The function of the class III histone deacetylase, Sir2, in promoting lifespan extension is well established in small model organisms. By analogy, SirT1, the mammalian orthologue of Sir2, is a candidate gene to slow down aging and forestall the onset of age-associated diseases. We have used SirT1-null mice to study the function of SirT1 in susceptibility to tumorigenesis. The number of intestinal polyps induced in mice carrying the Apcmin mutation was unaffected by the SirT1 genotype although the average polyp size was slightly smaller in the SirT1-null animals. Similarly, the presence or absence of SirT1 had no effect on incidence and tumor load of skin papillomas induced by the classical two-stage carcinogenesis protocol. We found that resveratrol topically applied to the skin profoundly reduced tumorigenesis. This chemoprotective effect was significantly reduced but not ablated in SirT1-null mice, suggesting that part of the protection afforded by resveratrol requires the SirT1-encoded protein. Thus, our results suggest that SirT1 does not behave like a classical tumor-suppressor gene but the antitumor activity of resveratrol is mediated at least in part by SirT1.
Silent information regulator 2 (Sir2) is a yeast gene encoding a nicotinamide adenine dinucleotide-dependent histone deacetylase. In small model organisms, Sir2 orthologues have been reported to promote lifespan extension (Kaeberlein et al., 1999; Tissenbaum and Guarente, 2001; Rogina and Helfand, 2004) and to be required for calorie restriction (CR) to extend lifespan (Lin et al., 2000; Rogina and Helfand, 2004). We have recently reported evidence that the orthologue of Sir2 in mammals, SirT1, is also required for many of the physiological effects of CR (Boily et al., 2008). These data suggest that SirT1 could modulate biological aging and hopes are that modulation of its activity can be used to protect against age-associated diseases.
Published evidence suggests that SirT1 prevents age-associated diseases such as metabolic disorders (Picard et al., 2004; Kitamura et al., 2005; Moynihan et al., 2005; Bordone et al., 2006; Gerhart-Hines et al., 2007; Rodgers and Puigserver, 2007), neurodegenerative diseases (Araki et al., 2004; Parker et al., 2005; Chen et al., 2005a; Qin et al., 2006; Kim et al., 2007) and atherosclerosis (Zhang et al., 2008). Accumulating evidence also point to SirT1 as having a function in cancer; however, inferences from the literature are that SirT1 has both oncogenic and tumor-suppressor activities. SirT1 was reported to be overexpressed in acute myeloid leukemia, non-melanoma skin cancer, breast cancer, colorectal carcinoma and prostate cancer (Bradbury et al., 2005; Kuzmichev et al., 2005; Hida et al., 2007; Huffman et al., 2007; Stunkel et al., 2007) and to be downregulated in glioblastoma, prostate cancer, bladder carcinoma, breast cancer, hepatocellular carcinoma and ovarian cancer (Wang et al., 2008a). It is also expressed at a high level in diffuse large B-cell lymphomas and is associated to bad prognosis (Jang et al., 2008). In cultured cancer cells, SirT1 can inhibit apoptosis (Ford et al., 2005) but also has an inhibitory effect on proliferation of certain cancer cell types (Fu et al., 2006). Several reports show data consistent with SirT1 having an inhibitory effect (Langley et al., 2002; Ota et al., 2006; Abdelmohsen et al., 2007; van der Veer et al., 2007; Huang et al., 2008), a promoting effect (Chua et al., 2005) or no effect (Michishita et al., 2005; Kamel, Boily and McBurney, unpublished data) on cellular senescence. SirT1 expression can be repressed by HIC1 (Chen et al., 2005b), activated by BRCA1 (Wang et al., 2008b), and both suppressed (in fed cells) and activated (with FoxO3a in starved cells) by p53 (Nemoto et al., 2004), three tumor-suppressor proteins. Moreover, some targets of SirT1 deacetylation, including p53 (Luo et al., 2001; Vaziri et al., 2001), p73 (Dai et al., 2007), nuclear factor-κB (NF-κB) (Yeung et al., 2004; Chen et al., 2005a) and AR (Fu et al., 2006), are important proteins involved in cancer. Finally, recent reports suggest that SirT1 has a function in maintaining genome stability (Oberdoerffer et al., 2008; Wang et al., 2008a) and compromise of the genome is a hallmark of cancer.
