Prooncogenic Factors miR-23b and miR-27b Are Regulated by Her2/Neu, EGF, and TNF-a in Breast Cancer

miRNAs (miR) are a critical class of small (21–25 nucleotides) noncoding endogenous RNAs implicated in gene expression regulation. We identified miR-23b and miR-27b as miRNAs that are highly upregulated in human breast cancer. We found that engineered knockdown of miR-23b and miR-27b substantially repressed breast cancer growth. Nischarin (NISCH) expression was augmented by knockdown of miR-23b as well as miR-27b. Notably, these miRNAs and Nischarin were inversely expressed in human breast cancers, underscoring their biologic relevance. We showed the clinical relevance of the expression of these miRNAs and showed that high expression of miR-23b and miR-27b correlates with poor outcome in breast cancer. Moreover, intraperitoneally delivered anti-miR-27b restoredNischarin expression anddecreased tumor burden in amouse xenograftmodel of humanmammary tumor. Also, we report for thefirst time thatHER2/neu (ERBB2), EGF, andTNF-apromotemiR23b/27b expression through the AKT/NF-kB signaling cascade. Nischarin was found to regulate miR-27b/23b expression through a feedback loop mechanism by suppressing NF-kB phosphorylation. Because anti-miR-27b compounds that suppress miR-27b inhibit tumor growth, the anti-miR-27b seems to be a good candidate for the development of new antitumor therapies. Cancer Res; 73(9); 2884–96. 2013 AACR. Introduction miRNAs are a class of small (21–25 nucleotides) noncoding endogenous RNAs that negatively regulate gene expression by binding to the 30-untranslated region (UTR) of target mRNAs, resulting in either transcript degradation or translational inhibition (1–3). Since the discovery of miRNAs in 1993, more than 1,500 human miRNAs have been identified. To date, miRNAs have been shown to regulate many cellular processes and pathways that are critical for neoplastic transformation and tumor progression (4–7). miRNAs have profound positive effects on cancer metastasis; examples include miR-31 and miR-200 family members (8, 9). Many miRNAs are upregulated or downregulated in tumors compared with normal tissues, supporting their dual role in carcinogenesis as either tumor suppressors or tumor promoters (the latter generally referred to as oncomirs; refs. 10, 11). Examples of tumor-suppressor miRNAs include the Let-7 family, which suppresses the Ras oncogene and is present at low levels in lung cancer, and miR-16, which suppresses the Bcl2 gene as well as multiple other cell-cycle genes and is downregulated in leukemia (12, 13). Conversely, the oncomirs miR-21 and miR-221/222 are upregulated in several cancers and modulate the expression of their targets (14, 15). Her2/neu (c-erbB-2) is a membrane receptor tyrosine kinase that is amplified or overexpressed in approximately 20% of breast cancers and is associated with increased disease recurrence, tumor invasion, and poor prognosis (16). Some oncomirs, such asmiR-21, can be upregulated byHer2/neu signaling and can promote cell invasion in breast cancer (17). Our previous in vitro and in vivo studies identified Nischarin, a novel intracellular protein, as a suppressor of tumor growth and lungmetastasis (18). Nischarin is a binding partner for the a5b1 integrin, interacts with members of the PAK family kinases, and thereby regulates the metastatic behavior of tumor cells (19, 20). Here, we show that Nischarin expression in breast cancer is regulated by miR-23b and miR-27b binding to the Nischarin 30-UTR region. Both miRNAs are highly expressed in human cancer at least in part due to elevated Her2, EGF, and TNF-a signaling. Moreover, suppression of miR-23b/27b activity upregulates Nischarin in breast cancer cells and diminishes tumor growth and metastasis in vivo. Materials and Methods Cell culture and reagents Human breast cancer cell lines MDA-MB-231-4175 (a gift from Dr. Joan Massague, Memorial Sloan-Kettering Cancer Center, New York, NY), MCF7, and MDA-MB-231 were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. MCF10A was maintained in MEBM medium supplemented with 5% fetal Authors' Affiliations: Department of Biochemistry andMolecular Biology and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana; Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; Regulus Therapeutics, San Diego, California; and Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Suresh K. Alahari, Biochemistry, Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, CSRB 406, 1901 Perdido Street, New Orleans, LA 70112. Phone: 504-568-4734; Fax: 504-568-2093; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-2162 2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(9) May 1, 2013 2884 on January 6, 2018. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 21, 2013; DOI: 10.1158/0008-5472.CAN-12-2162