Dominant-negative E-cadherin inhibits the invasiveness of inflammatory breast cancer cells in vitro
ABSTRACT E-cadherin is a transmembrane glycoprotein which mediates epithelial cell-to-cell adhesion function as a tumor suppressor and frequently loss of expression in a wide spectrum of human cancer. However, recent studies demonstrated that E-cadherin was always over-expressed in inflammatory breast cancer (IBC) specimen and cell lines, which is a clinical extreme malignancy of breast cancer. It is hypothesized that the gain and not the loss of the E-cadherin axis contributes to the IBC unique phenotype. To test this assumption, we generated dominant negative mutant E-cadherin high-expression inflammatory breast cancer cells by introduced dominant negative mutant E-cadherin (H-2kd-E-cad) cDNA into human IBC SUM149 cells. Our studies demonstrated that the ability of invasion of SUM149 cells was significantly inhibited by H-2kd-E-cad via down-regulation of MMP-1 and MMP-9 expression. The underlying signal pathway of MAPK phosphorylated Erk 1/2(P44/42) in H-2kd-E-cad-transfected SUM149 cells was significantly down-regulated compared to parental and mock contrast. Our studies provided further functional evidence as the gain of E-cadherin expression dedicated to the IBC malignant phenotype and the blockage of MAPK/Erk activation maybe a promising therapeutic target.
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ABSTRACT: Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with distinct molecular profiles. Gene expression profiling previously identified sonic hedgehog (SHH) as part of a gene signature that is differentially regulated in IBC patients. The effects of reducing GLI1 levels on protein expression, cell proliferation, apoptosis and migration were determined by immunoblots, MTT assay, Annexin-V/PI assay and conventional and automated cell migration assays. Evaluation of a panel of breast cancer cell lines revealed elevated GLI1 expression, typically a marker for hedgehog-pathway activation, in a triple-negative, highly invasive IBC cell line, SUM149 and its isogenic-derived counterpart rSUM149 that has acquired resistance to ErbB1/2 targeting strategies. Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis. Further, GLI1 suppression in these cell lines significantly inhibited cell migration as assessed by a wound-healing assay compared with MCF-7, a non-invasive cell line with low GLI1 expression. A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement. Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.British Journal of Cancer 05/2011; 104(10):1575-86. DOI:10.1038/bjc.2011.133 · 4.82 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via regulation of HOXD10. This report was soon challenged by another group who reported that miR-10b expression in a large patient cohort correlated inversely and significantly with tumor size, grade, and vascular invasion, but did not correlate with development of distant metastases or survival. These latter data suggest that miR-10b may impede specific functions associated with breast cancer progression. In this thesis, I present my analysis of miR-10b function in breast carcinoma cells, which revealed that it suppresses their migration and invasion. To define a mechanism that accounts for this suppressive function, I identified T-lymphoma invasion and metastasis 1 (TIAM1), a guanine nucleotide exchange factor for Rac1, as a miR-10b target and demonstrated that miR-10b inhibits TIAM1-dependent Rac1 activation, migration, and invasion. In addition, I identified the VEGF receptor fms-related tyrosine kinase 1 (FLT-1) as a second target of miR-10b and discovered a novel function for FLT-1 in promoting breast carcinoma cell migration and invasion. My results show, for the first time, that Rac activation can be regulated by a specific miRNA and provide a novel mechanism for the regulation of TIAM1 and FLT-1 in breast cancer. These data support the conclusion from clinical data that miR-10b expression correlates inversely with breast cancer progression, and suggest that miR-10b functions to impede breast carcinoma progression by regulating key target genes involved in cell motility.
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ABSTRACT: Inflammatory breast cancer (IBC) is the most lethal variant of locally advanced breast cancer. Although recognized as a distinct clinical entity, there have been few advances in the development of pre-clinical models of IBC, and a lack of IBC-specific therapeutic targets translated into clinical utility to increase overall survival, which is currently 40 % at three years. By use of newly developed pre-clinical models of IBC and patient tumor tissues, E-cadherin, anaplastic lymphoma kinase (ALK), and HSP90 have been identified as targets relevant to IBC that are matched by therapeutics that are either currently in clinical trials or will be tested in clinical trials within the next year. These exciting results illustrate the advances that have been made in recent years in defining the molecular basis of IBC as a distinct disease and the significant strides made in identifying more effective strategies for treatment of patients with IBC.Current Breast Cancer Reports 12/2012; 4(4). DOI:10.1007/s12609-012-0087-3