Jaime Abrego

MUC1 and HIF-1alpha Signaling Crosstalk Induces Anabolic Glucose Metabolism to Impart Gemcitabine Resistance to Pancreatic Cancer.

Poor response to cancer therapy due to resistance remains a clinical challenge. The present study establishes a widely prevalent mechanism of resistance to gemcitabine in pancreatic cancer, whereby increased glycolytic flux leads to glucose addiction in cancer cells and a corresponding increase in pyrimidine biosynthesis to enhance the intrinsic levels of deoxycytidine triphosphate (dCTP). Increased levels of dCTP diminish the effective levels of gemcitabine through molecular competition. We also demonstrate that MUC1-regulated stabilization of hypoxia inducible factor-1α (HIF-1α) mediates such metabolic reprogramming. Targeting HIF-1α or de novo pyrimidine biosynthesis, in combination with gemcitabine, strongly diminishes tumor burden. Finally, reduced expression of TKT and CTPS, which regulate flux into pyrimidine biosynthesis, correlates with better prognosis in pancreatic cancer patients on fluoropyrimidine analogs.

GOT1-mediated anaplerotic glutamine metabolism regulates chronic acidosis stress in pancreatic cancer cells

The increased rate of glycolysis and reduced oxidative metabolism are the principal biochemical phenotypes observed in pancreatic ductal adenocarcinoma (PDAC) that lead to the development of an acidic tumor microenvironment. The pH of most epithelial cell-derived tumors is reported to be lower than that of plasma. However, little is known regarding the physiology and metabolism of cancer cells enduring chronic acidosis. Here, we cultured PDAC cells in chronic acidosis (pH 6.9-7.0) and observed that cells cultured in low pH had reduced clonogenic capacity. However, our physiological and metabolomics analysis showed that cells in low pH deviate from glycolytic metabolism and rely more on oxidative metabolism. The increased expression of the transaminase enzyme GOT1 fuels oxidative metabolism of cells cultured in low pH by enhancing the non-canonical glutamine metabolic pathway. Survival in low pH is reduced upon depletion of GOT1 due to increased intracellular ROS levels. Thus, GOT1 plays an important role in energy metabolism and ROS balance in chronic acidosis stress. Our studies suggest that targeting anaplerotic glutamine metabolism may serve as an important therapeutic target in PDAC.

De Novo lipid synthesis facilitates gemcitabine resistance through endoplasmic reticulum stress in pancreatic cancer

Pancreatic adenocarcinoma is moderately responsive to gemcitabine-based chemotherapy, the most widely used single-agent therapy for pancreatic cancer. Although the prognosis in pancreatic cancer remains grim in part due to poor response to therapy, previous attempts at identifying and targeting the resistance mechanisms have not been very successful. By leveraging The Cancer Genome Atlas dataset, we identified lipid metabolism as the metabolic pathway that most significantly correlated with poor gemcitabine response in pancreatic cancer patients. Furthermore, we investigated the relationship between alterations in lipogenesis pathway and gemcitabine resistance by utilizing tissues from the genetically engineered mouse model and human pancreatic cancer patients. We observed a significant increase in fatty acid synthase (FASN) expression with increasing disease progression in spontaneous pancreatic cancer mouse model, and a correlation of high FASN expression with poor survival in patients and poor gemcitabine responsiveness in cell lines. We observed a synergistic effect of FASN inhibitors with gemcitabine in pancreatic cancer cells in culture and orthotopic implantation models. Combination of gemcitabine and the FASN inhibitor orlistat significantly diminished stemness, in part due to induction of endoplasmic reticulum (ER) stress that resulted in apoptosis. Moreover, direct induction of ER stress with thapsigargin caused a similar decrease in stemness and showed synergistic activity with gemcitabine. Our in vivo studies with orthotopic implantation models demonstrated a robust increase in gemcitabine responsiveness upon inhibition of fatty acid biosynthesis with orlistat. Altogether, we demonstrate that fatty acid biosynthesis pathway manipulation can help overcome the gemcitabine resistance in pancreatic cancer by regulating ER stress and stemness. Cancer Res; 77(20); 5503-17. ©2017 AACR.

