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The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth.

Firestein R, Blander G, Michan S, Oberdoerffer P, Ogino S, Campbell J, Bhimavarapu A, Luikenhuis S, de Cabo R, Fuchs C, Hahn WC, Guarente LP, Sinclair DA - PLoS ONE (2008)

Bottom Line: Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan.In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival.Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of beta-catenin in 81 human colon tumor specimens analyzed.

View Article: PubMed Central - PubMed

Affiliation: Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan. In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival. The Sir2/SIRT1 family of NAD(+)-dependent deacetylases is proposed to underlie the health benefits of calorie restriction (CR), a diet that broadly suppresses cancer in mammals. Here we show that CR induces a two-fold increase SIRT1 expression in the intestine of rodents and that ectopic induction of SIRT1 in a beta-catenin-driven mouse model of colon cancer significantly reduces tumor formation, proliferation, and animal morbidity in the absence of CR. We show that SIRT1 deacetylates beta-catenin and suppresses its ability to activate transcription and drive cell proliferation. Moreover, SIRT1 promotes cytoplasmic localization of the otherwise nuclear-localized oncogenic form of beta-catenin. Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of beta-catenin in 81 human colon tumor specimens analyzed. Taken together, these observations show that SIRT1 suppresses intestinal tumor formation in vivo and raise the prospect that therapies targeting SIRT1 may be of clinical use in beta-catenin-driven malignancies.

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SIRT1 represses β-catenin transcriptional activity by directly interacting with and deacetylating β-catenin.(A) Human 293T cells were transiently transfected with HA-S33Y-β-catenin in combination with either FLAG-tagged SIRT1 or vector control. Aliquots of total protein were subjected to immunoprecipitation with anti-FLAG antibody (IP FLAG). Immunoprecipitated proteins were immunoblotted with anti-HA (upper panel) and anti-FLAG (lower panel). Left lanes, unprocessed extracts (input). (B) Human 293T cells were transfected as in panel A. Proteins immunoprecipitated with anti-HA antibody and immunoblotted with anti-FLAG (upper panel), and anti-HA (lower panel). Left lanes, unprocessed extracts (input). (C) Immunoprecipitation of SIRT1 from LN-CAP cell extracts using anti-SIRT1 antibody or normal rabbit IgG as a control (IgG). 10% of the immunoprecipitated protein was then blotted with anti-SIRT1 (upper panel) while the remaining 90% was blotted with anti-β-catenin antibodies. (D) 293T cells were transfected as indicated and lysed 48 hr later. Comparable levels of β-catenin were immunoprecipitated and blotted for acetylated-lysine residues (IP: HA IB: Ac-K; upper panel). The blot was reprobed for HA to demonstrate approximately equal levels of the HA-β-catenin (IP: HA IB: HA; lower panel). (E–F) 293T cells were transfected as indicated together with the TOP-FLASH luciferase and PRL-TK Renilla luciferase construct. Nicotinamide (NAM) or retroviral SIRT1 shRNA (RNAi) was added as indicated. Data are normalized with respect to Renilla luciferase activity. The data are means±s.d. from samples performed in triplicate. (G) Indirect immunofluorescence staining of DLD-1 colon cancer cells infected with empty retrovirus or virus containing SIRT1 shRNA (RNAi) or SIRT1 cDNA (overexpression, O/E). Percent of cells with high, medium, or low levels of nuclear β-catenin staining for untreated: 6.5, 80.6, 12.9; SIRT1 O/E: 0, 29.4, 70.5; SIRT1 RNAi: 60.0, 32.0, 0.8. Images were taken at 100× magnification.
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pone-0002020-g004: SIRT1 represses β-catenin transcriptional activity by directly interacting with and deacetylating β-catenin.(A) Human 293T cells were transiently transfected with HA-S33Y-β-catenin in combination with either FLAG-tagged SIRT1 or vector control. Aliquots of total protein were subjected to immunoprecipitation with anti-FLAG antibody (IP FLAG). Immunoprecipitated proteins were immunoblotted with anti-HA (upper panel) and anti-FLAG (lower panel). Left lanes, unprocessed extracts (input). (B) Human 293T cells were transfected as in panel A. Proteins immunoprecipitated with anti-HA antibody and immunoblotted with anti-FLAG (upper panel), and anti-HA (lower panel). Left lanes, unprocessed extracts (input). (C) Immunoprecipitation of SIRT1 from LN-CAP cell extracts using anti-SIRT1 antibody or normal rabbit IgG as a control (IgG). 10% of the immunoprecipitated protein was then blotted with anti-SIRT1 (upper panel) while the remaining 90% was blotted with anti-β-catenin antibodies. (D) 293T cells were transfected as indicated and lysed 48 hr later. Comparable levels of β-catenin were immunoprecipitated and blotted for acetylated-lysine residues (IP: HA IB: Ac-K; upper panel). The blot was reprobed for HA to demonstrate approximately equal levels of the HA-β-catenin (IP: HA IB: HA; lower panel). (E–F) 293T cells were transfected as indicated together with the TOP-FLASH luciferase and PRL-TK Renilla luciferase construct. Nicotinamide (NAM) or retroviral SIRT1 shRNA (RNAi) was added as indicated. Data are normalized with respect to Renilla luciferase activity. The data are means±s.d. from samples performed in triplicate. (G) Indirect immunofluorescence staining of DLD-1 colon cancer cells infected with empty retrovirus or virus containing SIRT1 shRNA (RNAi) or SIRT1 cDNA (overexpression, O/E). Percent of cells with high, medium, or low levels of nuclear β-catenin staining for untreated: 6.5, 80.6, 12.9; SIRT1 O/E: 0, 29.4, 70.5; SIRT1 RNAi: 60.0, 32.0, 0.8. Images were taken at 100× magnification.

