SIRT1 regulates Dishevelled proteins and promotes transient and constitutive Wnt signaling Kimberly R. Hollowaya,b,1, Tara N. Calhouna,b,1, Madhurima Saxenaa,b, Cheynita F. Metoyerb, Ethan F. Kandlera,b, Chantal A. Riveraa,b, and Kevin Pruitta,b,c,2 aDepartment of Molecular and Cellular Physiology, bSchool of Medicine, and cFeist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA 71130 Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved April 16, 2010 (received for review October 2, 2009) Sirtuin 1 (SIRT1) is a class III histone deacetylase that deacetylates tively regulated by NEDL1 (18) and the Naked cuticle-PR72 histone and nonhistone proteins to regulate gene transcription and complex (19), and Dsh-2 and Dsh-3 were nicely shown to be neg- protein function. Because SIRT1 regulates very diverse responses atively regulated by the KLHL12-Cullin-3 complex (20). Recently, such as apoptosis, insulin sensitivity, autophagy, differentiation, and reports have revealed an intriguing requirement of nuclear entry of stem cell pluripotency, it has been a challenge to reconcile how it Dvl for canonical signaling (21, 22). However, why the nuclear entry orchestrates such pleiotropic effects. Here we show that SIRT1 serves of Dvl is required for canonical signaling is not known. Here we as an important regulator of Wnt signaling. We demonstrate that show that SIRT1 positively regulates the protein levels of all three SIRT1 loss of function leads to a significant decrease in the levels of Dvl family members. We demonstrate that SIRT1 and Dvl proteins all three Dishevelled (Dvl) proteins. Furthermore, we demonstrate complex in vivo and that inhibition of SIRT1 leads to changes in that SIRT1 and Dvl proteins complex in vivo and that inhibition of gene expression of classic Wnt/Dvl target genes. Moreover, we SIRT1 leads to changes in gene expression of Wnt target genes. show that SIRT1 regulates Wnt3a-induced cell migration. Finally, Finally, we demonstrate that Wnt-stimulated cell migration is in- we propose that SIRT1-mediated regulation of Dvl proteins may hibited by a SIRT1 inhibitor. Because the three mammalian Dvl help explain the diverse SIRT1-dependent physiological responses proteins serve as key messengers for as many as 19 Wnt ligands, observed in different cellular contexts. SIRT1-mediated regulation of Dvl proteins may explain the diverse physiological responses observed in different cellular contexts. Results CELL BIOLOGY Previously, SIRT1 had only been shown to mediate the epigenetic The first clue that SIRT1 regulates Wnt signaling came from the silencing of Wnt antagonists. In contrast, here we report that SIRT1 observation that SIRT1 localizes to the promoter of the Wnt an- regulates Dvl protein levels and Wnt signaling in several cellular tagonist secreted frizzled related protein 1 (SFRP1) and directly contexts. These findings demonstrate that SIRT1 is a regulator of contributes to its aberrant epigenetic silencing (23). Inhibition of transient and constitutive Wnt signaling. SIRT1 was shown to cause a reexpression of SFRP1. SIRT1 was originally shown to mediate epigenetic silencing of SFRP1 and breast cancer | colon cancer | cambinol | β-catenin | sirtuin 2 SFRP2 (23), and later was extended to DKK1 (24), another Wnt antagonist. However, whether this connection of SIRT1 with Wnt irtuin 1 (SIRT1) is an NAD+-dependent histone deacetylase signaling extended beyond the repression of Wnt antagonists was S(HDAC) that regulates a very broad and complex array of unknown. To address this, we systematically analyzed the impact of physiological processes. As such, it has been the source of some loss of SIRT1 function on several key mediators of Wnt signaling. controversy, as it has been difficult to reconcile the role it plays in First, we determined the expression pattern of Wnt proteins in the coordination of cellular responses and gene expression in colorectal cancer, breast cancer, and HEK293 cell lines to assess both normal and pathophysiological settings. For example, SIRT1 the extent of autocrine Wnt signaling. We performed reverse has been shown to inhibit the maturation of preadipocytes (1, 2), transcription (RT) PCR analysis and observed a variable pattern of antagonize p53-dependent apoptosis in response to stress (3, 4), expression in Wnt ligands (Fig. S2A) indicating potentially different and promote chemoresistance to conventional chemotherapeutic states of constitutive autocrine signaling. We found that the four agents (5, 6), and is associated with microsatellite instability and colon cancer (HT-29, HCT116, RKO, and DLD-1), two breast CpG island methylator phenotype in human colorectal cancer (7). cancer (T-47D and MDA-MB-231), and HEK293 cell lines each expressed multiple but different Wnt ligands. Furthermore, reports demonstrate that SIRT1 