SirT3 suppresses hypoxia inducible factor 1α and tumor growth by inhibiting mitochondrial ROS production

Abstract

It has become increasing clear that alterations in cellular metabolism have a key role in the generation and maintenance of cancer. Some of the metabolic changes can be attributed to the activation of oncogenes or loss of tumor suppressors. Here, we show that the mitochondrial sirtuin, SirT3, acts as a tumor suppressor via its ability to suppress reactive oxygen species (ROS) and regulate hypoxia inducible factor 1α (HIF-1α). Primary mouse embryo fibroblasts (MEFs) or tumor cell lines expressing SirT3 short-hairpin RNA exhibit a greater potential to proliferate, and augmented HIF-1α protein stabilization and transcriptional activity in hypoxic conditions. SirT3 knockdown increases tumorigenesis in xenograft models, and this is abolished by giving mice the anti-oxidant N-acetyl cysteine. Moreover, overexpression of SirT3 inhibits stabilization of HIF-1α protein in hypoxia and attenuates increases in HIF-1α transcriptional activity. Critically, overexpression of SirT3 decreases tumorigenesis in xenografts, even when induction of the sirtuin occurs after tumor initiation. These data suggest that SirT3 acts to suppress the growth of tumors, at least in part through its ability to suppress ROS and HIF-1α.

Figure 1

SirT3 loss of function increases proliferation and HIF-1α activity. (a) Population doublings in primary SirT3 wild type (wt) and SirT3^−/− (KO) MEFs cultured in normal oxygen conditions (21% O₂). (b) Luciferase values from immortalized wt and KO MEFs transfected with HRE-luciferase in normoxia. SirT3 protein levels in 143B (c) and HCT116 (d) cells stably expressing a scrambled control shRNA (scr) or two different shRNAs that target SirT3. Both shRNA sequences increase the rate of cellular proliferation. Relative luciferase values of 143B (e) and HCT116 (f) cells in SirT3 stable knockdown cell lines (shRNA) or a scrambled control vector (scr) line transfected with HRE-luciferase. Error bars are s.e.m. and * indicates a P value <0.05 with two-tailed Student's t-test.

Figure 2

Knockdown of SirT3 augments the hypoxic response. (a) HIF-1α protein levels from whole cell lysates of indicated tumor cell lines incubated at normoxic (N, 21% O₂) or hypoxic (H, 1% O₂) conditions for 4 h. (b) Relative luciferase values of 143B cells in SirT3 stable knockdown cell lines (shRNA) or a scrambled control vector (scr) line transfected with HRE-luciferase cultured in normoxic (N, 21% O₂) or hypoxic (H, 1% O₂) conditions for 16 h. Quantitative PCR for VEGF-A (c), PGK-1 (d) and PDK-1 (e) on RNA isolated from 143B cells stably expressing SirT3 shRNA or a scrambled control vector (scr) cultured in normoxic (N, 21% O₂) or hypoxic (H, 1% O₂) conditions for 16hrs. Error bars are s.e.m. and * indicates a P value <0.05 with two-tailed Student's t-test.

Figure 3

SirT3 gain of function inhibits hypoxic activation of HIF-1α. (a) Western blot of 143B cells stably overexpressing SirT3 tagged with V5. (b) Western blots of HIF-1α using total cell lysates from 143B control (c) and SirT3 overexpressing cells in normoxic (N, 21% O₂) or hypoxic (H, 1% O₂) conditions or treated with DMOG (d) for 16 h. (c) Relative luciferase values in 143B SirT3 overexpressing cells transfected with HRE-luciferase. (d) PGK1 mRNA from 143B SirT3 overexpressing cells in normoxic (N21% O₂) or hypoxic (H, 1% O₂) conditions. Error bars are s.e.m. and * indicates a P value <0.05 with two-tailed Student's t-test.

Figure 4

ROS levels are regulated by SirT3. Relative levels of dihydroethidium (DHE) fluorescence in wild type (wt) or SirT3 ^−/− (KO) primary MEFs (a) and immortalized MEFs (b) treated with 10 μ DHE in normal oxygen conditions and then analyzed by flow cytometry. Each data point represents an independent culture of cells. (c) Relative fluorescence of 143B cells with SirT3 stably knocked down with two different SirT3 shRNAs or a scrambled control after incubation with DHE. (d) Western blots of whole cell lysates isolated from 143B cells stably overexpressing SirT3 Flag and their relative fluorescence after incubation with DHE with or without antimycin A (2 μg/ml). Error bars are s.e.m. and * indicates a P value <0.05 with two-tailed Student's t-test.

Figure 5

Increased HIF-1α activity in the absence of SirT3 is mediated by ROS from complex III. (a) Population doublings of two wild-type and two SirT3^−/− (KO) primary MEFs in the presence or absence of NAC (5 m). The 143B (b) and HCT116 (c) SirT3 stable knockdown cell lines transfected with HRE-luciferase with or without 24 h NAC (10 m) pretreatment. Luciferase activity in stigmatellin (1 μ) treated or not treated (NT) 143B (d) and HCT116 (e) stable cell lines with scrambled or SirT3 shRNA and transfected with HRE-luciferase. Error bars are s.e.m. and * indicates a P value <0.05 with two-tailed Student's t-test.

Figure 6

Loss of SirT3 in established human cancer cell lines increases tumor growth and is dependent on ROS. Tumor volume (a) and tumor mass (b) 24 days after subcutaneous injection of HCT116 scramble control and SirT3 knockdown cells in Nu/Nu mice with or without 40 m NAC supplementation in the drinking water. (c, d) Pictures of tumors from a and b after mice were euthanized. (e) Quantitative PCR for VEGF-A and SirT3 on RNA isolated from the tumors. Error bars are s.e.m. and * indicates a P value <0.05 with two-tailed Student's t-test.

Figure 7

Overexpression of SirT3 negatively regulates proliferation and tumorigenesis. Inducible overexpression of GFP and SirT3 in 143B (a) and HCT116 (b) decreases proliferation. Tumor volume (c) and tumor mass (d) of HCT116 GFP or SirT3 cells injected into the flanks of Nu/Nu mice. Doxycycline was added on day 9 to induce expression of GFP or SirT3. Error bars are s.e.m. and * indicates a P value <0.05 with two-tailed Student's t-test. (e) Pictures of tumors from c and d.