Exogenous Pyruvate Is Required for Cell Adaption to Chronic Hypoxia

Hypoxia is a common feature in solid tumors due to the imbalance between the poor development of vascularization and rapid proliferation of tumor cells. Tumor hypoxia is associated with poor clinical prognoses and is correlated with tumor progression and metastasis. The Pasteur and Warburg effects have been well described in which endogenous glycolytic pyruvate is converted to lactate to maintain the intracellular NAD+ levels. However, a role of exogenous pyruvate in cell adaptation to hypoxia has not been fully investigated. My thesis research focuses on exploring how exogenous pyruvate facilitates cellular adaptation to chronic hypoxia. In the thesis, I demonstrate that exogenous pyruvate is required for cellular adaption to chronic hypoxia through supporting ATP generation and NAD+ recycling, sustaining ER stress response and regulating autophagy and apoptosis. Specifically, by using both hypoxic and [rho]0 cells with defective mitochondrial electron transfer chain, I show that exogenous pyruvate is required to sustain the proliferation of cancer and non-cancer cells that cannot utilize oxygen. Particularly, I show that absence of pyruvate led to glycolysis inhibition, ATP depletion and decreased NAD+ levels in [rho]0 cells; and exogenous pyruvate increases lactate yield, elevates NAD+/NADH ratio and ATP production. Knockdown of lactate dehydrogenase A (LDHA) significantly inhibits the rescuing effects of exogenous pyruvate. On the other hand, pyruvate derived metabolites,including acetyl-CoA, [alpha]-ketoglutarate, succinate and alanine do not rescue [rho]0 cell proliferation. Knockdown of pyruvate carboxylase (PC), pyruvate dehydrogenase subunit (PDHA1) and citrate synthase (CS) do not impair exogenous pyruvate to rescue [rho]0 cells. Importantly, I show that exogenous pyruvate enhances ATP levels and promotes the proliferation of hypoxic cells, and that well-oxygenated cells release pyruvate, providing a potential in vivo source of pyruvate. These findings support a novel pyruvate cycle model in which oxygenated cells release pyruvate for hypoxic cells as an oxygen surrogate (Chapter 2). Autophagy and apoptosis are critical in cell fate determination. I show that hypoxia causes autophagy as demonstrated by LC3 conversion and autophagosome formation, and exogenous pyruvate further enhances autophagy. I also demonstrate that exogenous pyruvate protects tumor cells from chronic hypoxia-induced apoptosis (Chapter 3). ER stress response plays an important role in cell adaptation to the micro-environmental stresses including hypoxia and nutrient insufficiency. I show that although activated by acute hypoxia, the three ER stress response branches, the eIF2[alpha]-ATF4 pathway, the ATF6 pathway and the XBP1 pathway are inhibited by chronic hypoxia. This inhibition is blocked in the presence of exogenous pyruvate (Chapter 4). I also show that the master regulator and downstream target of ER stress response, GRP78, plays a critical role in hypoxic cell adaptation. GRP78 knockdown with CRISPR/cas9 gene editing technique impairs the exogenous pyruvate-mediated rescue of hypoxic tumor cells. Thus, my findings delineate that exogenous pyruvate activates ER stress response and promotes cell survival under hypoxia (Chapter 5). In summary, my data show that exogenous pyruvate prevents NAD+ exhausting and ATP depletion, and maintains ER stress responses under chronic hypoxia, which in turn promotes cellular adaption to hypoxia by coordinately regulating autophagy and apoptosis.