coordinates di- Next, we determined the pattern of expression of all three Dvl verse metabolic responses to changes in nutrient availability (8), proteins. We found that, in contrast to the pattern of mRNA ex- regulates autophagy (9), and controls key stages of spermato- pression of Wnts, Dvl mRNA levels did not vary considerably genesis and germ stem cell proliferation and function (10, 11). among the six lines (Fig. 1A). Interestingly, however, we observed Given the complex influence of SIRT1 on cell-fate decisions in a more varied pattern of Dvl protein expression (Fig. 1B). Whereas multiple physiological settings, it is reasonable to anticipate that Dvl-1 protein varied more among the cell lines, Dvl-2 and Dvl-3 SIRT1 regulates one or more signaling networks recognized for fl expression was consistently higher. Because of the pattern of Wnt their in uence on these diverse cellular and organismal responses. ligand expression, and because Dvl proteins translocate to the nu- It is well-established that Wnt signaling, likewise, regulates di- verse processes such as adipogenesis (12), tumorigenesis (13), and stem cell pluripotency (14, 15). When Wnt ligands are present, they fi Author contributions: K.R.H., T.N.C., M.S., C.F.M., and K.P. designed research; K.R.H., transmit signals through speci c Frizzled (Fzd) or Fzd/LRP5/6 T.N.C., M.S., C.F.M., E.F.K., and K.P. performed research; C.A.R. and K.P. contributed coreceptor complexes (16). This signal is then propagated via new reagents/analytic tools; K.R.H., T.N.C., M.S., C.F.M., E.F.K., C.A.R., and K.P. ana- Dishevelled (Dvl) proteins that will direct canonical (β-catenin- lyzed data; and K.R.H., T.N.C., M.S., and K.P. wrote the paper. dependent) or noncanonical (β-catenin-independent) signaling The authors declare no conflict of interest. (17). Most of the mechanistic insights into Wnt signaling have come This article is a PNAS Direct Submission. from extensive studies detailing how β-catenin is regulated, yet 1K.R.H. and T.N.C. contributed equally to this work. precisely how Dvl is regulated is poorly understood despite its 2To whom correspondence should be addressed. E-mail: [email protected]. crucial function in bridging cytoplasmic Wnt signals with nuclear This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. responses. Early observations demonstrated that Dsh-1 is nega- 1073/pnas.0911325107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0911325107 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 cleus and serve as key effectors of extracellular signals (21, 22, 25), that SIRT1 inhibition leads to robust decreases in Dvl proteins in we focused on their regulation with respect to SIRT1 loss of func- several cellular contexts. tion. Of the Dvl family members, we were particularly interested in Because cambinol inhibits SIRT1 and SIRT2 (28), we specifi- Dvl-3 because it has been reported to be overexpressed in 36% (33/ cally inhibited SIRT1 via shRNA to further verify the impact of 91) of colon tumors and displayed a marked increase in its nuclear SIRT1 inhibition on Dvl regulation. Consistent with the phar- accumulation (25). Additionally, Dvl-3 was shown to be overex- macological inhibition, we found that the shRNA that caused pressed in 30% of invasive ductal breast carcinomas (26) and Dvl-2 a knockdown of SIRT1 in T-47D cells led to a reduction in Dvl-2 was shown to contribute to pancreatic cancer malignancy (27). We and Dvl-3 protein (Fig. 2A). This trend was also observed in continued analysis of SIRT1 regulation in cell lines that display HEK293 cells, where the shRNA constructs that caused a re- constitutive Wnt signaling yet show different patterns of Wnt ligand duction in SIRT1 also caused a reduction in Dvl-3 proteins (Fig. autocrine stimulation. 2B). Finally, this trend was also observed in DLD-1 cells, as in- As the first loss-of-function approach, we treated multiple hibition of SIRT1 by siRNA led to a significant reduction in Dvl-3 cell lines with a range of doses of cambinol, an inhibitor of and a small reduction in Dvl-2 (Fig. 2C). Together, these data SIRT1 and SIRT2 (28), and continued analysis with two doses of suggest that SIRT1 is a positive regulator of Dvl proteins. Because cambinol. Strikingly, we observed a robust dose-dependent re- cambinol inhibits both SIRT1 and SIRT2, we also performed duction in the levels of Dvl-2 and Dvl-3 in T-47D cells upon siRNA-mediated knockdown of SIRT1 and SIRT2 individually treatment with cambinol for 18 h (Fig. 1C). The levels of Dvl-1 and together. We found that the individual knockdown of SIRT1 were too low to assess changes in T-47D cells (Fig. 1B). We and SIRT2 caused a decreased in Dvl-2 and Dvl-3 and the com- tested the role of SIRT1 in the regulation of Dvl proteins in bined knockdown caused a reduction in Dvl-2 and Dvl-3 (Fig.