CDK1-Mediated SIRT3 Activation Enhances Mitochondrial Function and Tumor Radioresistance

Abstract

Tumor adaptive resistance to therapeutic radiation remains a barrier for further improvement of local cancer control. SIRT3, a member of the sirtuin family of NAD(+)-dependent protein deacetylases in mitochondria, promotes metabolic homeostasis through regulation of mitochondrial protein deacetylation and plays a key role in prevention of cell aging. Here, we demonstrate that SIRT3 expression is induced in an array of radiation-treated human tumor cells and their corresponding xenograft tumors, including colon cancer HCT-116, glioblastoma U87, and breast cancer MDA-MB231 cells. SIRT3 transcriptional activation is due to SIRT3 promoter activation controlled by the stress transcription factor NF-κB. Posttranscriptionally, SIRT3 enzymatic activity is further enhanced via Thr150/Ser159 phosphorylation by cyclin B1-CDK1, which is also induced by radiation and relocated to mitochondria together with SIRT3. Cells expressing Thr150Ala/Ser159Ala-mutant SIRT3 show a reduction in mitochondrial protein lysine deacetylation, Δψm, MnSOD activity, and mitochondrial ATP generation. The clonogenicity of Thr150Ala/Ser159Ala-mutant transfectants is lower and significantly decreased under radiation. Tumors harboring Thr150Ala/Ser159Ala-mutant SIRT3 show inhibited growth and increased sensitivity to in vivo local irradiation. These results demonstrate that enhanced SIRT3 transcription and posttranslational modifications in mitochondria contribute to adaptive radioresistance in tumor cells. CDK1-mediated SIRT3 phosphorylation is a potential effective target to sensitize tumor cells to radiotherapy.

Figure 1

Radiation-induced SIRT3 expression results in reduced levels of acetylated proteins both in vitro and in vivo. A, enhanced SIRT3 protein expression in human cancer cell lines and their corresponding mouse xenograft tumors receiving in vitro and local in vivo irradiation. Left panel, western blot of SIRT3 in human colon cancer (HCT-116), glioblastoma (U87) and breast cancer (MDA-MB231) cells and their corresponding mouse xenograft tumors 24 h after sham (−) or 5 Gy IR (+). Right panel, the levels of SIRT3 expression in irradiated cells and tumors were estimated by measuring the band intensity using ImageJ software, normalized with β-actin and compared with the sham radiation controls (n=3, *P < 0.05). B, western blot of SIRT3 as well as the acetylated proteins of mitochondrial fractions isolated from HCT-116 cells and the corresponding xenograft tumors (C) at different times after 5 Gy IR. The total acetylated mitochondrial protein levels were estimated by densitometry and normalized with COX IV as SIRT3/Ac-K ratio (lower panels).

Figure 2

NF-κB is responsible for radiation induced SIRT3 expression. A, enhanced SIRT3 mRNA levels in irradiated HCT-116, U87 and MDA-MB231 cells measured by quantitative RT-PCR. B, NF-κB reporter activity measured 24 h after sham or 5 Gy IR. C, NF-κB responsive fragment (−352 to −146) containing NF-κB-RE consensus sequence (−184 to −171) was identified in the promoter region of human SIRT3 gene. Luciferase reporter plasmids with wild type (SIRT3 WT) or mutant (SIRT3 MUT; lacking the NF-κB fragment) SIRT3 promoter region or Mock (empty vector control) were transfected to HCT-116 cells and luciferase activity was measured 24 h after sham or 5 Gy IR. Data were normalized to β-gal activity (n = 3; *P < 0.05, **P < 0.01). D, ChIP assay of NF-κB in SIRT3 promoter region. Fragment A (−352 to −146): encompassing the NF-κB consensus sequence; Fragment B (−1311 to −1509): a non-relevant upstream sequence as the control. Protein-DNA complexes were extracted from IR (5 Gy)-treated HCT-116 cells and immunoprecipitated using anti-NF-κB (anti-p65, anti-p50) with precipitation of normal IgG as the negative control, and anti-C-Rel as the positive control; DNA fragments were amplified with primers specific for the SIRT3 promoter sequence of fragment A (SIRT3-A) or fragment B (SIRT3-B). Total chromatin was included as the input control for PCR. The IκB promoter region (IκB-α-NFκB) and GAPDH were included as positive and negative controls.

Figure 3

SIRT3 co-localizes with CyclinB1/CDK1 in the mitochondria. A, mitochondrial accumulation of Cyclin B1 and CDK1. a, Time-course analysis of mitochondrial Cyclin B1 and CDK1 in IR-treated HCT-116 cells detected by western blot. b, Cyclin B1 and CDK1 protein levels were quantified by measuring band intensity from three western blots using Image J software and normalized with COX IV. B, representative images of mitochondrial localization of cyclin B1 (green, upper panel) and CDK1 (green, lower panel), co-stained with mitochondria marker, COX IV (red), in HCT-116 cells by 3-D structured illumination super-resolution microscopy. Scale bar, 1 unit = 5 μm. C, mitochondrial proteins were immunoprecipated (IP) with anti-phosphoserine (phos-S/T) or anti-SIRT3 followed by immunoblotting (IB) with anti-SIRT3 or anti-phos-S/T, respectively. IP with normal IgG serves as negative control and COXIV serves as equal loading control. D, co-IP of mitochondrial CDK1 and SIRT3 using mitochondrial fractions isolated from 5 Gy-irradiated or sham-irradiated HCT-116 cells (n=3).