Mentions: Acetylation of β-catenin by p300/CBP potentiates its oncogenicity by increasing β-catenin/TCF avidity at target gene promoters [26]. To test whether SIRT1 modifies β-catenin, HEK293T cells were transfected with a mutant form of β-catenin that constitutively localizes to the nucleus (S33Y-β-catenin) [27]. In these cells SIRT1 co-immunoprecipitated with β-catenin (Fig. 4A) and vice versa (Fig. 4B). An interaction between endogenous SIRT1 and β-catenin was also apparent (Fig. 4C).


The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth.

Firestein R, Blander G, Michan S, Oberdoerffer P, Ogino S, Campbell J, Bhimavarapu A, Luikenhuis S, de Cabo R, Fuchs C, Hahn WC, Guarente LP, Sinclair DA - PLoS ONE (2008)

SIRT1 represses β-catenin transcriptional activity by directly interacting with and deacetylating β-catenin.(A) Human 293T cells were transiently transfected with HA-S33Y-β-catenin in combination with either FLAG-tagged SIRT1 or vector control. Aliquots of total protein were subjected to immunoprecipitation with anti-FLAG antibody (IP FLAG). Immunoprecipitated proteins were immunoblotted with anti-HA (upper panel) and anti-FLAG (lower panel). Left lanes, unprocessed extracts (input). (B) Human 293T cells were transfected as in panel A. Proteins immunoprecipitated with anti-HA antibody and immunoblotted with anti-FLAG (upper panel), and anti-HA (lower panel). Left lanes, unprocessed extracts (input). (C) Immunoprecipitation of SIRT1 from LN-CAP cell extracts using anti-SIRT1 antibody or normal rabbit IgG as a control (IgG). 10% of the immunoprecipitated protein was then blotted with anti-SIRT1 (upper panel) while the remaining 90% was blotted with anti-β-catenin antibodies. (D) 293T cells were transfected as indicated and lysed 48 hr later. Comparable levels of β-catenin were immunoprecipitated and blotted for acetylated-lysine residues (IP: HA IB: Ac-K; upper panel). The blot was reprobed for HA to demonstrate approximately equal levels of the HA-β-catenin (IP: HA IB: HA; lower panel). (E–F) 293T cells were transfected as indicated together with the TOP-FLASH luciferase and PRL-TK Renilla luciferase construct. Nicotinamide (NAM) or retroviral SIRT1 shRNA (RNAi) was added as indicated. Data are normalized with respect to Renilla luciferase activity. The data are means±s.d. from samples performed in triplicate. (G) Indirect immunofluorescence staining of DLD-1 colon cancer cells infected with empty retrovirus or virus containing SIRT1 shRNA (RNAi) or SIRT1 cDNA (overexpression, O/E). Percent of cells with high, medium, or low levels of nuclear β-catenin staining for untreated: 6.5, 80.6, 12.9; SIRT1 O/E: 0, 29.4, 70.5; SIRT1 RNAi: 60.0, 32.0, 0.8. Images were taken at 100× magnification.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2289879&req=5

