Loss of SirT3 increases ROS and allows for transformation (Kim et al., 2010
). Increases in ROS are also associated with senescence and apoptosis in primary cells. We thus set out to determine the effect of SirT3 deletion on replicative life span of primary MEFs. Surprisingly, we found that SirT3−/−
MEFS have increased population doublings compared with wt controls (). Another example of increased replicative life span is found when cells are incubated in hypoxic conditions (Parrinello et al., 2003
) thereby generating ROS, and this increase is dependent on HIF activity (Bell et al., 2007b
). Therefore, we explored whether loss of SirT3 activates HIF. Using a synthetic HIF-1α reporter construct (HRE-luc) that consists of three putative HRE sites upstream of firefly luciferase, we found that MEFs displayed mildly elevated HIF-1α transcriptional activity in the absence of SirT3 (). Next we examined whether SirT3 has a role in the maintenance and progression of cancer by using established cancer cell lines. We knocked down SirT3 in the human osteosarcoma cell line 143B () and the human colon carcinoma cell line HCT116 with two different short-hairpin (sh)RNAs (), and found that both knockdown cell lines displayed increased proliferation (). These data indicate that SirT3 has tumor suppressor functions in already established cancer cell lines in addition to its previously described role in inhibiting transformation of primary cells (Kim et al., 2010
). Both the 143B and HCT116 SirT3 knockdown cell lines showed a mild increase in luciferase activity when transfected with HRE-luc (). These data demonstrate that deleting SirT3 results in higher growth rates in normal and established cancer cell lines, and may increase normoxic HIF-1α activity.
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 (more ...)
We next explored whether SirT3 knockdown affected the hypoxic activation of HIF-1α. Culturing the SirT3 knockdown cells in hypoxic conditions (1% O2) resulted in a striking increase in the amount of HIF-1α protein stabilized when compared with the expected increase in the scrambled shRNA control (). To determine whether there is a concomitant increase in HIF-1α transcriptional activity we utilized the HRE-luc construct. In hypoxic conditions there was a 13-fold increase in luciferase activity in the scramble control compared with normoxic conditions, whereas the SirT3 knockdown cells demonstrated a 70-fold increase in luciferase activity under the same conditions (). To determine the effects on endogenous HIF-1α targets, we performed quantitative PCR on RNA isolated from cells incubated at ambient O2 levels or 1% O2. Levels of all putative HIF-1α targets tested, VEGF-A, PGK-1 and PDK-1 were increased in hypoxia in the control samples, and the loss of SirT3 further increased these HIF-1α targets, albeit to a lesser extent than HRE-luc (). Interestingly, there was no increase of these targets in the SirT3 knockdown cells cultured under normoxic conditions. This may be due to a requirement of co-activators that are only activated in hypoxia.
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 4h. (b) Relative (more ...)
Since loss of SirT3 augments hypoxic activation of HIF-1α, we wished to know whether SirT3 gain of function attenuates HIF-1α in the hypoxic response. Thus we stably expressed V5-tagged SirT3 in 143B cells (), incubated cells in 1% O2 and analyzed HIF-1α expression. The control cells had a significant increase of HIF-1α protein in hypoxic conditions whereas the cells expressing SirT3 failed to stabilize HIF-1α protein during hypoxia (). However, both control and SirT3-expressing cells stabilized HIF-1α protein in the presence of DMOG, a competitive inhibitor of the proline hydroxylation enzymes. The decrease in HIF-1α protein stability in hypoxic SirT3-expressing cells corresponded to an attenuated transcriptional response of HRE-luc (), as well as the endogenous HIF-1α responsive target gene PGK1 (). These data demonstrate that increased SirT3 can prevent HIF-1α stabilization in hypoxia, reciprocal to the phenotype of SirT3 knockdown cells.
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 (more ...)
