SIRT1 is a class III histone deacetylase and plays important roles in aging, obesity, and cancer (1, 2). Dramatic up-regulation of SIRT1 has been observed in various cancers including breast, prostate, and ovarian cancers, implicating a role for SIRT1 in tumorigenesis (3-5). SIRT1 functions by deacetylating histone (e.g. H3-Lys9 and H4-Lys16) and non-histone proteins (e.g. p300 and Ku70) in an NAD ϩ -dependent manner, thus modifying gene expression and modulating protein activity (1, 6). Previous studies have illustrated several mechanisms of SIRT1-dependent gene silencing in addition to histone deacetylation. It was shown that at sites of DNA damage, SIRT1 recruits DNA methyltransferases (DNMTs) 2 to promoter regions leading to hypermethylation and potential silencing of tumor suppressor genes (e.g. E-cadherin) (7). It is also known that SIRT1 facilitates transcriptional repression of tumor suppressor genes by modulating histone methyltransferase SUV39h1, the key enzyme responsible for histone H3 methylation (H3-Lys9-me3) in regions of heterochromatin (8). SIRT1 induction of tumor suppressor gene silencing promotes the initiation and progression of tumors as well as drug resistance (1, 9, 10). Studies from our laboratory and others show that inhibition of SIRT1 by pharmacological inhibitors or genetic depletion reduces estrogen-dependent signaling pathways in breast cancer cells (11,12). The inhibition of SIRT1 in breast and prostate cancer cell lines has resulted in acetylation of p53 and subsequent growth arrest and apoptosis, while not affecting viability of several non-cancer epithelial cell lines (13,14). Although several inhibitors of sirtuins have been described (reviewed in Ref. 15), and the potential value that SIRT1 inhibition may possess for cancer therapy has been recognized, there are no ongoing clinical trials of SIRT1 inhibitors for cancer therapy because of serious concerns, e.g. stability and toxicity. These deficiencies have lead to the search for new molecules that regulate SIRT1 expression. SIRT1 expression can be mediated at the transcriptional level and several mechanisms involved in dysregulation of SIRT1 in cancer cells have been proposed (16). Tumor suppressors p53 and HIC1 (hypermethylated in cancer 1) can bind to the SIRT1 promoter and form a complex with SIRT1, leading to inhibition of SIRT1 transcription (17,18). In cancer cells, inactivation of these tumor suppressor genes by genetic or epigenetic mechanisms leads to up-regulation of SIRT1 transcription. However, this is not the sole mechanism for overexpression of SIRT1 in tumors. For example, the RNA binding protein HuR, a potential oncoprotein, stabilizes SIRT1 mRNA through 3Ј-untranslated region (3Ј-UTR) interactions leading to elevated SIRT1 levels (19). This suggests that the 3Ј-UTR of SIRT1 mRNA may also be important in governing SIRT1 expression in tumors.

NF-E2-related factor 2 (Nrf2) is an important transcription factor that activates the expression of cellular detoxifying enzymes. Nrf2 expression is largely regulated through the association of Nrf2 with Kelch-like ECH-associated protein 1 (Keap1), which results in cytoplasmic Nrf2 degradation. Conversely, little is known concerning the regulation of Keap1 expression. Until now, a regulatory role for microRNAs (miRs) in controlling Keap1 gene expression had not been characterized. By using miR array-based screening, we observed miR200a silencing in breast cancer cells and demonstrated that upon re-expression, miR-200a targets the Keap1 3-untranslated region (3-UTR), leading to Keap1 mRNA degradation. Loss of this regulatory mechanism may contribute to the dysregulation of Nrf2 activity in breast cancer. Previously, we have identified epigenetic repression of miR-200a in breast cancer cells. Here, we find that treatment with epigenetic therapy, the histone deacetylase inhibitor suberoylanilide hydroxamic acid, restored miR-200a expression and reduced Keap1 levels. This reduction in Keap1 levels corresponded with Nrf2 nuclear translocation and activation of Nrf2-dependent NAD(P)H-quinone oxidoreductase 1 (NQO1) gene transcription. Moreover, we found that Nrf2 activation inhibited the anchorage-independent growth of breast cancer cells. Finally, our in vitro observations were confirmed in a model of carcinogen-induced mammary hyperplasia in vivo. In conclusion, our study demonstrates that miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium, and we find that epigenetic therapy can restore miR200a regulation of Keap1 expression, therefore reactivating the Nrf2-dependent antioxidant pathway in breast cancer.

NF-E2-related factor 2 (Nrf2) is an important transcription factor involved in antioxidant response. Nrf2 binds antioxidant response elements (ARE) within promoters of genes encoding detoxification enzymes (e.g., NAD (P) H-quinone oxidoreductase 1 (NQO1)) leading to their transcriptional activation. Nrf2 function is regulated post-translationally by its negative regulator Kelch-like ECH-associated protein 1 (Keap1) that binds Nrf2 and induces cytoplasmic Nrf2 degradation. Our present studies provide new evidence that Nrf2 expression can be regulated by a Keap1-independent mechanism. Here, we utilized breast epithelial cells to explore the impact of microRNA (miRNA) on Nrf2 expression. We found that Nrf2 mRNA levels are reversibly correlated with miR-28 expression and that ectopic expression of miR-28 alone reduces Nrf2 mRNA and protein levels. We further investigated the molecular mechanisms by which miR-28 inhibits Nrf2 mRNA expression. Initially, the ability of miR-28 to regulate the 3′ untranslated region (3′UTR) of Nrf2 mRNA was evaluated via luciferase reporter assay. We observed that miR-28 reduces wild-type Nrf2 3′UTR luciferase reporter activity and this repression is eliminated upon mutation of the miR-28 targeting seed sequence within the Nrf2 3′UTR. Moreover, over-expression of miR-28 decreased endogenous Nrf2 mRNA and protein expression. We also explored the impact of miR-28 on Keap1-Nrf2 interactions and found that miR-28 overexpression does not alter Keap1 protein levels and has no effect on the interaction of Keap1 and Nrf2. Our findings, that miR-28 targets the 3′UTR of Nrf2 mRNA and decreases Nrf2 expression, suggest that this miRNA is involved in the regulation of Nrf2 expression in breast epithelial cells.

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.