Breast Cancer Metastasis Suppressor Research Articles

Autocrine hGH stimulated miRNA 96‐182‐183 cluster promotes epithelial‐mesenchymal transition and invasion in breast cancer (LB201)

Human growth hormone (hGH) plays critical roles in pubertal mammary gland growth, development and sexual maturation. Accumulated studies reported that autocrine hGH is an orthotopically expressed oncogene and could induce normal mammary epithelial cell malignant transformation. Previously, our group reported autocrine hGH promotes mammary epithelial cell epithelial‐mesenchymal transition (EMT), facilitating invasion and increasing MMP activity. However, how hGH regulates EMT and invasion remains largely obscure. miRNAs are widely reported to involve in multiple processes of cancer. To determine whether autocrine hGH promotes EMT through miRNAs, we performed microarray profiling using MCF‐7 cells stably expressing normal or translation deficient hGH. We found that miR‐96‐182‐183 cluster exhibited highest response to autocrine hGH stimulation. We further observed that forced expression of miR‐96‐182‐183 conferred epithelial MCF‐7 cells with mesenchymal phenotype and invasive behaviors both in vitro and in vivo. Moreover, we revealed that miR‐96‐182‐183 promotes EMT and invasion by directly and simultaneously suppressing Breast Cancer Metastasis Suppressor 1‐like (BRMS1L) gene. In addition, miR‐96 and miR‐182 target GHR, introducing a potential negative feedback loop in the hGH‐GHR signaling pathway. Mechanically, we found that autocrine hGH stimulates miR‐96‐182‐183 expression and facilitates EMT and invasion via STAT3 and STAT5 signaling pathway. Clinically, we inspected that miR‐96‐182‐183 expression is remarkably enhanced in metastatic breast tumors. Together, we elucidate one novel hGH‐GHR‐STAT3/STAT5‐miR‐96‐182‐183 cluster‐BRMS1L‐ZEB1/E47‐EMT‐invasion axis, which sheds new insights into understanding the mechanism of autocrine hGH stimulated EMT and invasion of breast cancer cells.

Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer

Background:Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotropic tumour growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals.Methods:Methylation of BRMS1 promoter was examined in 57 NSCLC tumours and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals.Results:The BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and in corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals (0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan–Meier estimates were significantly different in favour of patients with non-methylated BRMS1 promoter in cfDNA, concerning both disease-free interval (DFI) (P=0.048) and overall survival (OS) (P=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (P=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients' DFI time and OS and on advanced NSCLC patients' PFS and OS.Conclusions:Methylation of BRMS1 promoter in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumour biomarker.

MicroRNA-149 targets GIT1 to suppress integrin signaling and breast cancer metastasis

Metastasis is the predominant cause of death in breast cancer patients. Several lines of evidence have shown that microRNAs (miRs) can have an important role in cancer metastasis. Using isogenic pairs of low and high metastatic lines derived from a human breast cancer line, we have identified miR-149 to be a suppressor of breast cancer cell invasion and metastasis. We also identified GIT1 (G-protein-coupled receptor kinase-interacting protein 1) as a direct target of miR-149. Knockdown of GIT1 reduced migration/invasion and metastasis of highly invasive cells. Re-expression of GIT1 significantly rescued miR-149-mediated inhibition of cell migration/invasion and metastasis. Expression of miR-149 impaired fibronectin-induced focal adhesion formation and reduced phosphorylation of focal adhesion kinase and paxillin, which could be restored by re-expression of GIT1. Inhibition of GIT1 led to enhanced protein degradation of paxillin and α5β1 integrin via proteasome and lysosome pathways, respectively. Moreover, we found that GIT1 depletion in metastatic breast cancer cells greatly reduced α5β1-integrin-mediated cell adhesion to fibronectin and collagen. Low level of miR-149 and high level of GIT1 was significantly associated with advanced stages of breast cancer, as well as with lymph node metastasis. We conclude that miR-149 suppresses breast cancer cell migration/invasion and metastasis by targeting GIT1, suggesting potential applications of the miR-149-GIT1 pathway in clinical diagnosis and therapeutics.

