TUMOR PROMOTING FUNCTIONS FOR THE METABOLIC REGULATOR SIRT5

Cancer is among the leading causes of death worldwide, highlighting the urgent need for identification of new targets and the development of new strategies to intervene against this disease. Recently, it was reported that several members of the sirtuin family (Sirt1-7), the mammalian orthologs of the silent information regulator 2 (Sir2) in Saccharomyces cerevisiae, play important roles in carcinogenesis. In mammals there are three mitochondrial sirtuins, SIRT3, SIRT4, and SIRT5. Among these, SIRT5 is the only known enzyme that catalyzes nicotinamide adenine dinucleotide-dependent demalonylation, desuccinylation and deglutarylation. SIRT5 impacts diverse metabolic pathways. One substrate of SIRT5 is carbamoyl phosphate synthase 1 (CPS1), an enzyme involved in the rate-limiting step of the urea cycle. By deacylating CPS1, SIRT5 promotes urea cycle function and clearance of toxic ammonia. Sirt5-deficient mice fail to up-regulate CPS1 activity and show elevated blood ammonia during fasting. SIRT5 regulates mitochondrial respiration by targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase M2 (PKM2), pyruvate dehydrogenase complex (PDH), isocitrate dehydrogenase 2 (IDH2) and succinate dehydrogenase (SDH). SIRT5 also controls glutamine metabolism by targeting glutaminase (GLS), and fatty acid metabolism by activating very long-chain acyl-CoA dehydrogenase (VLCAD), enoyl-CoA hydratase (ECHA) and 3-Hydroxy-3-Methylglutaryl-CoA Synthase 2 (HMGCS2). SIRT5 has also been implicated in regulating NADPH generation and redox potential by deglutarylating glucose 6-phosphate dehydrogenase (G6PD) and desuccinylating superoxide dismutase 1 (SOD1). Recent studies revealed that SIRT5 knock-down in human breast and lung cancer cells inhibited their transformed properties, whereas it had no significant effect on the growth of normal cells. Furthermore, it has been shown that the expression levels of SIRT5 are increased in several human cancers, often due to gene amplification. We hypothesize that SIRT5 is required by cancer cells to regulate the post-translational modifications of key enzymes involved in cancer metabolism. This dissertation aims to (A) elucidate the role of SIRT5 in cancer by using both, in vitro and in vivo approaches, (B) identify targets for desuccinylation catalyzed by SIRT5 in cancer cells, (C) evaluate the impact of Sirt5 loss on the expression of genes that might be critical for tumor progression, and (D) test the effect of SIRT5 inhibition in tumor progression. To study the effect of Sirt5 deficiency on mammary tumorigenesis, we used MMTV-PyMT transgenic mice, a genetically engineered model in which the animals develop metastatic mammary adenocarcinoma. We tested how mammary tumor latency, multiplicity, size and histological grade are affected by Sirt5 loss, and found that Sirt5 knockout (Sirt5-/-) mice had delayed mammary tumor onset, significantly increased overall survival, and decreased incidence of lung metastasis as compared to controls. Thus, SIRT5 loss significantly reduced cancer cell proliferation in vitro and mammary tumor growth in vivo. To extend our knowledge of the pro-tumorigenic roles of SIRT5, we used another in vivo model that recapitulates glutamine-dependency of human cancers. We used a genetic mouse model in which Sirt5 is knocked out and c-Myc is constitutively activated in B cells. Unexpectelly, Sirt5 loss in Eμ-Myc transgenic mice did not affect lymphoma incidence and mortality, even though it targets multiple key metabolic enzymes, suggesting that the pro-tumorigenic roles of SIRT5 are tissue or oncogene-specific. Immunoblotting for succinyl-lysine levels in mammary tumor tissues from Sirt5-/- PyMT and Sirt5+/+ PyMT mice revealed that there were several proteins with greatly increased succinylation in Sirt5-/- PyMT mammary tumors. To identify targets for desuccinylation catalyzed by SIRT5, we performed liquid chromatography-mass spectrometry (LC-MS) analysis on tumor tissues from Sirt5-/- PyMT and Sirt5+/+ PyMT mice. We identified many metabolic enzymes that were highly succinylated in Sirt5-/- PyMT mammary tumors, many of which are known to plays a critical role in the metabolic reprograming of cancer cells. To investigate how Sirt5 loss affects gene expression, we performed a comparative analysis of expression profiles in mammary tumors from Sirt5+/+ PyMT and Sirt5-/- PyMT mice. We identified significant dysregulation of 129 genes involved in multiple cellular processes such as cellular metabolism, circadian clock, and inflammation. Among these dysregulated genes, serum amyloid A (Saa), a gene that encodes for an acute phase protein linked to inflammation, was significantly downregulated in Sirt5-/- PyMT mammary tumors. Increased levels of SAA have been linked to tumor pathogenesis and metastasis. In light of the tumor promoting activity of SIRT5 and the grossly normal phenotype of Sirt5-/- mice, we tested SIRT5-selective small molecule inhibitors as cancer therapeutics. Pharmacological inhibition of SIRT5 repressed the anchorage independent growth of cancer cells and suppressed tumor progression in vivo. Together, these data demonstrate the importance of SIRT5-regulated post-translational modification in cancer metabolism and highlight novel regulatory events that are important for cancer progression