.0% and 0.5%, respectively. P. pastoris strains were grown at 30°C in an * This study was supported by Grant BT/PR3889/BRB/10/996/2011 from the Department of Biotechnology, New Delhi, India (to P. N. R.