We (McBurney et al., 2003) and others (Cheng et al., 2003; Wang et al., 2008a) have created mice carrying targeted mutations in the SirT1 gene. On an outbred genetic background, most of our SirT1-null mice survive to adulthood. We used normal and SirT1-null mice to test the idea that SirT1 prevents or forestalls the onset of cancer. We induced the development of tumors on the skin of mice using a two-step carcinogenesis protocol, consisting of the application of 7,12-dimethylbenz(a)anthracene (DMBA) as a tumor initiator and phorbol 12-myristate 13-acetate (TPA) as a promoter (Jang et al., 1997). Resveratrol has a protective role on tumorigenesis in this protocol (Jang et al., 1997), so we also set out to determine whether the presence of SirT1 is required for this effect. In a second tumor model, we evaluated the effect of SirT1 on intestinal polyp formation arising in mice carrying the Apcmin allele.
Two-stage skin carcinogenesis
The effect of SirT1 genotype on skin tumorigenesis was tested in two independent experiments. Lower backs of mice were shaved and the initiator DMBA applied. A week later, the promoter TPA was applied to this area of the skin and this was repeated once weekly for 22 or 15 weeks. Virtually all mice developed tumors (Figure 1a) and SirT1 genotype did not have any effect on the rate of tumor induction (Figures 2a and 3a). SirT1+/+ and SirT1+/− animals in these and all of our experiments were not distinguishable so these two genotypes were grouped together in our analyses. Tumors that formed were typical papillomas (Figure 1b). The histological characteristics of the tumors were the same in normal and in the SirT1-null animals, except that the region of TPA application often appeared more irritated in the SirT1-null mice (data not shown).
The effect of resveratrol treatment and SirT1 genotype was also tested in two independent experiments, using 1 and 25 μmol of resveratrol, respectively (see Materials and methods section). In the first experiment, 1 μmol of resveratrol was applied weekly along with TPA. This dose had no effect on the tumor incidence of either normal or SirT1-null mice (Figure 2b, dashed lines). However, in the second experiment, the application of 25 μmol of resveratrol weekly along with TPA reduced the tumor incidence (Figure 3b, dashed lines). The protective effect of resveratrol was profound on normal mice, only 20% had developed tumors after 15 weeks. By contrast, resveratrol afforded more modest protection to SirT1-null mice, with 75% of these mice developing tumors by 15 weeks (Fischer's exact test at end point, P-value=0.03) (Figure 2b). This result suggests that there is both SirT1-dependent and SirT1-independent components to the effects of resveratrol on tumor incidence.
Total number of tumors was counted every week during the experiment and our results showed that SirT1 genotype had no effect (Figures 2c and 3c). Similarly, application of 1 μmol of resveratrol along with TPA had no effect on number of tumors (Figure 2d, dotted lines). However, 25 μmol of resveratrol reduced the number of tumors in both normal and SirT1-null mice, though the effect was less pronounced in null mice (Figure 3d).
Measurements of the three dimensions of the tumors were taken at end point and tumor volumes were calculated. In the first experiment, SirT1 genotype did not affect total and average tumor volumes (Figures 2e–f). When treated with 1 μmol of resveratrol, total and average tumor volumes of SirT1-null mice tended to be higher in SirT1-null mice, but this difference was not statistically significant (Figures 2e–f). In the second experiment, total tumor volume in SirT1-null mice tended to be higher and average tumor volume was significantly higher than those in normal mice (Figures 3e–f). Treatment with 25 μmol of resveratrol strongly and significantly reduced total and average tumor volumes in both normal and SirT1-null mice, but tumor volumes remained higher in SirT1-null mice than in normal mice (Figures 3e–f).
