Abstract
Sirtuin 2 (SIRT2) is a sirtuin family deacetylase, which maintains genome integrity and prevents tumorigenesis. Although Sirt2 deficiency in mice leads to tumorigenesis, the functional significance of somatic SIRT2 mutations in human tumors is unclear. Using structural insight combined with bioinformatics and functional analyses, we show that naturally occurring cancer-associated SIRT2 mutations at evolutionarily conserved sites disrupt its deacetylation of DNA-damage response proteins by impairing SIRT2 catalytic activity or protein levels but not its localization or binding with substrate. We observed that these SIRT2 mutant proteins fail to restore the replication stress sensitivity, impairment in recovery from replication stress, and impairment in ATR-interacting protein (ATRIP) focus accumulation of SIRT2 deficiency. Moreover, the SIRT2 mutant proteins failed to rescue the spontaneous induction of DNA damage and micronuclei of SIRT2 deficiency in cancer cells. Our findings support a model for SIRT2's tumor-suppressive function in which somatic mutations in SIRT2 contribute to genomic instability by impairing its deacetylase activity or diminishing its protein levels in the DNA-damage response. In conclusion, our work provides a mechanistic basis for understanding the biological and clinical significance of SIRT2 mutations in genome maintenance and tumor suppression.
Highlights
Sirtuin 2 (SIRT2) is a sirtuin family deacetylase, which maintains genome integrity and prevents tumorigenesis
SIRT2 cancer mutations are functional roid, and uterine cancer, most of which overlap with the 66 somatic SIRT2 mutations found in the Catalogue of Somatic Mutations in Cancer (COSMIC) database [17]
We focused our investigation on nine SIRT2 mutations present in uterine, lung, and colorectal cancer, which were found in both data sets, predicted to be high impact mutations by cBioPortal, and occurred at appreciable frequencies within their study cohort (Fig. 1A)
Summary
To determine the functional significance of somatic SIRT2 mutations in human cancers, we analyzed sequencing data from patient-derived tumor samples reported in The Cancer Genome Atlas through the cBioPortal for Cancer Genomics [16, 25]. Even the small addition of two methyl groups is likely enough to blow out this pocket and upset the orientation of His-187, thereby inhibiting deacetylation of substrate Overall, these four amino acids are heavily involved in proper deacetylase activity and/or cofactor binding. The other five mutations for this study lie outside of the active site and binding pocket of SIRT2 Four of these five amino acids (Glu-203, Arg-153, Leu-341, and Ser-73) have been verified for orientation and structure as the current crystal structures of SIRT2 lack a portion of the N terminus where Arg-42 lies (Fig. 3D). SIRT2-FLAG P140H and A186V failed to show the same degree of impairment in deacetylation of ATRIP Lys-32 and CDK9 Lys-48 (Fig. 4, A and B) compared with p53 Lys-320 (Fig. 2C), suggesting that these mutants are less impactful on SIRT2 deacetylase activity and/or impair deacetylation of specific substrates. These data suggest that P128L, P140H, and A186V impair SIRT2’s activity in the DDR, which leads to genomic instability
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