MUC1-Mediated Metabolic Alterations Regulate Response to Radiotherapy in Pancreatic Cancer

Purpose: MUC1, an oncogene overexpressed in multiple solid tumors, including pancreatic cancer, reduces overall survival and imparts resistance to radiation and chemotherapies. We previously identified that MUC1 facilitates growth-promoting metabolic alterations in pancreatic cancer cells. The present study investigates the role of MUC1-mediated metabolism in radiation resistance of pancreatic cancer by utilizing cell lines and in vivo models. Experimental Design: We used MUC1-knockdown and -overexpressed cell line models for evaluating the role of MUC1-mediated metabolism in radiation resistance through in vitro cytotoxicity, clonogenicity, DNA damage response, and metabolomic evaluations. We also investigated whether inhibition of glycolysis could revert MUC1-mediated metabolic alterations and radiation resistance by using in vitro and in vivo models. Results: MUC1 expression diminished radiation-induced cytotoxicity and DNA damage in pancreatic cancer cells by enhancing glycolysis, pentose phosphate pathway, and nucleotide biosynthesis. Such metabolic reprogramming resulted in high nucleotide pools and radiation resistance in in vitro models. Pretreatment with the glycolysis inhibitor 3-bromopyruvate abrogated MUC1-mediated radiation resistance both in vitro and in vivo, by reducing glucose flux into nucleotide biosynthetic pathways and enhancing DNA damage, which could again be reversed by pretreatment with nucleoside pools. Conclusions: MUC1-mediated nucleotide metabolism plays a key role in facilitating radiation resistance in pancreatic cancer and targeted effectively through glycolytic inhibition. Clin Cancer Res; 23(19); 5881–91. ©2017 AACR.

Glucose Limitation Alters Glutamine Metabolism in MUC1-Overexpressing Pancreatic Cancer Cells

Pancreatic cancer cells overexpressing Mucin 1 (MUC1) rely on aerobic glycolysis and, correspondingly, are dependent on glucose for survival. Our NMR metabolomics comparative analysis of control (S2–013.Neo) and MUC1-overexpressing (S2–013.MUC1) cells demonstrates that MUC1 reprograms glutamine metabolism upon glucose limitation. The observed alteration in glutamine metabolism under glucose limitation was accompanied by a relative decrease in the proliferation of MUC1-overexpressing cells compared with steady-state conditions. Moreover, glucose limitation induces G1 phase arrest where S2–013.MUC1 cells fail to enter S phase and synthesize DNA because of a significant disruption in pyrimidine nucleotide biosynthesis. Our metabolomics analysis indicates that glutamine is the major source of oxaloacetate in S2–013.Neo and S2–013.MUC1 cells, where oxaloacetate is converted to aspartate, an important metabolite for pyrimidine nucleotide biosynthesis. However, glucose limitation impedes the flow of glutamine carbons into the pyrimidine nucleotide rings and instead leads to a significant accumulation of glutamine-derived aspartate in S2–013.MUC1 cells.

Erratum: MUC1 and HIF-1alpha Signaling Crosstalk Induces Anabolic Glucose Metabolism to Impart Gemcitabine Resistance to Pancreatic Cancer (Cancer Cell (2017) 32(1) (71–87.e7) (S1535610817302544) (10.1016/j.ccell.2017.06.004))

