Ozkan Ozden

SIRT3 Is a Mitochondria-Localized Tumor Suppressor Required for Maintenance of Mitochondrial Integrity and Metabolism during Stress

The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3(-/-) mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3(-/-) MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3(-/-) MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3(-/-) mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.

Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress.

Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3⁻/⁻ livers at 3 months of age. Livers of Sirt3⁻/⁻ mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3⁻/⁻ MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD(K122-R)), increased MnSOD activity when expressed in MnSOD⁻/⁻ MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3⁻/⁻ MEFs with lenti-MnSOD(K122-R) inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3⁻/⁻ livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.

SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells.

Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321), and a PDHA1 mutant mimicking a deacetylated lysine (PDHA1(K321R)) increases PDH activity, compared to the K321 acetylation mimic (PDHA1(K321Q)) or wild-type PDHA1. Finally, PDHA1(K321Q) exhibited a more transformed in vitro cellular phenotype compared to PDHA1(K321R). These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration.

Sirt3, mitochondrial ROS, ageing, and carcinogenesis.

One fundamental observation in cancer etiology is that the rate of malignancies in any mammalian population increases exponentially as a function of age, suggesting a mechanistic link between the cellular processes governing longevity and carcinogenesis. In addition, it is well established that aberrations in mitochondrial metabolism, as measured by increased reactive oxygen species (ROS), are observed in both aging and cancer. In this regard, genes that impact upon longevity have recently been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. Interestingly, three of the seven sirtuin proteins are localized into the mitochondria suggesting a connection between the mitochondrial sirtuins, the free radical theory of aging, and carcinogenesis. Based on these results it has been hypothesized that Sirt3 functions as a mitochondrial fidelity protein whose function governs both aging and carcinogenesis by modulating ROS metabolism. Sirt3 has also now been identified as a genomically expressed, mitochondrial localized tumor suppressor and this review will outline potential relationships between mitochondrial ROS/superoxide levels, aging, and cell phenotypes permissive for estrogen and progesterone receptor positive breast carcinogenesis.

SIRT2 is a tumor suppressor that connects aging, acetylome, cell cycle signaling, and carcinogenesis

One long standing observation in clinical oncology is that age increase is the single most statistically significant factor/variable that predicts for the incidence of solid tumors. This observation suggests that the cellular and molecular processes and mechanisms that direct an organisms life span may be used to determine the clinical connection between aging and carcinogenesis. In this regard, the genes that impact upon longevity have been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. We have recently shown that the primary cytoplasmic sirtuin, Sirt2 appears to meet the criteria as a legitimate tumor suppressor protein. Mice genetically altered to delete Sirt2 develop gender-specific tumorigenesis, with females primarily developing mammary tumors, and males developing multiple different types of gastrointestinal malignancies. Furthermore human tumors, as compared to normal samples, displayed significant decreases in SIRT2 levels suggesting that SIRT2 may also be a human tumor suppressor.

Exploring the electrostatic repulsion model in the role of Sirt3 in directing MnSOD acetylation status and enzymatic activity

Mitochondrial oxidative metabolism is the major site of ATP production as well as a significant source of reactive oxygen species (ROS) that can cause damage to critical biomolecules. It is well known that mitochondrial enzymes that scavenge ROS are targeted by stress responsive proteins to maintain the fidelity of mitochondrial function. Manganese superoxide dismutase (MnSOD) is a primary mitochondrial ROS scavenging enzyme, and in 1983 Irwin Fridovich proposed an elegant chemical mechanism/model whereby acetylation directs MnSOD enzymatic activity. He christened it the electrostatic repulsion model. However, the biochemical and genetic mechanism(s) determining how acetylation directs activity and the reasons behind the evolutionarily conserved need for several layers of transcriptional and posttranslational MnSOD regulation remain unknown. In this regard, we and others have shown that MnSOD is regulated, at least in part, by the deacetylation of specific conserved lysines in a reaction catalyzed by the mitochondrial sirtuin, Sirt3. We speculate that the regulation of MnSOD activity by lysine acetylation via an electrostatic repulsion mechanism is a conserved and critical aspect of MnSOD regulation necessary to maintain mitochondrial homeostasis.

SIRT2-Mediated Deacetylation and Tetramerization of Pyruvate Kinase Directs Glycolysis and Tumor Growth

Sirtuins participate in sensing nutrient availability and directing metabolic activity to match energy needs with energy production and consumption. However, the pivotal targets for sirtuins in cancer are mainly unknown. In this study, we identify the M2 isoform of pyruvate kinase (PKM2) as a critical target of the sirtuin SIRT2 implicated in cancer. PKM2 directs the synthesis of pyruvate and acetyl-CoA, the latter of which is transported to mitochondria for use in the Krebs cycle to generate ATP. Enabled by a shotgun mass spectrometry analysis founded on tissue culture models, we identified a candidate SIRT2 deacetylation target at PKM2 lysine 305 (K305). Biochemical experiments including site-directed mutants that mimicked constitutive acetylation suggested that acetylation reduced PKM2 activity by preventing tetramerization to the active enzymatic form. Notably, ectopic overexpression of a deacetylated PKM2 mutant in Sirt2-deficient mammary tumor cells altered glucose metabolism and inhibited malignant growth. Taken together, our results argued that loss of SIRT2 function in cancer cells reprograms their glycolytic metabolism via PKM2 regulation, partially explaining the tumor-permissive phenotype of mice lacking Sirt2 Cancer Res; 76(13); 3802-12. ©2016 AACR.

Additional file 4: Table S1. of Activin and TGFβ use diverging mitogenic signaling in advanced colon cancer

Characteristics of colon cancer patient cohort randomly selected from Northwestern University for p21, TGFBR2, ACVR2, pERK, and pAkt staining. Ten patients did not have stage information available (X). (DOC 170 kb)