Coordinate Regulation of FOXO1 by miR-27a, miR-96, and miR-182 in Breast Cancer Cells

Abstract

The FOXO1 transcription factor orchestrates the regulation of genes involved in the apoptotic response, cell cycle checkpoints, and cellular metabolism. FOXO1 is a putative tumor suppressor, and the expression of this gene is dysregulated in some cancers, including prostate and endometrial cancers. However, the molecular mechanism resulting in aberrant expression of human FOXO1 in cancer cells is poorly understood. We show here that FOXO1 mRNA is down-regulated in breast tumor samples as compared with normal breast tissue. Silencing of the microRNA processing enzymes, Drosha and Dicer, led to an increase in FOXO1 expression. We also identified functional and specific microRNA target sites in the FOXO1 3-untranslated region for miR-27a, miR-96, and miR-182, microRNAs that have previously been linked to oncogenic transformation. The three microRNAs, miR-27a, miR-96 and miR-182, were observed to be highly expressed in MCF-7 breast cancer cells, in which the level of FOXO1 protein is very low. Antisense inhibitors to each of these microRNAs led to a significant increase in endogenous FOXO1 expression and to a decrease in cell number in a manner that was blocked by FOXO1 siRNA. Overexpression of FOXO1 resulted in decreased cell viability because of inhibition of cell cycle traverse and induction of cell death. We have identified a novel mechanism of FOXO1 regulation, and targeting of FOXO1 by microRNAs may contribute to transformation or maintenance of an oncogenic state in breast cancer cells.The Forkhead Box O subfamily of transcription factors (FOXO) regulates a variety of important cellular processes including metabolism, cellular differentiation, apoptosis, and cell-cycle progression (1, 2). Acting as master cellular regulators, FOXO transcription factors can both activate and repress target gene expression (3).The three predominant members of the FOXO family (FOXO1, FOXO3a, and FOXO4) were first implicated in tumorigenesis based on the observation that fusion proteins resulting from chromosomal breakpoints exist in certain types of cancers (2). Recent evidence suggests that FOXO proteins function as tumor suppressors based on their role in regulating cell-cycle progression and inducing apoptosis (4).One regulatory mechanism of FOXO1 activity is through phosphorylation, primarily downstream of the insulin-stimulated phosphatidylinositol 3-kinase/AKT/protein kinase B signaling pathway, which results in nuclear exclusion (5-7). FOXO1 activity can also be regulated by acetylation (8) and ubiquitination (9, 10). In addition to insulin, FOXO1 can be down-regulated by other growth factors including estrogen (11, 12) and epidermal growth factor (13). The estrogen receptor ␣ also complexes with phosphorylated FOXO1 and mediates its export from the nucleus. These mitogens are important for the growth and survival of breast cancer cells and may contribute to maintaining low levels of FOXO1 in breast cancer cells.Although the activity of FOXO1 has been well characterized, very little is known about the regulation of FOXO1 ...

Efficient in vivo microRNA targeting of liver metastasis

“…In addition to our human tissue studies showing that miR-182 is upregulated in a subset of malignant melanomas (Segura et al, 2009), this miRNA has also been found to be a prognostic marker for glioma progression (Jiang et al, 2010), and to be highly expressed in colon (Bandres et al, 2007) and breast cancer cell lines (Guttilla and White, 2009). Therefore, anti-miR-182 may have additional antitumoral applications beyond melanoma.…”

Upregulation of miR-27a contributes to the malignant transformation of human bronchial epithelial cells induced by SV40 small T antigen

“…For example, miR-27a accelerates breast cancer cell growth by targeting mRNAs of the cell-cycle-associated protein ZBTB10 and myelin transcription factor 1 (Mertens-Talcott et al, 2007). Recent studies have shown that miR-27a contributes to the transformation or maintenance of a malignant state in breast cancer cells or gastric adenocarcinoma by targeting mRNAs of the transcription factor FOXO1 (Guttilla and White, 2009) and the membrane protein prohibitin (Liu et al, 2009). In hepatocellular carcinoma cells, upregulation of miR-27a confers the cells resistant to apoptosis induced by transforming growth factor-b (Huang et al, 2008).…”

“…These programs suggested a great number of genes as possible targets of miR-27a. Among the predicted targets, several genes including FOXO1 (forkhead box O1), Myt-1 (myelin transcription factor 1), prohibitin, sprouty2 and ZBTB10 (zinc finger and BTB domain containing 10) have been previously identified as the targets of miR-27a (Mertens-Talcott et al, 2007;Guttilla and White, 2009;Liu et al, 2009;Ma et al, 2010). We focused on genes meeting the following criteria: (1) cancer suppressor genes; (2) ability to bind miR-27a (the free energy (Mfe) of binding of the target gene's mRNA with miR-27a oÀ13.4 kcal/mol (Zhao et al, 2007)); and (3) genes that function in the regulation of cell proliferation.…”

miR-495 is upregulated by E12/E47 in breast cancer stem cells, and promotes oncogenesis and hypoxia resistance via downregulation of E-cadherin and REDD1

“…Some of these potential miR-495 targets, such as the forkhead box O transcription factor 1 and circadian clock gene Per2, are known to have tumor suppressor functions. Downregulated forkhead box O transcription factor 1 expression has been observed in several cancers including breast tumors, and overexpression of forkhead box O transcription factor 1 in breast cancer cells led to decreased cell number through both inhibition of cell cycle and increased cell death (Guttilla and White, 2009). Likewise, downregulation of Per2 has also been observed in breast cancers (Winter et al, 2007) and overexpression of Per2 inhibited tumor growth, whereas Per2 suppression increased cancer cell proliferation and tumorigenesis (Yang et al, 2009).…”