Cancer cells elevate nutrient catabolism, causing excess production and accumulation of metabolic waste, especially lactate and ammonia. Using stable isotope tracing and mass spectrometry, we discovered that breast cancer cells scavenged ammonia generated by amino acid catabolism as a re-purposed nitrogen source for biomass (Spinelli et al. Science, 2017). Ammonia recycling accelerated proliferation in 3D cell culture and in vivo mouse xenograft models. These studies lead to two follow-up questions that will be addressed in Specific Aim 1 (F99 phase) of this proposal.
First (Aim 1 A), what is the mechanism by which ammonia stimulates breast cancer proliferation? Preliminary data supports the hypothesis that subcellular compartmentalization of ammonia metabolism is required for its effect on proliferation rate. Using metabolic tracing and rapid immunoprecipiation, we tracked the localization of ammonia assimilation to the mitochondria and the subsequent efflux of metabolites to the cytosolic fraction, showing a role in ammonia-stimulated proliferation.
Second (Aim 1 B), what is the effect of this novel ammonia-recycling pathway on response to therapy? Preliminary data shows that ammonia assimilation circumvents the effect of glutaminase (GLS) inhibitors through replenishing glutamate levels via glutamate dehydrogenase (GDH). Our proposed studies will determine if GDH and GLS inhibition are synergistic in vivo and in primary breast cancer tumors to elucidate a mechanism by which breast cancer cells are resistant to GLS inhibition. Beyond ammonia, mitochondria promote tumor growth and proliferation through numerous metabolic pathways. However, the mechanisms by which nutrients traverse the inner mitochondrial membrane are little studied, particularly because 45% of the mitochondria nutrient transporter family is uncharacterized. Therefore, using my expertise in metabolite tracing and mass spectrometry in addition to new skills gained in genetic (CRISPR) screening and bioinformatics, the proposed studies (Aim 2, K00 phase) will systematically assess the role of mitochondrial nutrient transporters in tumor growth and survival. These data will be the first to globally evaluate the essentiality of metabolic compartmentalization in cancer cells harboring oncogenic drivers such as KRAS, IDH and PI3K mutations. Furthermore, this study will elucidate the function of uncharacterized mitochondrial nutrient transporters. In addition to the proposed studies, the fellowship training plan includes gaining experience with mentorship, taking courses on responsible conduct of research, team management, and budgeting, and attending scientific conferences such as the Tumor Metabolism Keystone meeting to develop a network of scientific collaborators. The proposed studies will occur at Harvard Medical School and The Broad Institute, which have environments with the equipment, technology, core facilities, potential for collaboration, and the resources for research and career development needed to complete the proposed training plan.
There is a strong body of research that demonstrates a critical role of mitochondrial metabolism (such as ammonia metabolism), in tumor initiation, growth, and survival. The proposed research will fill a major gap in the fundamental understanding of the mechanisms in which metabolites transport across the mitochondrial inner membrane, and inevitably will elucidate new targets for cancer therapy development. Identification of the essential transporters for tumor growth and development of new treatments will improve the life expectancy and outcomes of clinically challenging cancer subtypes.