Abstract #5092: Inhibition of Sirt1 by sirtinol causes p53-independent activation of FoxO1 in human prostate cancer cells.

Prostate Cancer (PCa), a major age-related malignancy, is rarely seen in men younger than 40 years and its incidence significantly rises with each decade thereafter. Because of marked improvement in life expectancy, Americans are living longer and more cases of PCa are expected to be diagnosed. According to a prediction, by the year 2010, the number of annual PCa cases will skyrocket to 330,000. Therefore, it is important to identify the causal mechanistic connection between aging and this age-related malignancy. It is believed that sirtuins (Sirt proteins), which are nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases, could serve as a connection between aging and certain cancers. We have earlier shown that Sirt1 is over-expressed in PCa and the inhibition of Sirt1 enzyme by nicotinamide, as well as knockdown of Sirt1 gene via short hairpin RNA (shRNA)-mediated RNA interference imparts anti-proliferative effects in human PCa cells. In this study, in order to define the downstream targets of Sirt1 inhibition in PCa cells, we assessed the effect of sirtinol (a specific Sirt1 inhibitor)- mediated inhibition of Sirt1 on FoxO function. Our data demonstrated that sirtinol (30µM and 120µM for 24 hours) treatment to human PCa cells viz. 22R\#957;1, DU145 and PC3 resulted in a significant i) decrease in Sirt1 protein in the nucleus and cytoplasm, ii) decrease in the growth and viability of cells, and iii) increase in the acetylation of FoxO1 in the nucleus as well as in the cytoplasm. Further, as shown by luciferase activity, we found that inhibition of Sirt1 resulted in an increase in FoxO1 transcription of all PCa cells tested. Thus our data suggested that Sirt1 may in part be promoting PCa growth via inhibiting FoxO1 acetylation and transcription. Since, an intricate interplay between FoxOs, Sirt1, and p53 has been shown to exist; employing isogenic cell lines, PC3 and PC3-p53, we determined whether or not the Sirt1-inhibition-mediated activation of FoxO1 was dependent on p53. We found that sirtinol treatment resulted in a decrease in growth and viability of the cells that was accompanied with an increase in FoxO1 acetylation and subsequent transcriptional activation, irrespective of the p53 status of the cells; thereby suggesting that Sirt1-mediated deacetylation of FoxO1 is independent of p53 in PCa cells. Interestingly, we also found that Sirt1 inhibition resulted in an induction of apoptosis in PC3 (p53 null) cells; whereas in PC3-p53 (wild-type p53) cells, Sirt1 inhibition caused an induction of senescence. This finding suggested that Sirt1 inhibition-mediated activation of FoxO1 imparts anti-proliferative effects via two distinct mechanisms based on p53 status of the cells viz. apoptosis in absence of p53 and senescence in presence of p53. However, detailed studies in appropriate in vitro and in vivo systems are needed to delineate the mechanism(s) by which Sirt1 imparts a growth advantage to PCa cells.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5092.