An In Vivo Functional Screen Identifies ST6GalNAc2 Sialyltransferase as a Breast Cancer Metastasis Suppressor

To interrogate the complex mechanisms involved in the later stages of cancer metastasis, we designed a functional in vivo RNA interference (RNAi) screen combined with next-generation sequencing. Using this approach, we identified the sialyltransferase ST6GalNAc2 as a novel breast cancer metastasis suppressor. Mechanistically, ST6GalNAc2 silencing alters the profile of O-glycans on the tumor cell surface, facilitating binding of the soluble lectin galectin-3. This then enhances tumor cell retention and emboli formation at metastatic sites leading to increased metastatic burden, events that can be completely blocked by galectin-3 inhibition. Critically, elevated ST6GALNAC2, but not galectin-3, expression in estrogen receptor-negative breast cancers significantly correlates with reduced frequency of metastatic events and improved survival. These data demonstrate that the prometastatic role of galectin-3 is regulated by its ability to bind to the tumor cell surface and highlight the potential of monitoring ST6GalNAc2 expression to stratify patients with breast cancer for treatment with galectin-3 inhibitors.

Sticking to Sugars at the Metastatic Site: Sialyltransferase ST6GalNAc2 Acts as a Breast Cancer Metastasis Suppressor

O-linked glycans on plasma membrane proteins are altered in cancer cells, leading to changes in cell adhesive properties and contributing to metastasis. Mechanisms of how these carbohydrates alter tumor spread remain vague. In this issue of Cancer Discovery, Murugaesu and colleagues, using an in vivo functional RNA interference metastasis screen, identified sialyltransferase ST6GalNAc2 as a novel metastasis suppressor gene. Aggressive estrogen receptor-negative breast cancers with reduced ST6GalNAc2 expression alter O-linked glycans on tumor cell surface, increasing soluble lectin galectin-3 binding and tumor cell clustering at metastatic sites.

The Activating Transcription Factor 3 Protein Suppresses the Oncogenic Function of Mutant p53 Proteins

Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer.

Pharmacological Levels of Withaferin A (Withania somnifera) Trigger Clinically Relevant Anticancer Effects Specific to Triple Negative Breast Cancer Cells

Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer.

MiR181b is induced by the chemopreventive polyphenol curcumin and inhibits breast cancer metastasis via down-regulation of the inflammatory cytokines CXCL1 and -2

Chronic inflammation is a major risk factor for the development and metastatic progression of cancer. We have previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast and prostate cancer metastases. In the present study, we have analyzed the effects of Curcumin on miRNA expression and its correlation to the anti-tumorigenic properties of this natural occurring polyphenol.Using microarray miRNA expression analyses, we show here that Curcumin modulates the expression of a series of miRNAs, including miR181b, in metastatic breast cancer cells. Interestingly, we found that miR181b down-modulates CXCL1 and -2 through a direct binding to their 3′-UTR. Overexpression or inhibition of miR181b in metastatic breast cancer cells has a significant impact on CXCL1 and -2 and is required for the effect of Curcumin on these two cytokines. miR181b also mediates the effects of Curcumin on inhibition of proliferation and invasion as well as induction of apoptosis. Importantly, over-expression of miR181b in metastatic breast cancer cells inhibits metastasis formation in vivo in immunodeficient mice. Finally, we demonstrated that Curcumin up-regulates miR181b and down-regulates CXCL1 and -2 in cells isolated from several primary human breast cancers.Taken together, these data show that Curcumin provides a simple bridge to bring metastamir modulation into the clinic, placing it in a primary and tertiary preventive, as well as a therapeutic, setting.

Down-regulation of cyclooxygenase-2 (COX-2) by cannabidiolic acid in human breast cancer cells.

Metastases are known to be responsible for approximately 90% of breast cancer-related deaths. Cyclooxygenase-2 (COX-2) is involved not only in inflammatory processes, but also in the metastasis of cancer cells; it is expressed in 40% of human invasive breast cancers. To comprehensively analyze the effects of cannabidiolic acid (CBDA), a selective COX-2 inhibitor found in the fiber-type cannabis plant (Takeda et al., 2008), on COX-2 expression and the genes involved in metastasis, we performed a DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are invasive breast cancer cells that express high levels of COX-2, treated with CBDA for 48 hr at 25 µM. The results obtained revealed that COX-2 and Id-1, a positive regulator of breast cancer metastasis, were down-regulated (0.19-fold and 0.52-fold, respectively), while SHARP1 (or BHLHE41), a suppressor of breast cancer metastasis, was up-regulated (1.72-fold) and CHIP (or STUB1) was unaffected (1.03-fold). These changes were confirmed by real-time RT-PCR analyses. Taken together, the results obtained here demonstrated that i) CBDA had dual inhibitory effects on COX-2 through down-regulation and enzyme inhibition, and ii) CBDA may possess the ability to suppress genes that are positively involved in the metastasis of cancer cells in vitro.

