Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that differentially regulates expression of multiple genes, leading to suppression of metastasis without blocking orthotopic tumor growth in multiple human and murine cancer cells of diverse origins. We hypothesized that miR-146 may be involved in the ability of BRMS1 to supress metastasis because miR-146 expression is altered by BRMS1 and because BRMS1 and miR-146 are both associated with decreased signaling through the nuclear factor-KB pathway. BRMS1 significantly up-regulates miR-146a by 6-to 60-fold in metastatic MDA-MB-231 and MDA-MB-435 cells, respectively, and miR-146b by 40-fold in MDA-MB-435 as measured by real-time quantitative reverse transcription-PCR. Transduction of miR-146a or miR-146b into MDA-MB-231 down-regulated expression of epidermal growth factor receptor, inhibited invasion and migration in vitro, and suppressed experimental lung metastasis by 69% and 84%, respectively (mean F SE: empty vector = 39 F 6, miR-146a = 12 F 1, miR-146b = 6 F 1). These results further support the recent notion that modulating the levels of miR-146a or miR-146b could have a therapeutic potential to suppress breast cancer metastasis.

Despite advancements in knowledge from more than a century of metastasis research, the genetic programs and molecular mechanisms required for cancer metastasis are still incompletely understood. Genes that specifically regulate the process of metastasis are useful tools to elucidate molecular mechanisms and may become markers and/or targets for antimetastatic therapy. Recently, several noncoding regulatory RNA genes, microRNA (miRNA), were identified, which play roles in various steps of metastasis, some without obvious roles in tumorigenesis. Understanding how these metastasis-associated miRNA, which we term metastamir, are involved in metastasis will help identify possible biomarkers or targets for the most lethal attribute of cancer: metastasis.

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Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple tumor types without blocking tumorigenesis. BRMS1 forms complexes with SIN3, histone deacetylases and selected transcription factors that modify metastasis-associated gene expression (e.g., EGFR, OPN, PI4P5K1A, PLAU). microRNA (miRNA) are a recently discovered class of regulatory, noncoding RNA, some of which are involved in neoplastic progression. Based on these data, we hypothesized that BRMS1 may also exert some of its antimetastatic effects by regulating miRNA expression. Micro-RNA arrays were done comparing small RNAs that were purified from metastatic MDA-MB-231 and MDA-MB-435 and their nonmetastatic BRMS1-transfected counterparts. miRNA expression changed by BRMS1 were validated using SYBR Green RT-PCR. BRMS1 decreased metastasis-promoting (miR-10b, -373 and -520c) miRNA, with corresponding reduction of their downstream targets (e.g., RhoC which is downstream of miR-10b). Concurrently, BRMS1 increased expression of metastasis suppressing miRNA (miR-146a, -146b and -335). Collectively, these data show that BRMS1 coordinately regulates expression of multiple metastasisassociated miRNA and suggests that recruitment of BRMS1-containing SIN3:HDAC complexes to, as yet undefined, miRNA promoters might be involved in the regulation of cancer metastasis. ' UICCKey words: metastasis suppression; microRNA; BRMS1 Gene regulation by microRNA (miRNA) is a conserved mechanism in animals and plants.1 Endogenous, miRNA range from 15 to 28 nucleotides (nt) in Homo sapiens and, most commonly, negatively regulate gene expression, although some gene expression is positively regulated by miRNA. miRNA acts as templates for RNA-induced silencing complexes (RISC) to target mRNA. Animal miRNA differs functionally from plant miRNA in that they have imperfect base pairing with their target mRNA and more commonly inhibit protein translation than degrade mRNA. 36,40,41 which are involved in chromatin structure and selective regulation of gene expression.Collectively, these factors lead to the hypothesis that BRMS1 suppresses metastasis by altering expression of metastasis-associated genes. Previous studies have identified selective regulation of the epidermal growth factor receptor, 42 osteopontin, 35,36,43,44 connexins 45 and urokinase plasminogen activator. 46 We hypothesized that BRMS1 might also regulate recently discovered, metastasisassociated miRNA.To determine whether BRMS1 regulates miRNA expression, we compared miRNA expression patterns in nonexpressing and BRMS1 re-expressing breast carcinoma cells. miRNA expression was compared using microarrays imprinted with 328 known human miR probes and a selected common subset was further validated using quantitative real-time PCR (RTQ). In this article, we report that BRMS1 alters miRNA expression in metastatic breast carcinoma cells and notably down-regulated 3 of the 4 published metastasis-promoting miRNA 22,23,47 and up-regulated all 3 of the known metastasis-suppressing miRNA. 16,24Material and met...