Surendra K. Shukla, Vinee Purohit, Kamiya Mehla, Venugopal Gunda, Nina V. Chaika, Enza Vernucci, Ryan J. King, Jaime Abrego, Gennifer D. Goode, Aneesha Dasgupta, Alysha L. Illies, Teklab Gebregiworgis, Bingbing Dai, Jithesh J. Augustine, Divya Murthy, Kuldeep S. Attri, Oksana Mashadova, Paul M. Grandgenett, Robert Powers, Quan P. Ly, Audrey J. Lazenby, Jean L. Grem, Fang Yu, José M. Matés, John M. Asara, Jung-whan Kim, Jordan H. Hankins, Colin Weekes, Michael A. Hollingsworth, Natalie J. Serkova, Aaron R. Sasson, Jason B. Fleming, Jennifer M. Oliveto, Costas A. Lyssiotis, Lewis C. Cantley, Lyudmyla Berim, and Pankaj K. Singh* *Correspondence: pankaj.singh@unmc.edu http://dx.doi.org/10.1016/j.ccell.2017.08.008

Evaluating the Metabolic Impact of Hypoxia on Pancreatic Cancer Cells

Hypoxia is frequently observed in human cancers and induces global metabolic reprogramming that includes an increase in glucose uptake and glycolysis, alterations in NAD(P)H/NAD(P)+ and intracellular ATP levels, and increased utilization of glutamine as the major precursor for fatty acid synthesis. In this chapter, we describe in detail various physiological assays that have been adopted to study the metabolic shift propagated by exposure to hypoxic conditions in pancreatic cell culture model that includes glucose uptake, glutamine uptake, and lactate release by pancreatic cancer cell lines. We have also elaborated the assays to evaluate the ratio of NAD(P)H/NAD(P)+ and intracellular ATP estimation using the commercially available kit to assess the metabolic state of cancer cells.

Abstract 1059: Pancreatic cancer cells acclimatize to low pH by increasing glutamine metabolism

Pancreatic cancer (PC) is the fourth leading cause of cancer related deaths in the United States. PC has a five-year survival rate of only 6%; this is due to the lack of specific symptoms at the earliest stages of disease progression. Currently, surgery is the only treatment option with a reasonable hope of cure; however, due to late detection of the disease, only 15-25% of patients are eligible for surgery. Pancreatic tumors are characterized by increased glucose uptake, high glycolysis rate, and reduced flux to the TCA cycle. This metabolic phenotype, also known as the Warburg effect, is typical of rapidly dividing tumor cells and forms the basis of imaging by utilizing [18F]-FDG-PET (glucose analog). The consequence of this metabolic behavior is the continuous acidification of the tumor microenvironment. Acidification of the tumor microenvironment (TME) and its effects in cancer cell metabolism are not well defined. In this study we analyzed metabolic adaptations of PC cells experiencing physiological pH 7.4 versus cells cultured in 6.8∼7.0 pH that is similar to the pH range observed in the pancreatic TME. Using high performance liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis to determine the metabolite levels of PC cells cultured in these TME conditions, we observed a marked reduction in glycolysis metabolites in cells cultured at low pH. We observed reduced glucose uptake, as well as, reduced lactate secretion in low pH culture conditions. Furthermore, we observed that cells in low pH microenvironment were able to survive upon glucose deprivation, but not upon glutamine deprivation. In contrast, cells in normal physiologic pH culture could not survive upon glucose deprivation. Based on the up regulation of metabolites in the glutaminolysis pathway identified through LC/MS/MS analysis, and the increase in metabolic enzyme mRNA levels, as well as the increased sensitivity to inhibitors of this pathway, we conclude that glutamine metabolism is essential for survival of pancreatic cancer cells under low pH conditions. Furthermore, due to the increased levels of ATP and sensitivity of low-pH cultured cells to oligomycin, we conclude that oxidative phosphorylation is essential for the maintenance of cellular homeostasis at low pH. Such changes result in reduced Warburg Effect, denoted by reduced lactate release. This is the first study to establish glutamine dependence of PC cells under low pH conditions. Our results may provide novel therapeutic opportunities for targeting metabolic adaptations in pancreatic cancer cells in response to changes in the microenvironment. Citation Format: Jaime Abrego, Venugopal Gunda, Pankaj K. Singh, Gennifer Goode. Pancreatic cancer cells acclimatize to low pH by increasing glutamine metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1059.