Original ArticleGOT1-mediated anaplerotic glutamine metabolism regulates chronic acidosis stress in pancreatic cancer cells
Introduction
Metabolic alterations represent an important hallmark of cancer cells [1]. Metabolic reprogramming allows cancer cells to sustain uncontrolled proliferation by rapid generation of ATP, biosynthesis of macromolecules, and maintenance of redox status [2]. Cancer cells can also reprogram the major metabolic pathways (carbohydrates, proteins, lipids, and nucleic acids) to meet these basic demands for uncontrolled proliferation [3], [4]. The characteristic metabolic phenotype seen in cancer cells is the Warburg effect, which operates by enhancing glucose uptake and flux into glycolysis, while simultaneously diminishing the glucose carbon flux that enters the TCA cycle in the mitochondria, even in the presence of oxygen [5], [6]. Although ATP generation through substrate level phosphorylation is very rapid, this mechanism is far less efficient than oxidative phosphorylation in generating energy from glucose. Thus, the metabolic phenotype observed in the Warburg effect demands very high glucose uptake to meet the energetic, biosynthetic, and redox needs of cancer cells. For these reasons, the increased glucose uptake of cancer cells is useful for diagnosing cancer using radiolabeled glucose analog 18F-fluorodeoxyglucose and positron emission tomography (FDG-PET) to image and evaluate tumor progression without the need of a biopsy [7], [8].
Because of the enhanced metabolic rate of rapidly proliferating tumor cells, the glucose that is metabolized through substrate level phosphorylation produces lactic acid as the end product. Lactic acid is a weak acid, and it quickly dissociates and loses a hydrogen ion to produce lactate [9]. Lactate is transported outside of the cell by monocarboxylate symporters along with protons resulting in decreased pH in the extracellular milieu [10], [11]. Intracellular hydrogen ions can also be removed by sodium hydrogen exchangers that import sodium ions and extrude hydrogen ions, thereby acidifying the extracellular environment [12], [13]. Similarly, vacuolar ATPases extrude hydrogen ions against their concentration gradient to the extracellular space, and hence, lower the extracellular pH [14]. In vitro studies have shown that rapidly growing cells, which exhibit the Warburg effect, increase the expression of these cell surface proteins to maintain an alkaline intracellular pH environment [15], [16]. Indeed, increased intracellular pH is an established permissive signal for cellular proliferation promoting survival by limiting apoptosis, a process that is associated with intracellular acidification [17], [18]. The role of low extracellular pH in carcinogenesis is thus paradoxical: on one hand, alkaline intracellular pH promotes proliferation and survival, while at the same time, low extracellular pH promotes invasion and metastasis at the cost of inducing stress, senescence, and apoptosis [12], [19], [20].
In addition to glucose, glutamine metabolism is also essential for the proliferation of cancer cells. Recent studies have demonstrated that glutamate derived from glutamine is utilized by highly proliferative cells to generate non-essential amino acids (NEAAs) through the glutamic-oxaloacetic transaminase enzymes (GOT1 and GOT2), while quiescent cells metabolize glutamate through GLUD1 (glutamate dehydrogenase 1) and subsequent decarboxylation reactions in the TCA cycle [21], [22]. Thus, glutamine can be metabolized through both anabolic (anaplerotic) and catabolic pathways.
Several oncogenes are implicated in reprogramming tumor cell metabolism. One such gene is KRAS, which upon accumulating activating mutations serves as a key signature oncogene that plays a prominent role in malignant transformation and tumor progression in PDAC [23], [24]. PDAC cells with oncogenic KRAS have reprogrammed glucose and glutamine metabolism to serve anabolic processes [25], [26]. Canonical glutamine metabolism occurs through glutamate synthase (GLS)-mediated conversion of cytoplasmic glutamine into glutamate. Glutamate is then metabolized in the mitochondria through GLUD1 into alpha-ketoglutarate that enters the TCA cycle [27]. The non-canonical pathway metabolizes glutamate to aspartate and alpha-ketoglutarate through GOT2; aspartate is subsequently metabolized to oxaloacetate by GOT1 in the cytosolic compartment. Aspartate is metabolized by malate dehydrogenase (MDH) to malate, which is then metabolized by malic enzyme (ME) to produce pyruvate. These anaplerotic reactions increase the NADPH/NADP ratio thereby maintaining reactive oxygen species (ROS) balance. PDAC cells are dependent on these reactions for maintenance of intracellular ROS levels as it is evidenced by the decrease in cell survival upon knockdown of enzymes in the pathway [26].
Due to metabolic reprogramming by oncogenic KRAS present in 90% of PDAC cases, extracellular acidification is highly abundant. While the regulation of pH in cancer cells has been studied thoroughly, the metabolic adaptations to chronic acidosis induced stress are not well defined. Therefore, in the current study, we investigated the metabolic basis of adaptation to chronic low pH stress in PDAC cells, which exhibit high glycolytic capacity, by subjecting them to chronic acidosis. We utilized PDAC cells with oncogenic KRAS to identify the metabolomic alterations under chronic acidosis and identify vulnerabilities for therapy. Here, we report a pronounced increase in non-canonical anaplerotic glutamine metabolism, which serves the bioenergetic needs and maintains ROS balance in cells undergoing acidosis stress.
Section snippets
Cell culture
Cell culture of PDAC cell lines S2-013 and Capan-1 have been described previously [28], [29]. Cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich D5648) containing 4.5 g/L of glucose and 0.584 g/L of glutamine (Hyclone); additionally, the media was supplemented with 5% FBS. Low pH of the media was set at 6.9–7.1 by adding 1 g/L NaHCO3 and control pH was set by using 3.7 g/L NaHCO3. To establish chronic low pH exposure, we cultured the cells in pH 6.9–7.0
Pancreatic cancer cell growth is diminished under low pH conditions
Intracellular pH is known to have a significant role in conveying proliferation and death signals [16]. For example, it has been observed that proliferating cells require an intracellular alkaline pH value greater than 7.2, to allow growth-factor stimulated cells to enter the S-phase of the cell cycle at a faster rate, and proceed to the G2 and M phases more rapidly [33], [34]. Furthermore, a higher pH is known to suppress mitotic arrest due to activated DNA damage checkpoints; therefore,
Discussion
Acidification of the tumor microenvironment is a common feature of PDAC (and of most epithelial tumors). Understanding the metabolic changes that PDAC cells undergo due to acidosis stress is extremely critical in designing more effective treatments. Our metabolomic analysis shows that cells in low pH depart from Warburg effect metabolism and that they increase anaplerotic glutamine metabolism to allow cells to generate vast amounts of ATP, which in turn allows for maintaining cellular
Acknowledgements
This work was supported in part by funding from the National Institutes of Health grant (R01 CA163649, R01 CA210439, and R01 CA216853, NCI) to PKS, American Association for Cancer Research (AACR)—Pancreatic Cancer Action Network (PanCAN) Career Development Award (30-20-25-SING) to PKS, and the Specialized Programs of Research Excellence (SPORE, 2P50 CA127297, NCI) to PKS. We would also like to acknowledge the Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727, NCI) for supporting
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