Identification of a Novel Compound That Suppresses Breast Cancer Invasiveness by Inhibiting Transforming Growth Factor-β Signaling via Estrogen Receptor α.

Breast cancer is the most frequently diagnosed cancer and the leading cause of death by cancer among females worldwide. An overwhelming majority of these deaths is because of metastasis. Estrogen stimulates and promotes growth of breast tumors, whereas transforming growth factor-beta (TGF-β) signaling promotes invasion and metastasis. We previously reported that estrogen and estrogen receptor alpha (ERα) suppressed breast cancer metastasis by inhibiting TGF-β signaling, whereas antiestrogens that suppress breast cancer growth, such as the selective ER modulator tamoxifen (TAM) or the pure antiestrogen fulvestrant (ICI 182,780), cannot suppress TGF-β signaling or breast cancer invasiveness. Therefore, we predicted that a compound that inhibits TGF-β signaling but does not facilitate ERα signaling would be ideal for suppressing breast cancer invasiveness and growth. In the present study, we identified an ideal candidate compound, N-23. Like estrogen, N-23 strongly decreased expression of TGF-β/Smad target gene plasminogen activator inhibitor-1 (PAI-1), but it did not increase the expression of ERα target gene pS2. While estrogen decreased the levels of phosphorylated Smad2 and Smad3, N-23 had no effect. In addition, TGF-β-dependent recruitment of Smad3 to the PAI-1 gene promoter was inhibited in the presence of estrogen or N-23. We also investigated the effects of N-23 on proliferation, migration, and invasion of breast cancer cells. In contrast to estrogen, N-23 inhibited the cellular proliferation of breast cancer cells. Moreover, we showed that N-23 suppressed the migration and invasion of breast cancer cells to the same extent as by estrogen. Taken together, our findings indicate that N-23 may be a candidate compound that is effective in inhibiting breast cancer progression.

Osteopontin Knockdown Inhibits αv,β3 Integrin-Induced Cell Migration and Invasion and Promotes Apoptosis of Breast Cancer Cells by Inducing Autophagy and Inactivating the PI3K/Akt/mTOR Pathway

Background: Osteopontin (OPN) is associated with tumor formation, progression and metastasis, and increased OPN levels have been associated with poor survival in breast cancer. We investigated the mechanisms responsible for OPN activity, and the relationships between OPN expression and clinical parameters in breast cancer. Methods: OPN mRNA and protein levels were compared in malignant and benign breast tumors by polymerase chain reaction (PCR) and immunohistochemistry, respectively, and levels in breast cancer cells were determined by PCR and western blotting. The effects of lentiviral-mediated knockdown of OPN on OPN and α_v,β₃ integrin expression, cell invasion and migration, autophagy and apoptosis were analyzed in MDA-MB-231 cells. Results: OPN expression increased with aggressiveness of breast cancer phenotype. OPN knockdown inhibited α_v,β₃ integrin expression in MDA-MB-231 cells, with subsequent inhibition of cell migration and invasion. Knockdown also inhibited the PI3K/Akt/mTOR pathway, promoted expression of the autophagy-related gene products LC3 and Beclin 1, and increased apoptosis. OPN expression was positively associated with tumor grade and lymph node metastasis. Conclusion: These results suggest that knockdown of OPN may inhibit breast cancer metastasis by regulating α_v,β₃ integrin expression and inducing autophagy and subsequent inhibition of PI3K/Akt/mTOR signaling, thus providing further insights into the complex mechanisms regulating tumor growth and metastasis.

Novel guanide-substituted compounds bind to CXCR4 and inhibit breast cancer metastasis

CXCR4 has been shown to be overexpressed on breast cancer cells including the human MDA-MB-231 cell line. Cancer cells overexpressing the CXCR4 receptor are capable of undergoing metastasis to organs expressing high levels of CXCL12. We have synthesized numerous guanide, biguanide, phenylguanide, and naphthylguanide compounds that bind to CXCR4 at the CXCL12-binding site and thus should prevent CXCR4-facilitated cancer metastasis. The novel compounds presented here were tested for CXCR4 affinity, toxicity, receptor activation, and for their ability to prevent breast cancer metastases. Three of the compounds bound to CXCR4 at IC50 values of 0.06-0.2 μmol/l, with no associated cell toxicity or receptor activation at these concentrations. These high CXCR4 affinity compounds also showed inhibition of in-vitro wound migration. They were then tested in an in-vivo mouse breast cancer lung colony model. All of these compounds showed reductions in the number of MDA-MB-231 lung metastases compared with mock-treated control mice without evidence of cardiac, liver, or kidney toxicity in treated mice.

