Pancreatic ductal adenocarcinomas (PDACs) are the second leading cause of cancer death in the United States, with a survival rate of 3% once it spreads to distant organs. PDAC cells within a tumor are frequently starved for oxygen due to their high proliferation rate and insufficient vasculature. This degree of hypoxia in the tumor triggers strong metabolic adaptations on cancer cells that allow their survival and proliferation. Among these adaptations are altered uptake and utilization of major nutrients, such as glutamine. However, the precise mechanisms through which pancreas cancer metabolism adapts to hypoxia, and whether this could be exploited for therapy, remain unknown. In preliminary studies, we found that, when pancreatic and lung cancers are exposed to low levels of oxygen, the amino acid aspartate, required for protein and nucleotide synthesis, becomes limiting. Simply increasing the uptake of exogenous aspartate by expression of a plasma membrane aspartate transporter strongly promoted the growth rate of cancer cells under hypoxia and in tumors, as well as enhanced their metastatic potential. These findings provide evidence that aspartate can be a cancer growth-limiting metabolite in vivo. Furthermore, a CRISPR/Cas9 screen using a library of sgRNAs targeting rate-limiting metabolic enzymes revealed that GOT2, one of the two enzymes that de novo synthesizes cellular aspartate from glutamine, is essential for in vitro proliferation under hypoxia of a KRAS/TP53 mutant PDAC cell line. Building upon these results, I propose to test the hypothesis that targeting de novo aspartate synthesis may have therapeutic potential in pancreatic tumors at the level of primary tumor growth and metastasis. Throughout the initial phase of this award, we will define the essentiality of GOT2-mediated aspartate synthesis in a panel of PDAC cell lines upon being exposed to low tensions of oxygen and, importantly, when grown as tumors and during colonization of distant organs. Additionally, we will determine the contribution and impact on cancer proliferation of the two divergent metabolic routes of glutamine conversion into aspartate: oxidative and reductive metabolism. Building upon the obtained results, we will target aspartate synthesis in pre-clinical patient-derived models and KRAS/TP53 mutant mouse models and, by using isotope-labeling metabolomic analysis, we will show which aspartate synthesis route is used by pancreatic tumors in vivo. Finally, by using in vivo metabolomics and mitochondrial pull-downs in primary and metastatic tumors, I propose to define whether hypoxia-triggered metabolic rewiring is a determinant of the metastatic potential of PDAC cells. These analysis will identify which metabolic changes PDAC cells undergo during metastasis, unveiling potential liabilities that could be targeted in order to decrease spread of PDAC to distant organs. Altogether, the proposed experiments will define the role of aspartate synthesis in PDAC tumor growth and metastasis, and will test whether any other metabolic changes are required for PDAC cells to metastasize.
Pancreatic ductal adenocarcinoma, frequently subjected to oxygen-limiting environments, is highly refractory to all forms of treatment and novel approaches to inhibit their primary growth and colonization of distant organs are urgently needed. In this proposal, as a potential therapeutic strategy, we will identify and inhibit the metabolic route and enzyme that pancreatic cancers rely on to synthesize aspartate, a metabolite that we previously found to be limiting under hypoxia and whose supplementation increases primary tumor growth and metastatic potential of pancreatic cancer cells. Additionally, the analysis of which metabolic changes pancreatic cancer cells undergo during metastasis will increase understanding of the metabolic rewiring this process triggers and will identify potential metabolic liabilities of metastasis.
