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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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Generation of the conditional SIRT1 transgenic mice that mimic calorie restriction induced SIRT1 overexpression.(A) Western blot analysis showing expression levels in the gut epithelium of SIRT1 in ad libitum-fed (AL) or calorie restricted (CR) rats. β-actin served as the loading control in all lanes. (B) Schematic representation of the strategy used for the generation of the floxed SIRT1 mouse embryonic stem (MES) cells. SIRT1 was cloned downstream of a constitutive CAGGS promoter followed by a transcriptional loxP-STOP-loxP cassette. This construct was specifically targeted in the 3′ UTR of the collagen A1 locus (ColA1) of mouse embryonic stem cells (MES) cells by FLP recombination. The targeted MES cells were injected into blastocysts. Red arrows indicate location of the SIRT1-Tg genotyping primers. (C) Southern blot showing the confirmation of the SIRT1STOP single integration into the Col1A locus of MES cells. (D) PCR confirming the germline transmission of the SIRT1STOP transgene to the chimaeras' offspring. (E) Western blot showing the levels of SIRT1 in the triple transgenic mice overexpressing SIRT1 (SIRT1ΔSTOP) and controls (SIRT1STOP). β-actin served as the loading control in all lanes. (F) Mucin stain and immunohistochemistry of SIRT1 in the small intestine of experimental (SIRT1ΔSTOP) and controls (SIRT1STOP) animals.
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pone-0002020-g001: Generation of the conditional SIRT1 transgenic mice that mimic calorie restriction induced SIRT1 overexpression.(A) Western blot analysis showing expression levels in the gut epithelium of SIRT1 in ad libitum-fed (AL) or calorie restricted (CR) rats. β-actin served as the loading control in all lanes. (B) Schematic representation of the strategy used for the generation of the floxed SIRT1 mouse embryonic stem (MES) cells. SIRT1 was cloned downstream of a constitutive CAGGS promoter followed by a transcriptional loxP-STOP-loxP cassette. This construct was specifically targeted in the 3′ UTR of the collagen A1 locus (ColA1) of mouse embryonic stem cells (MES) cells by FLP recombination. The targeted MES cells were injected into blastocysts. Red arrows indicate location of the SIRT1-Tg genotyping primers. (C) Southern blot showing the confirmation of the SIRT1STOP single integration into the Col1A locus of MES cells. (D) PCR confirming the germline transmission of the SIRT1STOP transgene to the chimaeras' offspring. (E) Western blot showing the levels of SIRT1 in the triple transgenic mice overexpressing SIRT1 (SIRT1ΔSTOP) and controls (SIRT1STOP). β-actin served as the loading control in all lanes. (F) Mucin stain and immunohistochemistry of SIRT1 in the small intestine of experimental (SIRT1ΔSTOP) and controls (SIRT1STOP) animals.

Mentions: Earlier studies have shown a dramatic tumor suppressive effect of CR but the molecular mechanism(s) are currently unknown. We observed that rats on a CR diet have ∼2-fold higher levels of SIRT1 in the gut epithelium relative to ad lib-fed controls (Fig. 1A). To test the effect of increasing SIRT1 expression in intestinal epithelial cells, we generated a floxed SIRT1 transgenic mouse (Fig. 1B). SIRT1 transgenic mice were crossed to APCmin/+ mice followed by breeding to the Villin-Cre strain [25]. Thus, we generated triple transgenic mice (SIRT1ΔSTOP; Vil-Cre; APCmin/+) which overexpress SIRT1 specifically in the gut villi (referred to as SIRT1ΔSTOP). The SIRT1 levels in the gut of SIRT1ΔSTOP mice were approximately 7-fold (Fig. 1E) and the morphology of villi appeared otherwise normal (Fig. 1F).


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)

Generation of the conditional SIRT1 transgenic mice that mimic calorie restriction induced SIRT1 overexpression.(A) Western blot analysis showing expression levels in the gut epithelium of SIRT1 in ad libitum-fed (AL) or calorie restricted (CR) rats. β-actin served as the loading control in all lanes. (B) Schematic representation of the strategy used for the generation of the floxed SIRT1 mouse embryonic stem (MES) cells. SIRT1 was cloned downstream of a constitutive CAGGS promoter followed by a transcriptional loxP-STOP-loxP cassette. This construct was specifically targeted in the 3′ UTR of the collagen A1 locus (ColA1) of mouse embryonic stem cells (MES) cells by FLP recombination. The targeted MES cells were injected into blastocysts. Red arrows indicate location of the SIRT1-Tg genotyping primers. (C) Southern blot showing the confirmation of the SIRT1STOP single integration into the Col1A locus of MES cells. (D) PCR confirming the germline transmission of the SIRT1STOP transgene to the chimaeras' offspring. (E) Western blot showing the levels of SIRT1 in the triple transgenic mice overexpressing SIRT1 (SIRT1ΔSTOP) and controls (SIRT1STOP). β-actin served as the loading control in all lanes. (F) Mucin stain and immunohistochemistry of SIRT1 in the small intestine of experimental (SIRT1ΔSTOP) and controls (SIRT1STOP) animals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2289879&req=5

pone-0002020-g001: Generation of the conditional SIRT1 transgenic mice that mimic calorie restriction induced SIRT1 overexpression.(A) Western blot analysis showing expression levels in the gut epithelium of SIRT1 in ad libitum-fed (AL) or calorie restricted (CR) rats. β-actin served as the loading control in all lanes. (B) Schematic representation of the strategy used for the generation of the floxed SIRT1 mouse embryonic stem (MES) cells. SIRT1 was cloned downstream of a constitutive CAGGS promoter followed by a transcriptional loxP-STOP-loxP cassette. This construct was specifically targeted in the 3′ UTR of the collagen A1 locus (ColA1) of mouse embryonic stem cells (MES) cells by FLP recombination. The targeted MES cells were injected into blastocysts. Red arrows indicate location of the SIRT1-Tg genotyping primers. (C) Southern blot showing the confirmation of the SIRT1STOP single integration into the Col1A locus of MES cells. (D) PCR confirming the germline transmission of the SIRT1STOP transgene to the chimaeras' offspring. (E) Western blot showing the levels of SIRT1 in the triple transgenic mice overexpressing SIRT1 (SIRT1ΔSTOP) and controls (SIRT1STOP). β-actin served as the loading control in all lanes. (F) Mucin stain and immunohistochemistry of SIRT1 in the small intestine of experimental (SIRT1ΔSTOP) and controls (SIRT1STOP) animals.
Mentions: Earlier studies have shown a dramatic tumor suppressive effect of CR but the molecular mechanism(s) are currently unknown. We observed that rats on a CR diet have ∼2-fold higher levels of SIRT1 in the gut epithelium relative to ad lib-fed controls (Fig. 1A). To test the effect of increasing SIRT1 expression in intestinal epithelial cells, we generated a floxed SIRT1 transgenic mouse (Fig. 1B). SIRT1 transgenic mice were crossed to APCmin/+ mice followed by breeding to the Villin-Cre strain [25]. Thus, we generated triple transgenic mice (SIRT1ΔSTOP; Vil-Cre; APCmin/+) which overexpress SIRT1 specifically in the gut villi (referred to as SIRT1ΔSTOP). The SIRT1 levels in the gut of SIRT1ΔSTOP mice were approximately 7-fold (Fig. 1E) and the morphology of villi appeared otherwise normal (Fig. 1F).

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