While previous studies reported aberrant expression of microRNAs (miRNAs) in non-small cell lung cancer (NSCLC), little is known about which miRNAs play central roles in NSCLC's pathogenesis and its regulatory mechanisms. To address this issue, we presented a robust computational framework that integrated matched miRNA and mRNA expression profiles in NSCLC using feed-forward loops. The network consists of miRNAs, transcription factors (TFs), and their common predicted target genes. To discern the biological meaning of their associations, we introduced the direction of regulation. A network edge validation strategy using three independent NSCLC expression profiling data sets pinpointed reproducible biological regulations. Reproducible regulation, which may reflect the true molecular interaction, has not been applied to miRNA-TF coregulatory network analyses in cancer or other diseases yet. We revealed eight hub miRNAs that connected to a higher proportion of targets validated by independent data sets. Network analyses showed that these miRNAs might have strong oncogenic characteristics. Furthermore, we identified a novel miRNA-TF co-regulatory module that potentially suppresses the tumor suppressor activity of the TGF-β pathway by targeting a core pathway molecule (TGFBR2). Follow-up experiments showed two miRNAs (miR-9-5p and miR-130b-3p) in this module had increased expression while their target gene TGFBR2 had decreased expression in a cohort of human NSCLC. Moreover, we demonstrated these two miRNAs directly bind to the 3 ′ untranslated region of TGFBR2. This study enhanced our understanding of miRNA-TF co-regulatory mechanisms in NSCLC. The combined bioinformatics and validation approach we described can be applied to study other types of diseases.

Background/Aims: To determine breast cancer metastasis suppressor 1 (BRMS1) expression in breast cancers and the efficacy of BRMS1 as a prognostic indicator, BRMS1 expression was assessed in two sets of breast cancer tissues. Methods: Epithelial cells from 36 frozen samples of breast cancers and corresponding normal breast were collected by laser capture microdissection and assessed for BRMS1 by quantitative RT-PCR and immunohistochemistry. BRMS1 was also evaluated by immunohistochemistry in a tissue microarray of 209 breast cancers and correlated with indicators of prognosis [estrogen receptor (ER), progesterone receptor (PR), ErbB2, p53, p27^Kip1, Bcl2 and Ki-67]. Results: BRMS1 mRNA and protein were higher in 94 and 81%, respectively, of breast cancers than in corresponding normal epithelium. BRMS1 localization was predominantly nuclear, but 60–70% of cancers also exhibited cytoplasmic immunostaining. Breast cancers with lower nuclear than cytoplasmic BRMS1 (nuclear score – cytoplasmic score ≤0; 11% of cancers) had lower ER, lower PR and higher Ki-67 expression. There was also a trend toward poorer overall survival in this group of cancers, but this was only of borderline significance (p = 0.073). In Cox proportional hazards models, loss of nuclear BRMS1 was not a significant predictor of overall survival. Conclusions: Loss of nuclear BRMS1 was associated with ER-negative cancers and a high rate of proliferation, but was not an independent indicator of prognosis.

Clinical studies evaluating the mRNA expression level of the BRMS1 metastasis suppressor in the progression of breast cancer have not been consistent. The purpose of this study was to characterize endogenous BRMS1 mRNA and protein in a model of the progression of breast cancer. BRMS1 protein expression was evaluated in the genetically related MCF10 cell lines representing 'normal' breast epithelial cells (MCF10A), pre-malignant breast disease (MCF10AT), comedo ductal carcinoma in situ (MCF10DCIS.com), and metastatic carcinoma (MCF10CAa.1 and MCF10CAd. 1α) with two antibodies that recognize distinct epitopes in the BRMS1 protein. Nuclear expression of the characteristic ∼35 kDa BRMS1 protein was detected in all cell lines. Because BRMS1 was expressed in the metastatic MCF10 variants, the BRMS1 exons were sequenced to scan for possible genetic mutations. BRMS1 was wild-type with the exception of a synonymous T/C transition in exon 7. However, alternatively spliced variants were detected by RT-PCR. Two variants, BRMS1.v2 and BRMS1.v4 were only detected in the MCF10A and AT cell lines, while BRMS1 and BRMS1.v3 were detected in all lines. These results demonstrate that expression of the characteristic ∼35 kDa BRMS1 protein is not sufficient to prevent metastasis. The differential expression of alternative splice variants suggests caution should be taken when evaluating BRMS1 mRNA in clinical samples.

Metastatic lung cancer is common in lung adenocarcinoma patients, but the molecular mechanisms of metastasis remain incompletely resolved. MicroRNA (miR) regulate gene expression and contribute to cancer development and progression. This report identifies miR-576–3p and its mechanism of action in lung cancer progression. miR-576–3p was determined to be significantly decreased in clinical specimens of late-stage lung adenocarcinoma. Overexpression of miR-576–3p in lung adenocarcinoma cells decreased mesenchymal marker expression and inhibited migration and invasion. Inhibition of miR-576–3p in non-malignant lung epithelial cells increased migration and invasion as well as mesenchymal markers. Serum/glucocorticoid regulated kinase 1 (SGK1) was a direct target of miR-576–3p, and modulation of miR-576–3p levels led to alterations in SGK1 protein and mRNA as well as changes in activation of its downstream target linked to metastasis, N-myc downstream regulated 1 (NDRG1). Loss of the ability of miR-576–3p to bind the 3’-UTR of SGK1, rescued the inhibition in migration and invasion observed with miR-576–3p overexpression. Additionally, increased SGK1 levels were detected in lung adenocarcinoma patient samples expressing mesenchymal markers, and pharmacological inhibition of SGK1 resulted in a similar inhibition of migration and invasion of lung adenocarcinoma cells as observed with miR-576–3p overexpression. Together, these results reveal miR-576–3p downregulation is selected for in late-stage lung adenocarcinoma due to its ability to inhibit migration and invasion by targeting SGK1. Furthermore, these results also support targeting SGK1 as a potential therapeutic for lung adenocarcinoma.