Phd in MOLECULAR MEDICINE

Thesis title: Glutaminase role in cancer cell

Metabolic reprogramming is recognized as an essential characteristic of tumors. Indeed, cancer cells are characterized by an increased glucose and glutamine consumption. Literature suggests that metabolic reprogramming may be key to disease progression and a promising target in the development of new therapies. Specifically, glutamine catabolism affects tumor cell proliferation, redox balance, biosynthesis of other nonessential amino acids, and maintenance of cancer stem cell. The glutaminase (GLS) enzyme, which is involved in hydrolytic deamination of glutamine to glutamate and ammonium, controls a limiting step in the glutamine catabolism process and its inhibition has been considered as a therapeutic approach for different types of tumors. Recent studies have affirmed that breast tumors, especially the triple negative subtype, have a high expression of GLS1. Our recent study demonstrated that GLS activity and transformation of glutamine to glutamate with ammonia production is inhibited by Sirtuin 5 (SIRT5). Sirtuins (SIRTs) are a family of NAD-dependent protein deacetylases and/or ADP ribosylases controlling metabolic homeostasis. In particular, SIRT5 is a global regulator of lysine succinylation in mitochondria. For this reason, we have developed MC3138, a selective SIRT5 activator. Indeed, treatment with MC3138 mimicked the deacetylation effect mediated by SIRT5 overexpression. Cancer cells treated with MC3138 show decreased levels of metabolites such as glutamine and glutamate. In addition, mammalian glutaminase activity is regulated by inorganic phosphate (Pi). In fact, it has been hypothesized that the accumulation of inorganic phosphate in the mitochondria is the trigger for increased glutaminase C-based activity. Considering the important and interconnected role of glutaminase, SIRT5 and Pi for cancer growth and progression, our hypothesis is that activation of SIRT5 and reduction of Pi could represent a valid anti-tumoral strategy. By treating the cells with MC3138 and lanthanum acetate, a Pi chelator, individually or in combination, we observed a decrease in cell viability and clonogenicity. Treatments with the SIRT5 activator MC3138 and lanthanum acetate also altered the levels of autophagy and mitophagy, especially after 48 hours. Indeed if, after 24h, the expression of proteins such as LC3 II, Beclin1 and ULK1 remains almost unchanged, after 48h of treatment important variations of expression are observed in these autophagic markers as well as in mitophagy markers such as BNIP3 and Parkin. We also observed an increase of reactive oxygen species in cancer cells treated with MC3138 alone or in combination with lanthanum acetate. Finally, since the hypoxic tumor microenvironment increases the mitochondrial Pi content, we conducted the same studies under hypoxic conditions showing that our treatments are potentiated under hypoxia.