We have studied the therapeutic effect of resveratrol on BRCA1 mutant breast cancer cells. There are a number of findings that can be concluded from this study. First, we demonstrated that BRCA1 is involved in modulating expression of SIRT1 and Survivin. The absence of BRCA1 results in low levels of SIRT1 and high levels of Survivin. Second, we found that resveratrol treatment decreases expression of Survivin through increasing SIRT1 activity. We further demonstrated that SIRT1 negatively regulates Survivin expression through its deacetylase activity, which epigenetically modifies the Survivin promoter and turns the promoter into a transcription silent configuration. Third, we demonstrated that resveratrol is a potent inhibitor for the initiation and progression of BRCA1 mutant cancer both in vitro and in vivo. These findings suggest that resveratrol may serve as an excellent reagent for targeted therapy for BRCA1 associated breast cancers.
It has been shown that BRCA1 acts as a transcription factor that positively or negatively regulates expression of many important genes, including p21, Gadd45, Mad2, and IGF axis members (Harkin et al., 1999
; Shukla et al., 2006
; Wang et al., 2004
). Here we found that BRCA1 is involved in maintaining SIRT1 expression as reflected by reduced expression of SIRT1 in BRCA1 mutant MEFs, cultured cancer cells, and primary tumors. We further revealed that BRCA1 positively regulates SIRT1 promoter activity. Currently, several factors have been reported to affect SIRT1 expression, including p53, HIC1 and Foxo3a (Chen et al., 2005b
; Nemoto et al., 2004
). The potential relationship between BRCA1 and these factors in relation to SIRT1 expression is unclear, and remains to be addressed in future studies.
In yeast, Sir2 is involved in aging and longevity (Guarante, 2005); in mice, SIRT1 is responsible for an increased physical activity caused by caloric restriction (Chen et al., 2005a
). Our previous study reveals that BRCA1 mutant mice undergo premature aging and suffer from high risk of spontaneous tumor formation (Cao et al., 2006
; Cao et al., 2003
; Cao et al., 2007
). Thus, reduced SIRT1 expression upon BRCA1 deficiency is consistent with the premature aging phenotype exhibited in the BRCA1 mutant mice. On the other hand, the role of SIRT1 in cancer formation is currently under active discussion. It was shown that activation of SIRT1 down-regulates stress-induced p53 and Foxo pathways and protects cells from apoptosis (anti-apoptosis) (Brunet et al., 2004
; Motta et al., 2004
). In contrast, it was shown that activation of SIRT1 by resveratrol treatment inhibits NF-kappaB activity and sensitized cells to TNFα-induced apoptosis (pro-apoptosis) (Yeung et al., 2004
). Based on the fact that SIRT1 deacetylates p53 to decrease its activity, it was hypothesized that increased SIRT1 activity may elevate the risk of cancer in mammals (Chen et al., 2005b
). However, recent studies revealed that SIRT1 might serve as a tumor suppressor for certain tumor types in mice (Firestein et al., 2008
, Wang et al. 2008
These observations, which are contradictory on the surface, suggest that SIRT1 may play different or opposite roles in different populations of cells. This perhaps is because SIRT1 can affect multiple pathways with different biological functions, whereas the availability of these pathways in different cells may cause different phenotypes upon activation of SIRT1. For example, the previous finding that activation of SIRT1 might reduce p53 activity and therefore, elevate the risk of cancer in mammals (Chen et al., 2005b
) should not be a concern in our study as virtually all BRCA1 mutant cancer cells are p53 deficient (Brodie et al., 2001
; Xu et al., 1999
). This may account for the reason that activation of SIRT1 does not cause proliferation, but rather induces apoptosis through inhibition of Survivin expression. More importantly, we showed that ectopically over-expression of SIRT1 in BRCA1 mutant cells inhibits tumor formation in nude mice and reduces cell growth in cultured cells. Thus, our data indicate that SIRT1 serves as a tumor suppressor in the context of BRCA1 deficiency, although it remains elusive whether SIRT1 has a general tumor suppressor role of SIRT1 in other types of tumors.
Survivin is a well-known anti-apoptotic protein (Zaffaroni et al., 2005
). We demonstrated that all BRCA1 mutant primary tumors and cultured tumor cell lines express Survivin at levels significantly higher than controls. Thus, we concluded that escaping apoptosis is one of the major events that BRCA1 tumor cells adapt to survive and proliferate. This is primarily because loss of BRCA1 triggers apoptosis due to extensive DNA damage and abnormal cell cycle progression (reviewed in (Deng, 2006
)). Apparently, an increase in Survivin raises the apoptotic threshold, providing a suitable environment for cells with genetic instability to proliferate. This may explain why inhibition of Survivin kills BRCA1 mutant cells more profoundly than cells driven by activated ErbB2/Neu signaling tested in this study. We further demonstrated that resveratrol treatment could significantly decrease levels of Survivin, reduce cell growth and increase apoptosis. Our data indicates that SIRT1 binds to the Survivin promoter and inhibits Survivin expression. Furthermore, it was recently reported that NF-kB also plays a positive role in Survivin expression (Kawakami et al., 2005
). Therefore, the decreased levels of Survivin upon resveratrol treatment could be due to a direct inhibitory role of SIRT1 on Survivin and/or an indirect role of SIRT1 through decreased NF-kB activity. In all three protocols tested, the resveratrol treatment clearly delays the onset of tumor initiation and retards their progression. We also noticed that the resveratrol treatment alone does not completely block tumor development. This may be due to the fact that BRCA1 associated cancers have undergone many additional molecular/genetic alterations (Brodie et al., 2001
). Furthermore, some other factors, besides SIRT1, may also regulate Survivin expression (Sato et al., 2006
; Xia et al., 2006
). These observations suggest that targeting Survivin specifically in addition to a combination of traditional chemotherapeutic drugs is needed to eliminate the cells with genetic mutations for tumorigenesis and minimize the frequency of tumor re-occurrence.
In conclusion, our data indicates that BRCA1 plays an important tumor suppressor function through maintenance of SIRT1 expression, which in turn inhibits expression of Survivin directly and/or indirectly (). The absence of BRCA1 causes reduced levels of SIRT1 and increased expression of Survivin, allowing BRCA1 deficient cells to overcome growth defects and apoptosis and finally undergo malignant transformation. Our analysis reveals that resveratrol strongly inhibits the growth of BRCA1 mutant tumors both in vitro and in vivo. This is accomplished through up-regulating SIRT1 activity followed by reduction of Survivin expression, leading to growth arrest and apoptosis of BRCA1 deficient cancer cells (). This provides a basis for the use of resveratrol on chemoprevention and therapeutic treatment of BRCA1 associated cancer patients.