Investigating the Fate of Ammonia in Breast Cancer

Abstract

Ammonia (NH3) is a ubiquitous by-product of cellular metabolism that is copiously generated by proliferating cells, especially cancer cells. Although NH3 is classically conveyed as toxic metabolic waste, it accumulates in the tumor microenvironment (TME) 10-fold higher than what is considered toxic to healthy tissue. Thus, we hypothesized that NH3 functions beyond a mere waste product in tumor biology. However, the fate of NH3 generated by tumor metabolism had never been explored. To address this, we developed a novel LC-MS assay to detect 15N and14N-isotopologues of NH3 (Spinelli et al., Sci. Rep. 2017), and performed stable isotope tracing experiments in breast cancer cells to track the fate of NH3. We discovered that breast cancer cells recycled NH3 generated by tumor metabolism with 60% efficiency through a “reverse” catalytic activity of glutamate dehydrogenase (GDH) to generate glutamate (Spinelli et al., Science. 2017). This nitrogen-scavenging pathway accelerated proliferation of breast cancer cells grown in 3D culture models and in vivo xenograft tumor models. Furthermore, using a novel method for rapid mitochondrial isolation for metabolomics, we tracked the compartmentalized nature of NH3 metabolism in breast cancer cells (Spinelli et al., submitted). Mitochondrial metabolomics revealed that NH3 is assimilated inside this compartment as a local nitrogen source for glutamate. Importantly, mitochondrial localization of nitrogen recycling is required for NH3 to stimulate proliferation, unveiling a limitation in the mitochondrial glutamate pool for breast cancer proliferation. We determined that the levels of mitochondrial glutamate dictate the rate of local translation. Thus, NH3 assimilation to produce mitochondrial glutamate increases cellular respiratory capacity to enable accelerated proliferation. Taken together, this thesis work reveals that breast cancer cells recycle the waste product NH3 to generate mitochondrial glutamate, which regulates local protein translation to accelerate proliferation.