Previous work has demonstrated that addition of exogenous ROS is sufficient to stabilize and activate HIF-1α in normoxic conditions, and ROS is necessary for its hypoxic stabilization (Chandel et al., 2000
). As has been previously published, we observed an increase in reactive oxygen species in SirT3−/−
primary and immortalized MEFs (). Interestingly, when we knocked down SirT3 in established cancer cell lines we also observed increased ROS ( and Supplementary Figure S1). Further, overexpression of SirT3 decreased basal ROS levels, as well as the increased ROS levels mediated by antimycin A (). These data suggest that the alteration of ROS levels may be the main mechanism by which SirT3 regulates HIF-1α. To determine whether the activation of HIF-1α in the absence of SirT3 is due to ROS, we treated cells with antioxidants and determined the extent of HIF-1α activation. The 143B and HCT116 cells that have SirT3 stably knocked down were treated with N
-acetyl-cysteine (NAC), and the increase in ROS was prevented (Supplementary Figure S1). Strikingly, addition of NAC to primary SirT3−/−
MEFs abolished the increase in population doublings observed in the primary SirT3−/−
MEFs (). NAC treatment also decreased the normoxic increase of HIF-1α transcriptional activity in 143B and HCT116 SirT3 knockdown cells ().
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 10μ DHE in normal oxygen conditions and (more ...)
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 (5m). The 143B (b) and (more ...)
Hypoxia or treatment with antimycin A induces ROS production from the Qo
site of mitochondrial complex III. The fact that SirT3 overerxpression inhibits hypoxic activation of HIF-1α as well as antimycin A induced ROS, thus indicates that SirT3 may be acting on complex III. To determine if the source of the increased ROS in the absence of SirT3 is complex III of mitochondria, we measured HIF-1α activity in the presence or absence of the complex III inhibitor stigmatellin. Stigmatellin binds to the Qo
site of complex III to inhibit electron transfer and therefore the ability of ROS to be generated from the Qo
site (Breyton, 2000
). Addition of stigmatellin attenuated the increase in HRE-luciferase in the SirT3 knockdown cells (), thereby demonstrating that the increase in HIF-1α activity is due to ROS generated by complex III. Knocking down SirT3 in both the 143B- and HCT116-established cancer cell lines did not alter protein levels of MnSOD or the cytosolic antioxidants catalase and SOD1 (Supplementary Figure S2).
To determine whether SirT3 modulates the progression and maintenance of tumors, we preformed studies taking advantage of the fact that HCT116 cells are dependent on HIF for the generation of tumors in xenograft models (Dang et al., 2006
). When HCT116 cells with stable knockdown of SirT3 were injected into the flanks of Nu/Nu mice, they formed tumors with an increased rate of growth and increased final mass compared with control HCT116 cells expressing a scrambled shRNA injected in the same mouse on the opposite flank (). The tumors formed by the injected cells maintained the SirT3 knockdown level of expression and also had increased mRNA levels of the pro-angiogenic HIF-1α target VEGF-A (), indicating that HIF-1α activity is upregulated in these tumors. To determine whether this difference in tumorigenesis is due to increased ROS in the SirT3 knockdown cells, we supplied NAC to the drinking water. In the presence of NAC, the tumors derived from the SirT3 shRNA cells did not display an increase in the rate of tumor growth (), final mass () or VEGF-A mRNA (). This data implies that SirT3 deficiency increases ROS to activate HIF-1α and facilitate tumorigenesis.
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 (more ...)
The data from suggests that overexpression of SirT3 might decrease the ability of cells to form tumors. However, we were not successful in generating HCT116 cell lines overexpressing SirT3, suggesting that constitutive SirT3 overexpression is selected against in this cell line. Therefore we turned to an inducible system in order to analyze the role of SirT3 gain of function on proliferation and tumorigenesis. We generated 143B and HCT116 cells stably expressing doxycycline-inducible SirT3, or GFP as a control (). When induced by the addition of doxycycline, both the 143B and HCT116 cells that expressed SirT3 had decreased proliferation compared with the GFP cells and the un-induced cells (). Next, we performed xenograft experiments with the HCT116 GFP or SirT3 inducible cell lines. Nine days after injecting the cells the food was switched in half the cages to food containing doxycycline. Induction of SirT3 led to smaller tumors compared with the induction of GFP or no induction (). The rate of tumorigenesis was decreased, as well as the final tumor mass, with the cells that stably induced SirT3 expression (). These data demonstrate for the first time that overexpression of SirT3 indeed can inhibit the growth of established cancer cells in xenograft models of tumorigenesis.
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 (more ...)