We compared the SirT1 expression levels in skin tumors and adjacent normal skin of wild-type mice. SirT1 was preferentially expressed in cells of the epidermis, sometimes in a mosaic fashion, and was similarly expressed in papillomas (Figure 4).
TPA treatment results in the activation of several signaling pathways, including NF-κB and MAP kinases, and eventually culminates in the induction of Cox2, a pro-inflammatory enzyme, whose expression is critical for skin tumor formation (Muller-Decker et al., 2002; Tiano et al., 2002; Kundu et al., 2006). Cox2 expression was extremely variable in skin tumors in both normal and SirT1-null mice. We selected the highest Cox2 expression regions of tumor samples for comparison. As shown in a representative selection of tumor samples in Figure 5a, areas of high and low expression were observed in similar proportions in wild-type and in SirT1-null tumors. In the groups treated with 25 μmol of resveratrol, tumors of the two wild-type mice that developed tumors had higher Cox2 expression than seen in any of the SirT1-null tumors investigated (Figure 5b).
A recent report has shown that SirT1 is involved in postnatal angiogenesis (Potente et al., 2007). The induction of tumors using DMBA and TPA leads to the formation of new vessels in the dermis located beneath the tumors, as shown by staining with the blood vessel marker CD31 (Figure 6a, columns 1–2). Vessels also developed in the skin of SirT1-null mice and the number of vessels per surface unit was not different from that of SirT1 wild-type mice (Figure 6a, columns 2–3 and Figure 6b). Moreover, the distribution of vessel size in SirT1-null mice was not different from that in wild-type mice (Figure 6c).
Intestinal polyp formation in SirT1-null/Apcmin mice
To evaluate the in vivo function of SirT1 in the susceptibility to develop intestinal tumors, we introduced the Apcmin gene into stock carrying the SirT1 knockout allele. Although Apcmin/+ mice on the inbred C57Bl/6 genetic background develop polyps in the first months of life and start to die at about 4 months of age, the mixed background obtained from our breeding retarded the development of polyps so that Apcmin/+ animals did not start to die before 9 months of age. We harvested our Apcmin/+ mice at 9–12 months (most of them at 12 months) of age and compared their polyp load. These mice developed polyps almost exclusively in their small intestines and very rarely in their colon. There was no notable difference in the morphology of the polyps, macroscopically or microscopically, arising in the different SirT1 genotypes (Figures 7a–b). SirT1-null Apcmin/+ mice developed similar numbers of polyps to normal Apcmin/+ mice (Figure 7c). SirT1-null Apcmin/+ mice tended to have a smaller total polyp surface relative to polyps in mice with normal SirT1, although this difference was not statistically significant (Figure 7d). However, when the average polyp surface (total surface per number of polyps) was calculated, polyps of SirT1-null mice were significantly smaller (Figure 7e).
Although expression of SirT1 in intestines has been previously reported (Firestein et al., 2008), we found very low levels of SirT1 protein in intestines, despite the fact that the immunohistochemistry protocol was the same as the one we used for skin and that exposure time to the detection substrate was eight times longer (Figure 7f). This low protein expression was confirmed by western blot (Figure 7g). We sometimes observed that SirT1 was expressed slightly more in the polyps compared to normal villi, though this expression was also very low in all cases (Figure 7f). Altogether, our data suggest that the absence of SirT1 does not modulate polyp formation but does affect the rate of growth of these tumors.
Effect of SirT1 and resveratrol on cell transformation in vitro
As a final test for SirT1 on oncogenic transformation, we used oncogene-mediated NIH3T3 cell focus formation (Ono et al., 1991). Cells were co-transfected with a plasmid encoding polyoma middle T antigen (midTAg) together with plasmids expressing GFP (ML8), SirT1 (KJ321 and MA4) or catalytically inactive (H355Y) SirT1 (KJ322 and MA5). Following transfection, cells were plated for focus formation in the presence of different concentrations of resveratrol. Overexpression of SirT1 or catalytically inactive SirT1 did not significantly affect focus formation (Figure 8). Treatment with 30 or 100 μM of resveratrol also did not reduce the number of foci (Figure 8).
