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ABSTRACT: Several reports have indicated that miR-140, a possible tumor suppressor microRNA (miR), is down-regulated in breast tumors compared to normal breast tissues. However, the role of miR-140 in breast tumorigenesis is unclear. We initiated studies that examined estrogen receptor alpha (ERα) signaling in the tissue-specific regulation of miR-140 in breast cancer. We found that estrogen stimulation of ERα positive breast cancer cells resulted in decreased miR-140 expression. We performed promoter analyses and examined predicted ERα binding elements in the miR-140 promoter using luciferase constructs of a miR-140 promoter deletion series. Our studies revealed that ERα binds to one specific estrogen response element flanking the miR-140 promoter and consequently suppresses miR-140 transcription. We found that the stem cell self-renewal regulator SOX2 is a novel target of miR-140, and that this miR-140/SOX2 pathway critically regulates breast tumor-initiating cell survival, providing a new link between ERα signaling and breast cancer stem cell maintenance.
Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
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ABSTRACT: 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 miR-200a 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 miR-200a regulation of Keap1 expression, therefore reactivating the Nrf2-dependent antioxidant pathway in breast cancer.
Journal of Biological Chemistry 09/2011; 286(47):40725-33. · 4.77 Impact Factor
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ABSTRACT: 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 over-expression 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.
Breast Cancer Research and Treatment 06/2011; 129(3):983-91. · 4.43 Impact Factor
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ABSTRACT: Evidence supports a critical role for microRNAs (miRNAs) in regulation of tissue-specific differentiation and development. Signifying a disruption of these programs, expression profiling has revealed extensive miRNA dysregulation in tumors compared with healthy tissue. The miR-200 family has been established as a key regulator of epithelial phenotype and, as such, is deeply involved in epithelial to mesenchymal transition (EMT) processes in breast cancer. However, the effects of the miR-200 family on transformation of normal mammary epithelial cells have yet to be fully characterized. By examining a TGF-β driven model of transformation of normal mammary epithelium, we demonstrate that the class III histone deacetylase silent information regulator 1 (SIRT1), a proposed oncogene in breast cancer, is overexpressed upon EMT-like transformation and that epigenetic silencing of miR-200a contributes at least in part to the overexpression of SIRT1. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3'-UTR. We also observed SIRT1 and miR-200a participation in a negative feedback regulatory loop. Restoration of miR-200a or the knockdown of SIRT1 prevented transformation of normal mammary epithelial cells evidenced by decreased anchorage-independent growth and decreased cell migration. Finally, we observed SIRT1 overexpression in association with decreased miR-200a in breast cancer patient samples. These observations provide further evidence for a critical tumor suppressive role of the miR-200 family in breast epithelium in addition to identifying a novel regulatory mechanism, which may contribute to SIRT1 up-regulation in breast cancer.
Journal of Biological Chemistry 05/2011; 286(29):25992-6002. · 4.77 Impact Factor
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ABSTRACT: Evidence supports a critical role for microRNAs (miRNAs) in regulation of tissue specific differentiation and development.
Signifying a disruption of these programs, expression profiling has revealed extensive miRNA dysregulation in tumors compared
with healthy tissue. The miR-200 family has been established as a key regulator of epithelial phenotype and as such, is deeply
involved in epithelial to mesenchymal transition (EMT) processes in breast cancer. However, the effects of the miR-200 family
on transformation of normal mammary epithelial cells have yet to be fully characterized. By examining a TGF-b driven model
of transformation of normal mammary epithelium, we demonstrate that the class III histone deacetylase silent information regulator
1 (SIRT1), a proposed oncogene in breast cancer, is overexpressed upon EMT-like transformation and that epigenetic silencing
of miR-200a contributes at least in part to the over-expression of SIRT1. We have established SIRT1 transcript as subject
to regulation by miR-200a, through miR-200a targeting of SIRT1 3 UTR. We also observed SIRT1 and miR-200a participation in
a negative feedback regulatory loop. Restoration of miR-200a or the knockdown of SIRT1 prevented transformation of normal
mammary epithelial cells evidenced by decreased anchorage independent growth and decreased cell migration. Finally, we observed
SIRT1 over-expression in association with decreased miR-200a in breast cancer patient samples. These observations provide
further evidence for a critical tumor suppressive role of the miR-200 family in breast epithelium in addition to identifying
a novel regulatory mechanism, which may contribute to SIRT1 up-regulation in breast cancer.
Journal of Biological Chemistry 05/2011; · 4.77 Impact Factor
