We have previously demonstrated that Sirt3 is an in vitro
and in vivo
and the knockout mice spontaneously form well differentiated, ER/PR-positive mammary tumors(Kim et al., 2010
). In addition, SIRT3 protein levels are decreased in human breast cancers as well as several other human malignancies. These results identified Sirt3 as a genomically expressed, mitochondrially localized TS and cells lacking Sirt3
may be a useful in vivo
model to investigate the subtype of breast cancer observed in post-menopausal or older women.
One intriguing finding from our previous work was that cells lacking Sirt3
exhibited altered mitochondrial metabolism as exhibited by increased mitochondrial superoxide levels during stress. These results suggested a connection between the increase in superoxide and the Sirt3
knockout mouse tumor permissive phenotype. However, one outstanding question from this previous work involved the observation that MnSOD transcription, via FOXO3a acetylation, was not decreased until one year—roughly the same time that the mammary tumors were first observed in the Sirt3
knockout mice. Thus, while the connection between the MnSOD and increased mitochondrial superoxide seemed strong, the mechanism appeared to be more complex than a decrease in FOXO3a driven MnSOD transcription (Kim et al., 2010
). Thus, additional wild-type and Sirt3
knockout mouse colonies were established and followed to more rigorously investigate the connection between superoxide levels and MnSOD as an early carcinogenic event in the Sirt3
knockout mouse tumor permissive phenotype. These results suggested a second potential mechanism for Sirt3 regulation of MnSOD activity that is independent of MnSOD
expression and potentially related to post-translational modification involving acetylation.
The connection between mitochondrial damage and carcinogenesis is well established; however, the mechanism appears to be complex (Singh, 2006
; Wallace, 2005
). In addition, it has been suggested that the mitochondria play a role in radiation induced malignancies, but the specific molecular steps are not completely understood (Du et al., 2009
). One proposed mechanism involves the accumulation of mitochondrial ROS; these reactive molecules can damage numerous cellular processes, creating an environment permissive for genomic instability as well as carcinogenesis (Oberley, 2005
). Since Sirt3 is the primary mitochondrial deacetylase, we hypothesized that the increase in mitochondrial superoxide levels in Sirt3
knockout mice and MEFs exposed to stress may be due to aberrant acetylation and regulation of MnSOD enzymatic activity.
Our results identify MnSOD amino acid 122 as a reversibly acetylated lysine residue that is deacetylated by 36 hours of fasting and MnSOD acetylation is significantly increased and enzymatic activity is decreased in Sirt3−/− cells. In addition, MnSOD is deacetylated by Sirt3, suggesting that mitochondrial acetylation plays a role, at least in part, in regulation of MnSOD function. This idea was validated by a pair of MnSOD mutants that demonstrated increased activity when lysine 122 was changed to arginine (to mimic the deacetylated state). MnSODK122-R also prevented IR-induced foci formation in MnSOD−/− cells and immortalization of Sirt3−/− MEFs by a single oncogene, as well as IR-induced genomic instability and loss of contact inhibition.
Finally, we show in vivo that IR induces deacetylation of MnSOD and increased enzymatic activity in irradiated wild-type mouse liver mitochondria but not in liver mitochondria of the Sirt3 knockout mice. While it is tempting to suggest this IR-induced damage permissive phenotype is primarily due to changes in MnSOD activity, it seems likely that other Sirt3 deacetylation targets may also play a role in this histological and biochemical phenotype. This idea would fit the well established data that IR-induced damage and cytotoxicity is a multi-factorial process involving several cellular pro-reparative pathways including redox scavenging, DNA repair, and stress responding proteins. Taken together the in vivo and in vitro results suggest MnSOD enzymatic activity is regulated by acetylation during stress and that Sirt3 regulates acetylation under specific conditions associated with neoplastic transformation, including IR-induced cellular damage.
A fundamental paradigm in biology is the presence of intracellular redox sensing proteins that recognize specific cellular conditions and initiate post-translational signaling cascades (Slane et al., 2006
), and these pathways activate the cellular machinery that maintains cellular homeostasis. The most common example of this is the cytoplasmic activation of kinases that phosphorylate a series of downstream targets in response to different environmental conditions, thereby minimizing any potentially permanent cellular detrimental effects (Slane et al., 2006
). In this regard, lysine acetylation has recently emerged as an important and perhaps primary post-translational modification employed to regulate mitochondrial proteins (Lombard et al., 2007
The results presented above support this hypothesis, and together with recent findings (Kim et al., 2010
), suggest that mitochondrial sirtuins, including Sirt3, may function as fidelity proteins whose loss of function may result in a damage permissive phenotype that leads to neoplastic transformation. The results presented above also suggest for the first time that the superoxide scavenging enzymatic function of MnSOD is regulated by changes in specific lysine acetylation. The connection between MnSOD and carcinogenesis, as well as tumor cell resistance to anti-cancer agents, is significant, however the mechanism of action appears to be complex (Oberley, 2005
; Aykin-Burns et al., 2009
). The current work represents a paradigm shift in our understanding of the post-translational regulation of MnSOD enzymatic function and the mechanism of its role in responses to stress. Finally, since Sirt3 is proposed to sense nutrient deprivation and oxidative stress, it also seems logical that exposure to IR, which has previously been shown to induce both oxidative stress and mitochondrial damage (Slane et al., 2006
) would activate Sirt3 as a signaling pathway to protect against persistent IR-induced metabolic stress and normal tissue damage.