If SirT1 has a general function in forestalling the onset of age-related diseases, we predicted that SirT1 would have tumor-suppressive activity and that SirT1-null mice would develop tumors at precocious rates. This prediction was not confirmed in two in vivo experimental tumor models or in one in vitro cell transformation assay. We found that SirT1-null mice did not differ from their normal littermates in terms of incidence or tumor load. However, resveratrol had a profound inhibitory effect on skin carcinogenesis in normal animals and a much less remarkable effect in the SirT1-null mice. Thus, the beneficial effects of resveratrol are partially though not entirely dependent on the presence of the SirT1 protein.
Several effects of resveratrol have been shown to be dependent on SirT1 (Howitz et al., 2003; Picard et al., 2004; Wood et al., 2004; Yeung et al., 2004), although the mechanism of resveratrol activity is controversial (Borra et al., 2005; Kaeberlein et al., 2005). In the present study, resveratrol-mediated reduction of tumor incidence was largely dependent on SirT1, yet the presence of SirT1 itself was not protective against tumorigenesis. One explanation might be that resveratrol activates SirT1 whereas SirT1 is inactive or has low activity in skin not treated with this phytochemical. Our study also showed a SirT1-independent chemoprotection by resveratrol. This result is not surprising because resveratrol has been reported to modulate the activities of a variety of enzymes and pathways including several that are germane to development of cancer—NF-κB, AMPK, NQO-1, prostaglandins, steroid hormones, MAP kinases and PKC isozymes (Cucciolla et al., 2007).
SirT1 has been reported to be a critical regulator of postnatal angiogenesis (Potente et al., 2007). However, we found that the density and size distribution of newly formed vessels in the skin tumors of SirT1-null mice were similar to those of normal mice. Thus, a function of SirT1 in angiogenesis was not evident in our carcinogenesis experiments.
Although the skin tumors in SirT1-null mice were on average slightly larger than those in normal littermates, the average Apcmin-induced polyps in SirT1-null mice were smaller than in normal animals. Insulin-like growth factors (IGFs) are important for development of a number of cancers. Notably, elevated levels of IGFs increase the number of intestinal tumors (Wu et al., 2002; Sakatani et al., 2005). We have previously shown that SirT1-null mice produce elevated levels of insulin-like growth factor binding protein 1 (IGFBP1) (Lemieux et al., 2005). IGFBPs bind to circulating IGFs and can limit their growth-promoting effect (Duan and Xu, 2005). Thus, high IGFBP1 levels generally found in SirT1-null background could have resulted in smaller average polyp surface in the SirT1-null mice.
Recently, two reports have appeared that suggest a tumor-suppressive function for Sirt1. Firestein et al. (2008) reported that Apcmin/+ mice overexpressing SirT1 specifically in the gut villi had fewer and smaller polyps in their intestines, suggesting that SirT1 has tumor-suppressive effects. This seems inconsistent with our observation that Apcmin/+ SirT1-null mice had similar numbers of polyps. However, we found a very low level of SirT1 protein in the intestine so perhaps the suppressive effect seen by Firestein et al. is conditional to the achievement of a SirT1 activity threshold reached by overexpression of the SirT1 transgene, which is not normally reached by the endogenous SirT1 expression level. Wang et al. (2008a) reported that SirT1 is responsible for chromosome stability and that SirT1+/− p53+/− mice are more cancer prone than SirT1+/+ p53+/− animals. We have not looked at chromosome stability but did found no difference between SirT1+/+ and SirT1+/− mice in susceptibility to skin carcinogenesis. Were there a significant effect of SirT1 on chromosome stability, we would predict that SirT1-null mice would be cancer prone. That our SirT1-null mice are not cancer prone is at odds with the conclusions from Wang et al. and an explanation for this discrepancy is not apparent at this time.
