Phosphatidic acid (PA) is a critical metabolite at the heart of membrane phospholipid biosynthesis. However, PA also serves as a critical lipid second messenger that regulates several proteins implicated in the control of cell cycle progression and cell growth. Three major metabolic pathways generate PA: phospholipase D (PLD), diacylglycerol kinase (DGK), and lysophosphatidic acid acyltransferase (LPAAT). The LPAAT pathway is integral to de novo membrane phospholipid biosynthesis, whereas the PLD and DGK pathways are activated in response to growth factors and stress. The PLD pathway is also responsive to nutrients. A key target for the lipid second messenger function of PA is mTOR, the mammalian/mechanistic target of rapamycin, which integrates both nutrient and growth factor signals to control cell growth and proliferation. Although PLD has been widely implicated in the generation of PA needed for mTOR activation, it is becoming clear that PA generated via the LPAAT and DGK pathways is also involved in the regulation of mTOR. In this minireview, we highlight the coordinated maintenance of intracellular PA levels that regulate mTOR signals stimulated by growth factors and nutrients, including amino acids, lipids, glucose, and Gln. Emerging evidence indicates compensatory increases in one source of PA when another source is compromised, highlighting the importance of being able to adapt to stressful conditions that interfere with PA production. The regulation of PA levels has important implications for cancer cells that depend on PA and mTOR activity for survival.

mTOR, the mammalian target of rapamycin, integrates growth factor and nutrient signals to promote a transformation from catabolic to anabolic metabolism, cell growth, and cell cycle progression. Phosphatidic acid (PA) interacts with the FK506-binding protein-12-rapamycin-binding (FRB) domain of mTOR, which stabilizes both mTOR complexes: mTORC1 and mTORC2. We report here that mTORC1 and mTORC2 are activated in response to exogenously supplied fatty acids via the synthesis of PA, a central metabolite for membrane phospholipid biosynthesis. We examined the impact of exogenously supplied fatty acids on mTOR in KRas-driven cancer cells, which are programmed to utilize exogenous lipids. The induction of mTOR by oleic acid was dependent upon the enzymes responsible for synthesis of PA. Suppression of the synthesis of PA resulted in G cell cycle arrest. Although it has long been appreciated that mTOR is a sensor of amino acids and glucose, this study reveals that mTOR also senses the presence of lipids via production of PA.

ObjectiveIn multicellular organisms, cell division is regulated by growth factors (GFs). In the absence of GFs, cells exit the cell cycle at a site in G1 referred to as the restriction point (R) and enter a state of quiescence known as G0. Additionally, nutrient availability impacts on G1 cell cycle progression. While there is a vast literature on G1 cell cycle progression, confusion remains – especially with regard to the temporal location of R relative to nutrient-mediated checkpoints. In this report, we have investigated the relationship between R and a series of metabolic cell cycle checkpoints that regulate passage into S-phase.MethodsWe used double-block experiments to order G1 checkpoints that monitor the presence of GFs, essential amino acids (EEAs), the conditionally essential amino acid glutamine, and inhibition of mTOR. Cell cycle progression was monitored by uptake of [3H]-thymidine and flow cytometry, and analysis of cell cycle regulatory proteins was by Western-blot.ResultsWe report here that the GF-mediated R can be temporally distinguished from a series of late G1 metabolic checkpoints mediated by EAAs, glutamine, and mTOR – the mammalian/mechanistic target of rapamycin. R is clearly upstream from an EAA checkpoint, which is upstream from a glutamine checkpoint. mTOR is downstream from both the amino acid checkpoints, close to S-phase. Significantly, in addition to GF autonomy, we find human cancer cells also have dysregulated metabolic checkpoints.ConclusionThe data provided here are consistent with a GF-dependent mid-G1 R where cells determine whether it is appropriate to divide, followed by a series of late-G1 metabolic checkpoints mediated by amino acids and mTOR where cells determine whether they have sufficient nutrients to accomplish the task. Since mTOR inhibition arrests cells the latest in G1, it is likely the final arbiter for nutrient sufficiency prior to committing to replicating the genome.

tative of at least 3 independent experiments. Statistical differences between groups were determined using 2-tailed unpaired Student's t test, Mann-Whitney U test, or 1-way ANOVA with post hoc Tukey's honestly significant difference test. Correlation was tested using Pearson's method. Statistical analysis was performed using Microsoft Office and Prism 6.0 software (Graph-Pad).

During G 1 -phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G 1 to the presence of nutrients that indicate sufficient raw material to generate two daughter cells. Dividing cells rely on the "conditionally essential" amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G 1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G 1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to ␣-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers.

Glutamine is a key nutrient required for sustaining cell proliferation, contributing to nucleotide, protein, and lipid synthesis. The mTOR complex 1 (mTORC1) is a highly conserved protein complex that acts as a sensor of nutrients, relaying signals for the shift from catabolic to anabolic metabolism. Although glutamine plays an important role in mTORC1 activation, the mechanism is not clear. Here we describe a leucine- and Rag-independent mechanism of mTORC1 activation by glutamine that depends on phospholipase D and the production of phosphatidic acid, which is required for the stability and activity of mTORC1. The phospholipase D-dependent activation of mTORC1 by glutamine depended on the GTPases ADP ribosylation factor 1 (Arf1), RalA, and Rheb. Glutamine deprivation could be rescued by α-ketoglutarate, a downstream metabolite of glutamine. This mechanism represents a distinct nutrient input to mTORC1 that is independent of Rag GTPases and leucine.

Abbreviations: 4E-BP1, eIF4E binding protein-1; eIF4E, eukaryotic initiation factor 4E; Gln, glutamine; GOT, glutamateoxaloacetate-transaminase; mTOR, mammalian target of rapamycin; mTORC1/2, mTOR complex 1/2; PARP, poly-ADP-ribose polymerase; PI3K, phosphatidylinositol-3-kinase; S6K, S6 kinase; TGF-b, transforming growth factor-b.Mutations in genes encoding regulators of mTOR, the mammalian target of rapamycin, commonly provide survival signals in cancer cells. Rapamycin and analogs of rapamycin have been used with limited success in clinical trials to target mTOR-dependent survival signals in a variety of human cancers. Suppression of mTOR predominantly causes G1 cell cycle arrest, which likely contributes to the ineffectiveness of rapamycin-based therapeutic strategies. While rapamycin causes the accumulation of cells in G1, its effect in other cell cycle phases remains largely unexplored. We report here that when synchronized MDA-MB-231 breast cancer cells are allowed to progress into S-phase from G 1 , rapamycin activates the apoptotic machinery with a concomitant increase in cell death. In Calu-1 lung cancer cells, rapamycin induced a feedback increase in Akt phosphorylation at Ser473 in S-phase that mitigated rapamycin-induced apoptosis. However, sensitivity to rapamycin in S-phase could be reestablished if Akt phosphorylation was suppressed. We recently reported that glutamine (Gln) deprivation causes K-Ras mutant cancer cells to aberrantly arrest primarily in S-phase. Consistent with observed sensitivity of S-phase cells to rapamycin, interfering with Gln utilization sensitized both MDA-MB-231 and Calu-1 K-Ras mutant cancer cells to the apoptotic effect of rapamycin. Importantly, rapamycin induced substantially higher levels of cell death upon Gln depletion than that observed in cancer cells that were allowed to progress through S-phase after being synchronized in G 1 . We postulate that exploiting metabolic vulnerabilities in cancer cells such as S-phase arrest observed with K-Ras-driven cancer cells deprived of Gln, could be of great therapeutic potential.

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