Breast cancer metastasis suppressor-1 inhibits human glioma cells invasion by tissue inhibitor of metalloproteinase-2/matrix metalloproteinase-2 pathway

Objective To investigate the effect and mechanism of breast cancer metastasis suppressor-1 (BRMS1) on the human glioma cells invasion.Methods Transfection of the pEGFP-BRMS1 plasmids into the U251 and U87 glioma cells were carried out using Lipofectamine 2000.We studied the role of BRMS1 in glioma cell invasion by matrigel cell invasion assay.We performed gelatin zymography to measure the matrix metalloproteinase (MMP)-2 activities in glioma cells and Western blotting to examine the tissue inhibitorof metalloproteinase-2 (TIMP-2) and MMP-2 proteins expression after BRMS1 restoration.Results Compared to the control plasmids,pEGFP-BRMS1 plasmids could increase the expression of BRMS1 in both glioma cells significantly.In cell invasion assay,BRMS1 inhibits cell invasion ability of U251 and U87 cells in matrigel-coated Boyden chamber by 43％ and 52％,respectively.BRMS1 also suppress the MMP-2 activity.Increasing of BRMS1 expression upregulated TIMP-2 protein expression and inhibited MMP-2 protein expression.Conclusion Our data indicate that BRMS1 may be reduced glioma cells invasion abilities by the imbalance between TIMP-2 and MMP-2. Key words: Breast cancer metastasis suppressor-1; Glioma; Invasion; Tissue inhibitor of metalloproteinase-2 ; Matrix metalloproteinase-2

Inactivation of a RASGAP Promotes Breast Cancer Progression

Abstract RAS protein activator-like 2 (RASAL2) is a tumor and metastasis suppressor in breast cancer.

Infrequent Loss of Luminal Differentiation in Ductal Breast Cancer Metastasis

Lymph node involvement is a major prognostic variable in breast cancer. Whether the molecular mechanisms that drive breast cancer cells to colonize lymph nodes are shared with their capacity to form distant metastases is yet to be established. In a transcriptomic survey aimed at identifying molecular factors associated with lymph node involvement of ductal breast cancer, we found that luminal differentiation, assessed by the expression of estrogen receptor (ER) and/or progesterone receptor (PR) and GATA3, was only infrequently lost in node-positive primary tumors and in matched lymph node metastases. The transcription factor GATA3 critically determines luminal lineage specification of mammary epithelium and is widely considered a tumor and metastasis suppressor in breast cancer. Strong expression of GATA3 and ER in a majority of primary node-positive ductal breast cancer was corroborated by quantitative RT-PCR and immunohistochemistry in the initial sample set, and by immunohistochemistry in an additional set from 167 patients diagnosed of node-negative and –positive primary infiltrating ductal breast cancer, including 102 samples from loco-regional lymph node metastases matched to their primary tumors, as well as 37 distant metastases. These observations suggest that loss of luminal differentiation is not a major factor driving the ability of breast cancer cells to colonize regional lymph nodes.

Inhibition of Breast Cancer Metastasis by Resveratrol-Mediated Inactivation of Tumor-Evoked Regulatory B Cells

We reported previously that tumor-evoked regulatory B cells (tBregs) play an essential role in breast cancer lung metastasis by inducing TGF-β-dependent conversion of metastasis-promoting Foxp3(+) regulatory T cells (Tregs). In this article, we show that resveratrol (RSV), a plant-derived polyphenol, at low and noncytotoxic doses for immune cells, can efficiently inhibit lung metastasis in mice. The mechanism of this process is that RSV inactivates Stat3, preventing the generation and function of tBregs, including expression of TGF-β. As a result, it frees antitumor effector immune responses by disabling tBreg-induced conversion of Foxp3(+) Tregs. We propose that low doses of RSV may also benefit humans by controlling cancer escape-promoting tBregs/Tregs without nonspecific inactivation of effector immune cells.