In conclusion, the present work suggests that SirT1 encoded by the endogenous gene does not modulate susceptibility to tumor formation. However, SirT1 is required for effective chemoprotection by resveratrol. This latter finding, along with the observation that overexpression of SirT1 inhibits polyp formation in the Apcmin/+ mouse (Firestein et al., 2008), suggests that SirT1 might be a conditional tumor-suppressor whose antitumor activity is dependent on a threshold of enzymatic activity reached, either by gene overexpression or by drug exposure.
Materials and methods
All animal procedures were carried out in accordance with the guideline of the Canadian Council for Animal Care. The mutant mice used in this study carried the sirt1-null allele previously described by McBurney et al. (2003) maintained on a mixed genetic background derived from intercrosses between the CD1 out bred strain and 129/J. SirT1-null animals were created by crossing heterozygotes and were identified at weaning by a characteristic eyelid defect. Genotypes of animals were determined by a PCR-based test carried out on DNA isolated from tail tip biopsy. Primers IndexTermTTCACATTGCATGTGTGTGG and IndexTermTAGCCTGCGTAGTGTTGGTG amplify a 423-bp fragment from the normal sirt1 allele whereas a 526-bp fragment from the null allele is amplified from the first primer and IndexTermATTTGGTAGGGACCCAAAGG, a sequence derived from the pgk-1 gene inserted to create the null allele by homologous recombination. sirt1-null mice were normally housed in cages with littermates of the same sex.
SirT1+/− mice from the inbred 129/J genetic background (see above) were interbred with C57BL/6J-Apcmin/J mice (Jax Mice; The Jackson Laboratory, Bar Harbor, ME, USA) (Moser et al., 1990). SirT1+/− of the first generation and bearing the Apcmin allele were interbred with SirT1+/− mice of the inbred 129/J genetic background but no SirT1-null mice were obtained. Mice of this second generation were then interbred with SirT1+/− mice of the 129/J-CD1 outbred background. SirT1-null mice were born from these matings and mice of all SirT1 genotypes bearing the Apcmin allele were used for the analyses. Mice of this third generation were also intercrossed to give rise to a fourth generation, represented by about a dozen individuals that were also used for the analyses because they were phenotypically similar to the third generation. Apc genotypes of mice were determined by a PCR-based test (http://jaxmice.jax.org/pub-cgi/protocols/protocols.sh?objtype=protocol&protocol_id=529) carried out on DNA isolated from tail tip biopsy. Primers IndexTermTTCCACTTTGGCATAAGGC and IndexTermGCCATCCCTTCACGTTAG amplify a fragment of ∼600 bp of the wild-type Apc allele whereas a fragment of ∼340 bp of the Apcmin allele is amplified by the first primer and IndexTermTTCTGAGAAAGACAGAAGTTA. Sirt1/Apcmin mice were normally housed in cages with littermates of the same sex. Mice started to die from polyps at about 9 months so all mice were killed between 9 and 12 months (the large majority at 12 months). Small intestine and colon were opened by a longitudinal cut and feces were flushed with phosphate-buffered solution. Intestines were fixed flat on Whatman paper in 10% buffered formalin. Fixed intestine were stained in 0.1% methylene blue and examined under a dissection microscope to count polyps and to measure their size with a caliper.