Figure 4

SIRT3 is phosphorylated by mitochondrial CDK1 after radiation. A, CDK1-mediated SIRT3 phosphorylation was analyzed by IP with mitochondrial fractions isolated from HCT-116 cells harboring mitochondria-targeted wild type (pERFP-MTS-CDK1) or mutant (pERFP-MTS-CDK1-D146N, deficient phosphorylation activity) CDK1 with anti-phos-S/T followed by IB with anti-SIRT3 (Control, normal IgG; Mock, empty vector transfectants of HCT-116). IB of mitochondria fractions with anti-RFP antibody was used as equal IP loading control of exogenous wild type and mutant CDK1. B, phosphorylation of SIRT3 in mitochondria of irradiated HCT-116 cells harboring Flag tagged wild type or mutant SIRT3 were analyzed by IP using anti-phos-S/T followed by IB with anti-flag. IB of mitochondria fractions with anti-Flag antibody was used as equal IP loading control of exogenous wild type and mutant SIRT3. C, kinase assay using commercial Cyclin B1/CDK1 and total proteins isolated from HCT-116 cells harboring wild type, single mutant SIRT3/T150A or SIRT3/S159A and double mutant SIRT3/T150A/S159A. D, kinase assay using commercial Cyclin B1/CDK1 and total proteins isolated from HCT-116 cells harboring wild type or double mutant SIRT3 (IP/SIRT3 as input SIRT3; IP/Mock was the empty vector control). E, kinase assay using commercial (left) and immunoprecipitated mitochondrial (right) CDK1 with wild type (duplicate 1 and 2) or Thr150/Ser159 mutant SIRT3 proteins synthesized in E. coli (GST, negative control) (n=3). F, western blot of mitochondrial acetylated proteins of irradiated HCT-116 cells harboring wild-type or Thr150/Ser159 mutant SIRT3 (a). COX IV, mitochondrial protein equal loading control; Flag, equal loading control of mitochondria targeted expression of wild type and mutant SIRT3. The acetylated protein levels were estimated by densitometry and normalized with COX IV from three separated blots (b, *P < 0.05). G, SIRT3 targeted deacetylation of NDUFA9, MnSOD and p53 in irradiated HCT-116 cells harboring mock, wild type or Thr150/Ser159 mutant SIRT3 detected by IP with anti-Ac-K followed by IB with respective antibodies.

Figure 5

Expression of Thr150/Ser159 mutant SIRT3 decreases MnSOD activity and mitochondrial functions. HCT116 cells harboring mock, wild type SIRT3 or Thr150/Ser159 mutant SIRT3 were treated with sham or 5 Gy IR. MnSOD activity (A), ΔΨm (B), mitochondrial superoxide levels (C) and mitochondrial ATP production (D) were measured 24 h after radiation. HCT116 cells harboring Thr150/Ser159 mutant SIRT3 were treated with MnSOD mimic drug, WR1065 (40 μM, 24 h), followed by sham or 5 Gy IR. Mitochondrial ATP production (E) and mitochondrial membrane potential (F) were measured 24 h after radiation. ATP production (G), mitochondrial membrane potential (H), and mitochondrial superoxide levels (I) were measured 24 h post IR in HCT116 cells transfected with scrambled control or SIRT3 siRNA for 48 h. (n = 5, *, P < 0.05, **, P < 0.01).

Figure 6

Expression of Thr150/Ser159 mutant SIRT3 inhibits clonogenic survival and increases tumor radiosensitivity. A, western blot of cytochrome c and caspase 3 cleavage in HCT-116 cells harboring wild type or Thr150/Ser159 mutant SIRT3 24 h after 5 Gy IR. B, apoptosis assay in HCT-116 cells harboring wild type or Thr150/Ser159 mutant SIRT3 upon sham or 5 Gy IR (n = 3, *, P < 0.05). C, Clonogenic survival rates were measured in above HCT-116 stable transfectants after irradiation with 5 and 10 Gy. Survival fractions normalized to mock sham controls were shown (n = 5, *, P < 0.05, **, P < 0.01). D, Clonogenic survival of HCT116 cells that are transfected with scrambled control or SIRT3 siRNA for 48 h, with or without radiation (n=3, *p<0.01). E, HCT-116 cells stably transfected with mock, wild type or mutant SIRT3 were inoculated in mice and when tumor reached ~ 0.3 cm³, 5 Gy IR was delivered locally to tumors with surrounding tissue shielded. a, tumor volumes were measured post-irradiation every 3 days (mean ± SD; n = 6). b, Representative photographs of surgically removed tumors at the end of experiments were shown (see also Fig. S6).

Figure 7

A mitochondrial homeostasis model in tumor radioresistance. Findings from this study demonstrate a unique mechanism by which SIRT3 is upregulated in tumor cells by radiation via NF-κB-mediated SIRT3 promoter activation. SIRT3 enzymatic activity is further enhanced in mitochondria via Cyclin B1/CDK1-mediated SIRT3 Thr150/Ser159 phosphorylation. Expression of the mutant Thr150/Ser159 SIRT3 decreases the level of mitochondrial protein deacetylation, MnSOD activity and ATP generation whereas increases cell radiosensitivity.