Highlights of This Issue

The cellular response to DNA double-strand breaks (DSB) is commonly dysregulated in multiple human cancers. In this context, the ATM and 53BP1 tumor suppressors normally interact at chromatin surrounding DSBs to promote efficient repair. Rybanska-Spaeder and colleagues have examined the in vivo significance of this interaction using a mouse model of combined ATM and 53BP1 deficiencies. Significantly, loss of 53BP1 markedly decreased lymphoma latency and survival in ATM-deficient mice. Mechanistically, 53BP1 deficiency promoted oncogenic translocations in ATM-deficient lymphocytes by aggravating their defect in nonhomologous end-joining–mediated repair. Together, these findings demonstrate ATM-independent functions for 53BP1 in DSB repair and translocation suppression.Evidence of chronic inflammation is frequently associated with prostate cancer, though it remains unclear how signaling molecules associated with inflammation affect the tumor microenvironment. In this Rapid Impact article, Smith and colleagues report on their work with a dissociated prostate tissue recombination assay, which revealed that stromal expression of either interleukin-6 (IL6) or the related cytokine oncostatin-M (OSM) synergized with epithelial loss of PTEN to promote aggressive prostate malignancy. Critically, tumor progression was associated with invasive epithelial growth and increased activation of the JAK/STAT signaling axis. These results indicate that cytokines ordinarily present in inflammatory microenvironments can act in concert with existing genetic aberrations to promote tumor progression.Multiple endocrine neoplasia type 1 (MEN1) is an inherited tumor syndrome resulting from mutations in the gene encoding the protein menin. Previous work demonstrated that menin suppressed Hedgehog (Hh) signaling via repression of a critical cell surface protein cofactor, Gas1. However, the capacity and extent to which menin regulates other components of the Hh pathway is unclear. Here, Gurung and colleagues illustrate that menin directly suppresses the transcription factor GLI1 via repressive PRMT5 histone arginine methyltransferase, resulting in decreased GLI1 expression and cell proliferation. These data indicate that aberrant GLI1 regulation is, in part, responsible for MEN1 tumorigenesis and that targeting GLI1 may mitigate MEN1 tumors.Breast cancer is one of the most commonly diagnosed cancers in women, and generally the underlying cause of mortality is due to metastatic burden. Recent evidence suggests that detection of circulating tumor cells (CTC) has prognostic potential. Previously, it was shown that the breast cancer metastasis suppressor-1 (BRMS1) promoter was methylated in CTCs from clinical specimens. In the current study, Chimonidou and colleagues characterize the BRMS1 promoter methylation status and BRMS1 protein expression in primary breast cancer tumors and corresponding CTCs. Critically, BRMS1 promoter methylation status in primary disease was significantly correlated with disease-free survival. Combined, these observations reveal the clinical importance of evaluating BRMS1 promoter methylation in early stages of breast cancer.

Breast Cancer Metastasis Suppressor-1 Promoter Methylation in Primary Breast Tumors and Corresponding Circulating Tumor Cells

Breast cancer metastasis suppressor-1 (BRMS1) differentially regulates the expression of multiple genes, leading to metastasis suppression without affecting orthotopic tumor growth. For the first time, BRMS1 promoter methylation was evaluated as a prognostic biomarker in primary breast tumors and a subset of corresponding circulating tumor cells (CTC). Formalin-fixed paraffin embedded samples were analyzed for BRMS1 methylation status using methylation-specific PCR in a human specimen cohort consisting of noncancerous tissues, benign fibroadenomas, and primary breast tumors, including some with adjacent noncancerous tissues. Peripheral blood mononuclear cells from a large subset of these patients were fixed in cytospins and analyzed. In addition, BRMS1 expression in cytospins was examined by double-immunofluorescence using anti-BRMS1 and pan-cytokeratin antibodies. BRMS1 promoter methylation was not detected in noncancerous breast tissues or benign fibroadenomas; however, methylation was observed in more than a third of primary breast tumors. Critically, BRMS1 promoter methylation in primary tumors was significantly associated with reduced disease-free survival with a trend toward reduced overall survival. Similarly, a third of cytospin samples were positive for the presence of CTCs, and the total number of detected CTCs was 41. Although a large fraction of CTCs were negative or maintained low expression of BRSM1, promoter methylation was observed in a small fraction of samples, implying that BRSM1 expression in CTCs was either downregulated or heterogeneous. In summary, these data define BRMS1 promoter methylation in primary breast tumors and associated CTCs. This study indicates that BRSM1 promoter methylation status has biomarker potential in breast cancer.