Lower back skin of normal (SirT1+/+ or SirT1+/−) or SirT1-null (−/−) mice was shaved and on the day after, 100 μg of DMBA (catalog no. D3254; Sigma-Aldrich, Oakville, ON, Canada) in 100 μl of acetone was applied to a 1 × 1 cm2 area. After 1 week, 10 μg of TPA (no. P8139; Sigma-Aldrich) was applied on the same region along with 0, 1 or 25 μmol of resveratrol (no. R5010; Sigma-Aldrich), in a total volume of 100 μl of acetone. Because some of the mice showed slight ulceration on the following week, TPA dose was reduced to 4 μg and was applied once a week for the next 14–21 weeks (total number of TPA application 15 and 22, respectively). Before the weekly applications of TPA, tumors were counted and two dimensions of the tumors were measured using a caliper. Three independent experiments were performed: (1) DMBA and TPA but no resveratrol were applied (TPA for 21 weeks); (2) DMBA, TPA and 1 μmol of resveratrol were applied on all animals (TPA and resveratrol for 22 weeks) and (3) DMBA, TPA and 0 or 25 μmol of resveratrol were applied (TPA and/or resveratrol for 15 weeks). In the third experiment, we decided to apply TPA and/or resveratrol only for 15 weeks as in the previous experiments, some mice had to be killed starting around the 14th week because of severe skin ulceration. Consequently, results from the first and second experiments were plotted together (0 and 1 μmol of resveratrol) and those from the third were plotted separately (0 and 25 μmol of resveratrol).
Normal skin, skin tumors and intestines were fixed in phosphate-buffered 10% formalin and tissue samples were embedded in paraffin. After deparaffinization and rehydration, tissue sections were treated with 1.5% hydrogen peroxide for 15 min to quench endogenous peroxidases. Before immunodetection, antigen retrieval was performed by boiling sections for 15 min in Tris-EDTA buffer (Tris 10 mM, EDTA 1 mM, Tween-20 0.05% (pH 9.0)) when probing for SirT1 or for 10 min in Tris-EDTA buffer when probing for Cox2. Sections were blocked with 1.5% normal goat serum (no. sc-2043; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 5 min and probed with Sir2α antibody (no. 07-131; Upstate/Millipore, Billerica, MA, USA) or Cox2 (no. 160106; Cayman Chemical, Ann Harbor, MI, USA) antibody in blocking solution for 1 h. Horseradish peroxidase-coupled goat anti-rabbit secondary antibody (Envision no. K4002; Dako, Mississauga, ON, Canada) was incubated for 30 min and signal was detected using Dako Chromogen System (no. K3465; Dako). Tissues were counterstained with hematoxylin and coverslipped.
Number and size distribution of blood vessels
Tumor samples of SirT1-null and wild-type mice were immunostained with an antibody to the blood vessel marker CD31. Pictures ( × 400) of the regions underneath the tumors were taken and used to count the number of blood vessels and determine the size distribution. Areas of analysed regions and blood vessels were calculated using the ImageJ program (http://rsb.info.nih.gov/ij/docs/index.html).
Transfections and foci formation assay
On the day before transfections, 0.2 million NIH3T3 cells were plated in six-well dishes in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), at 37 °C under 5% CO2 atmosphere. Transfections were performed in triplicate using GeneJuice (Novagen, Gibbstown, NJ, USA) as recommended by the manufacturer (5 μl of GeneJuice and 1 μg total amount of DNA of the indicated plasmids). Plasmid pML8 was used as the control and carrier plasmid and encodes GFP driven from the murine pgk-1 promoter, pKJMT expresses polyoma midTAg, pKJ321 and pMA4 express WT SirT1 (under CMV and pgk-1 promoters, respectively), and pKJ322 and pMA5 express a catalytically inactive SirT1 version (under CMV and pgk-1 promoters, respectively). On the day following the transfections, cells were trypsinized and 0.15 million cells were re-plated in a 10 cm dish in DMEM containing 10% FBS. After 2 days, medium was replaced with DMEM containing 3% FBS and 0, 30 or 100 μM resveratrol (no. R5010; Sigma-Aldrich). Media were changed every 2–3 days and, after 2 weeks, foci were counted. The same procedure was carried out in four independent experiments.
Conflict of interest
The authors declare no conflict of interest.
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We thank Christine Pratt for allowing us to use some of her chemicals. The projects were funded by the Canadian Institutes of Health Research. GB is a recipient of fellowship from the Fonds de Recherche en Santé du Québec (FRSQ).
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Boily, G., He, X., Pearce, B. et al. SirT1-null mice develop tumors at normal rates but are poorly protected by resveratrol. Oncogene 28, 2882–2893 (2009) doi:10.1038/onc.2009.147
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