pone-0002020-g004: SIRT1 represses β-catenin transcriptional activity by directly interacting with and deacetylating β-catenin.(A) Human 293T cells were transiently transfected with HA-S33Y-β-catenin in combination with either FLAG-tagged SIRT1 or vector control. Aliquots of total protein were subjected to immunoprecipitation with anti-FLAG antibody (IP FLAG). Immunoprecipitated proteins were immunoblotted with anti-HA (upper panel) and anti-FLAG (lower panel). Left lanes, unprocessed extracts (input). (B) Human 293T cells were transfected as in panel A. Proteins immunoprecipitated with anti-HA antibody and immunoblotted with anti-FLAG (upper panel), and anti-HA (lower panel). Left lanes, unprocessed extracts (input). (C) Immunoprecipitation of SIRT1 from LN-CAP cell extracts using anti-SIRT1 antibody or normal rabbit IgG as a control (IgG). 10% of the immunoprecipitated protein was then blotted with anti-SIRT1 (upper panel) while the remaining 90% was blotted with anti-β-catenin antibodies. (D) 293T cells were transfected as indicated and lysed 48 hr later. Comparable levels of β-catenin were immunoprecipitated and blotted for acetylated-lysine residues (IP: HA IB: Ac-K; upper panel). The blot was reprobed for HA to demonstrate approximately equal levels of the HA-β-catenin (IP: HA IB: HA; lower panel). (E–F) 293T cells were transfected as indicated together with the TOP-FLASH luciferase and PRL-TK Renilla luciferase construct. Nicotinamide (NAM) or retroviral SIRT1 shRNA (RNAi) was added as indicated. Data are normalized with respect to Renilla luciferase activity. The data are means±s.d. from samples performed in triplicate. (G) Indirect immunofluorescence staining of DLD-1 colon cancer cells infected with empty retrovirus or virus containing SIRT1 shRNA (RNAi) or SIRT1 cDNA (overexpression, O/E). Percent of cells with high, medium, or low levels of nuclear β-catenin staining for untreated: 6.5, 80.6, 12.9; SIRT1 O/E: 0, 29.4, 70.5; SIRT1 RNAi: 60.0, 32.0, 0.8. Images were taken at 100× magnification.
Mentions: Acetylation of β-catenin by p300/CBP potentiates its oncogenicity by increasing β-catenin/TCF avidity at target gene promoters [26]. To test whether SIRT1 modifies β-catenin, HEK293T cells were transfected with a mutant form of β-catenin that constitutively localizes to the nucleus (S33Y-β-catenin) [27]. In these cells SIRT1 co-immunoprecipitated with β-catenin (Fig. 4A) and vice versa (Fig. 4B). An interaction between endogenous SIRT1 and β-catenin was also apparent (Fig. 4C).

Bottom Line: Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan.In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival.Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of beta-catenin in 81 human colon tumor specimens analyzed.

View Article: PubMed Central - PubMed

Affiliation: Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan. In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival. The Sir2/SIRT1 family of NAD(+)-dependent deacetylases is proposed to underlie the health benefits of calorie restriction (CR), a diet that broadly suppresses cancer in mammals. Here we show that CR induces a two-fold increase SIRT1 expression in the intestine of rodents and that ectopic induction of SIRT1 in a beta-catenin-driven mouse model of colon cancer significantly reduces tumor formation, proliferation, and animal morbidity in the absence of CR. We show that SIRT1 deacetylates beta-catenin and suppresses its ability to activate transcription and drive cell proliferation. Moreover, SIRT1 promotes cytoplasmic localization of the otherwise nuclear-localized oncogenic form of beta-catenin. Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of beta-catenin in 81 human colon tumor specimens analyzed. Taken together, these observations show that SIRT1 suppresses intestinal tumor formation in vivo and raise the prospect that therapies targeting SIRT1 may be of clinical use in beta-catenin-driven malignancies.

Show MeSH
Related in: MedlinePlus