Abstract B093: Discovery of microRNAs associated with breast cancer EMT using bioinformatics and next-generation sequencing

Abstract Breast cancer is the most common malignancy among women worldwide, with mortality often associated with distant metastasis. In recent years, microRNAs (miRs) have emerged as a new class of regulatory molecules that act as tumor suppressors or oncogenes and are capable of affecting metastatic processes such as epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). Our study aimed to identify miRs associated with breast cancer EMT through miR profiling of EMT in PMC42 (ET vs LA, -/+ EGF) and MDA-MB-468 (control vs EGF, hypoxia) human breast cancer cells using microarray and Next Generation Sequencing (miR-Seq). Several miRs were reproducibly different between the untreated cells, as well as up- or down-regulated in response to epidermal growth factor (EGF). Expression levels of 36 miRs were validated in six breast cancer cell lines comprising a range of epithelial to mesenchymal cell lines. Nine miRs were then selected and the effects of manipulating these miRs were investigated in vitro and in vivo. MDA-MB-468 cells stably overexpressing (OE) the miRs of interest were produced, validated and tested for changes in in vitro migratory potential using a monolayer wound healing assay on the Cellomics platform. We found that the rate of wound closure was significantly faster (p<0.05) in MDA-MB-468 breast cancer cells OE miR-34b/c upon EGF induction even though there were no significant differences in migrative potential using Transwell migration assay. Although these cells showed no proliferative differences, MDA-MB-468 miR-34b/c OE cells showed decreased clonogenicity (p<0.01) compared to control cells. We extended this by looking at these manipulated cells in the MDA-MB-468 xenograft model of in vivo EMT. Our data suggests that there might be a slightly slower rate of primary tumor growth and an inhibition of lymph node metastasis in vivo. Other groups have also identified miR-34 family as a p53 target gene regulating Snail1-driven EMT (Kim et al., 2011) and have shown miR-34 to suppress breast cancer metastasis by targeting Fra-1 (Yang et al., 2012). Fra-1 and several other MEK pathway components were upregulated in the PMC42 and MDA-MB-468 EMT as determined by corresponding RNA-Seq analysis, and integrative analysis of the miR-Seq and RNA-Seq data is ongoing. Our work on EMT-associated miRs is targeted to provide a better understanding of miRs for their potential uses in diagnostic and therapeutic approaches to breast cancer. Citation Format: Eliza Soo, Tony Blick, Mark Waltham, Gregory Goodall, Izhak Haviv, Kaylene Simpson, Erik Thompson, Bryce van Denderen. Discovery of microRNAs associated with breast cancer EMT using bioinformatics and next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B093.

Hsa-miR-206 inhibits the migration and invasion of breast cancer by targeting Cx43

To explore the effects of hsa-miR-206 on the proliferation, migration and invasion of breast cancer. The hsa-miR-206 mimics, inhibitors and their paired negative controls were transfected into human breast cancer cell line MDA-MB-231 by liposome. The proliferation of cell was evaluated by CCK-8 and the migration and invasion was detected by Transwell. Matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), breast cancer metastasis-suppressor 1 (BRMS-1) and connexin 43 (Cx43) were detected by both quantitative polymerase chain reaction (qPCR) and Western blot. The expression of miR-206 was detected by qPCR. Dual luciferase assay was detected to confirm the specific binding sites of miR-206 and Cx43. (1) The proliferation activity of 206m-group cell (0.74 ± 0.16) was significantly lower than that of control group (1.12 ± 0.23) (t = -3.066, P = 0.037) while that of 206i-group cell (1.43 ± 0.26) was higher than that of control group (0.98 ± 0.14) (t = 3.635, P = 0.022). (2) Transwell tests showed the migration and invasion of 206m-group cell decreased significantly (migration:0.56 ± 0.01 vs 0.63 ± 0.01, t = -23.00, P = 0.002; invasion:0.79 ± 0.01 vs 0.99 ± 0.01, t = -21.200, P = 0.002), but that of 206i-group cell increased significantly (migration:0.97 ± 0.11 vs 0.61 ± 0.09, t = 32.787, P = 0.001; invasion:1.10 ± 0.01 vs 0.93 ± 0.05, t = 5.167, P = 0.035). (3) The expressions of MMP-2,MMP-9 and Cx43 decreased and the expression of BRMS-1 increased in 206m-group cell and vice versa in 206i group. (4) The expression of miR-206 in lymph node-negative group of clinical breast cancer sample was higher than that of lymph node-positive one. And there was statistical difference (Z = -2.098, P = 0.003). And the expression of Cx43 was opposite. (5) Dual luciferase reporter assay confirmed the specific binding sites of hsa-miR-206 and Cx43. Hsa-miR-206 has negative controls of proliferation, migration and invasion of breast cancer cell by targeting Cx43.