Faculty Bibliography

As oxygen is essential for many metabolic pathways, tumour hypoxia may impair cancer cell proliferation^1-4. However, the limiting metabolites for proliferation under hypoxia and in tumours are unknown. Here, we assessed proliferation of a collection of cancer cells following inhibition of the mitochondrial electron transport chain (ETC), a major metabolic pathway requiring molecular oxygen ⁵ . Sensitivity to ETC inhibition varied across cell lines, and subsequent metabolomic analysis uncovered aspartate availability as a major determinant of sensitivity. Cell lines least sensitive to ETC inhibition maintain aspartate levels by importing it through an aspartate/glutamate transporter, SLC1A3. Genetic or pharmacologic modulation of SLC1A3 activity markedly altered cancer cell sensitivity to ETC inhibitors. Interestingly, aspartate levels also decrease under low oxygen, and increasing aspartate import by SLC1A3 provides a competitive advantage to cancer cells at low oxygen levels and in tumour xenografts. Finally, aspartate levels in primary human tumours negatively correlate with the expression of hypoxia markers, suggesting that tumour hypoxia is sufficient to inhibit ETC and, consequently, aspartate synthesis in vivo. Therefore, aspartate may be a limiting metabolite for tumour growth, and aspartate availability could be targeted for cancer therapy.

Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for T cell function and adaptive immunity. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels that are activated by stromal interaction molecule (STIM) 1 and STIM2. SOCE regulates many Ca2+-dependent signaling molecules, including calcineurin, and inhibition of SOCE or calcineurin impairs antigen-dependent T cell proliferation. We here report that SOCE and calcineurin regulate cell cycle entry of quiescent T cells by controlling glycolysis and oxidative phosphorylation. SOCE directs the metabolic reprogramming of naive T cells by regulating the expression of glucose transporters, glycolytic enzymes, and metabolic regulators through the activation of nuclear factor of activated T cells (NFAT) and the PI3K-AKT kinase-mTOR nutrient-sensing pathway. We propose that SOCE controls a critical "metabolic checkpoint" at which T cells assess adequate nutrient supply to support clonal expansion and adaptive immune responses.

During tumorigenesis, the high metabolic demand of cancer cells results in increased production of reactive oxygen species. To maintain oxidative homeostasis, tumor cells increase their antioxidant production through hyperactivation of the NRF2 pathway, which promotes tumor cell growth. Despite the extensive characterization of NRF2-driven metabolic rewiring, little is known about the metabolic liabilities generated by this reprogramming. Here, we show that activation of NRF2, in either mouse or human cancer cells, leads to increased dependency on exogenous glutamine through increased consumption of glutamate for glutathione synthesis and glutamate secretion by xc- antiporter system. Together, this limits glutamate availability for the tricarboxylic acid cycle and other biosynthetic reactions creating a metabolic bottleneck. Cancers with genetic or pharmacological activation of the NRF2 antioxidant pathway have a metabolic imbalance between supporting increased antioxidant capacity over central carbon metabolism, which can be therapeutically exploited.

Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Proliferative cells have an increased need for macromolecular precursors required to sustain proliferation. Therefore, alteration of metabolic pathway flux and metabolite consumption is a hallmark of the transformed state. We have begun to investigate the specific metabolic pathway rewiring that occurs in the transformation of human breast cells, and the different metabolic phenotypes exhibited in breast cancers of specific molecular subtypes. These efforts have focused on the contribution of proper amino acid management in supporting breast tumor metabolism. For example, we have observed that ER-negative breast cancers exhibit activation of the serine biosynthetic pathway, controlled primarily by the expression of the enzyme PHGDH. ER-negative breast cancer cell lines with high PHGDH expression depend on its continued expression to maintain viability, and PHGDH expression permits cells to survive in the absence of extracellular serine. We have also identified the enzyme SHMT2, responsible for catabolizing serine for use in cellular methylation reactions, as an important determinant for cellular survival in low oxygen conditions. Finally, we have uncovered a key enzyme required to catabolize cysteine for the biosynthesis of iron-sulfur clusters, as being required for breast cancer cells to proliferate in high-oxygen environments, or under conditions of oxidative damage. We will discuss our development of mouse models to both investigate the contribution of these metabolic pathways to breast tumorigenesis and develop strategies to impact breast cancer growth and development

Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based short hairpin RNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.

Amino acids have a dual role in cellular metabolism, as they are both the building blocks for protein synthesis and intermediate metabolites which fuel other biosynthetic reactions. Recent work has demonstrated that deregulation of both arms of amino acid management are common alterations seen in cancer. Among the most highly consumed nutrients by cancer cells are the amino acids glutamine and serine, and the biosynthetic pathways that metabolize them are required in various cancer subtypes and the object of current efforts to target cancer metabolism. Also altered in cancer are components of the machinery which sense amino acid sufficiency, nucleated by the mechanistic target of rapamycin (mTOR), a key regulator of cell growth via modulation of key processes including protein synthesis and autophagy. The precise ways in which altered amino acid management supports cellular transformation remain mostly elusive, and a fuller mechanistic understanding of these processes will be important for efforts to exploit such alterations for cancer therapy.