Diversity of matriptase expression level and function in breast cancer

Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent.     

FilGAP,a Rho/Rho-associated protein kinase–regulated GTPase-activating protein for Rac,controls tumor cell migration

Tumor cells exhibit two interconvertible modes of cell motility referred to as mesenchymal and amoeboid migration. Mesenchymal mode is characterized by elongated morphology that requires high GTPase Rac activation, whereas amoeboid mode is dependent on actomyosin contractility induced by Rho/Rho-associated protein kinase (ROCK) signaling. While elongated morphology is driven by Rac-induced protrusion at the leading edge, how Rho/ROCK signaling controls amoeboid movement is not well understood. We identified FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/ROCK-dependent amoeboid movement of carcinoma cells. We show that depletion of endogenous FilGAP in carcinoma cells induced highly elongated mesenchymal morphology. Conversely, forced expression of FilGAP induced a round/amoeboid morphology that requires Rho/ROCK-dependent phosphorylation of FilGAP. Moreover, depletion of FilGAP impaired breast cancer cell invasion through extracellular matrices and reduced tumor cell extravasation in vivo. Thus phosphorylation of FilGAP by ROCK appears to promote amoeboid morphology of carcinoma cells, and FilGAP contributes to tumor invasion.     

An in vitro model that recapitulates the epithelial to mesenchymal transition (EMT) in human breast cancer

The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells down-regulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In human breast cancer, invasion into surrounding tissue is the first step in metastatic progression. Here, we devised an in vitro model using selected cell lines, which recapitulates many features of EMT as observed in human breast cancer. By comparing the gene expression profiles of claudin-low breast cancers with the experimental model, we identified a 9-gene signature characteristic of EMT. This signature was found to distinguish a series of breast cancer cell lines that have demonstrable, classical EMT hallmarks, including loss of E-cadherin protein and acquisition of N-cadherin and vimentin expression. We subsequently developed a three-dimensional model to recapitulate the process of EMT with these cell lines. The cells maintain epithelial morphology when encapsulated in a reconstituted basement membrane, but undergo spontaneous EMT and invade into surrounding collagen in the absence of exogenous cues. Collectively, this model of EMT in vitro reveals the behaviour of breast cancer cells beyond the basement membrane breach and recapitulates the in vivo context for further investigation into EMT and drugs that may interfere with it.     

S-Allylcysteine reduces breast tumor cell adhesion and invasion

Previous studies show that aqueous garlic extract and its derivatives (e.g. S-allylcysteine [SAC]) prevent carcinogen-induced breast tumorigenesis. However, investigations testing the effect of SAC on later stages of breast tumorigenesis and/or metastasis have produced mixed results. Here we show that SAC significantly reduced anchorage-dependent and -independent growth of MDA-MB-231 breast tumor cells in a dose- and time-dependent fashion, and sub-lethal SAC-treatment altered mammary tumor cell adhesion and invasion through components of the extracellular matrix. We provide evidence to suggest increased expression of E-cadherin and reduced MMP-2 expression and activity are partially responsible for inhibition of mammary tumor cell invasion by SAC. Because E-cadherin and MMP-2 are important in cancer metastasis, these results suggest a link between SAC induction of E-cadherin and reduction of MMP2 activity with the inhibition of cell motility and invasion; thus providing evidence that events leading to breast cancer metastasis are repressed by sub-lethal SAC-treatment.     

Methylation patterns of the E-cadherin 5' CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression

Metastatic progression of most common epithelial tumors involves a heterogeneous, transient loss of expression of the homotypic cell adhesion protein, E-cadherin, rather than the uniform loss of a functional protein resulting from coding region mutation. Indeed, whereas E-cadherin loss may promote invasion, reexpression may facilitate cell survival within metastatic deposits. The mechanisms underlying such plasticity are unclear. We now show that the heterogeneous loss of E-cadherin expression in primary human breast cancers reflects a heterogeneous pattern of promoter region methylation, which begins early prior to invasion. In cultured human tumor cells, such heterogeneous methylation is dynamic, varying from allele to allele and shifting in relation to the tumor microenvironment. Following invasion in vitro, which favors diminished E-cadherin expression, the density of promoter methylation markedly increased. When these cells were cultured as spheroids, which requires homotypic cell adhesion, promoter methylation decreased dramatically, and E-cadherin was reexpressed. These data show that the methylation associated with E-cadherin loss in human breast cancer is heterogeneous and unstable and suggest that such epigenetic plasticity may contribute to the dynamic, phenotypic heterogeneity that drives metastatic progression.     

MDM2 promotes cell motility and invasiveness by regulating E-cadherin degradation

Gene amplification and protein overexpression of MDM2, which is often found in certain types of cancers, indicate that MDM2 plays an important role in tumorigenesis. Interestingly, several clinical reports have demonstrated that amplification of the MDM2 gene correlates with the metastatic stage. Using an antibody array assay, we identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin is a substrate for the MDM2 E3 ubiquitin ligase. We demonstrate that MDM2 interacts in vivo with E-cadherin, resulting in its ubiquitination and degradation. This regulation appears to be clinically relevant, as we found a significant correlation between high MDM2 and low E-cadherin protein levels in resected tumor specimens recovered from breast cancer patients with lymph node metastases. Ectopic expression of MDM2 in breast cancer cells was found to disrupt cell-cell contacts and enhance cell motility and invasive potential. We found that E-cadherin and MDM2 colocalized on the plasma membrane and in the early endosome, where ubiquitin moieties were attached to E-cadherin. Blocking endocytosis with dominant-negative mutants of dynamin abolished the association of MDM2 with E-cadherin, prevented E-cadherin degradation, and attenuated cell motility as observed by fluorescence microscopy. Thus, we provide evidence to support a novel role for MDM2 in regulating cell adhesions by a mechanism that involves degrading and down-regulating the expression of E-cadherin via an endosome pathway. This novel MDM2-regulated pathway is likely to play a biologically relevant role in cancer metastasis.     

TGF-β1对乳腺癌细胞系Snail表达的影响

目的通过TGF-β1诱导乳腺癌MCF-7发生上皮-间质转化(epithelial-mesenchymal transition,EMT)后检测锌指转录因子Snail表达的改变,探讨Snail在EMT及乳腺癌发生发展中的作用。方法常规培养乳腺癌细胞株MCF-7后,用TGF-β1诱导其发生EMT,用Transwell侵袭小室法进行细胞体外侵袭能力检测;用免疫组织化学方法及免疫荧光检测E-cadherin、Vi mentin、Snail的表达;用real ti me PCR检测E-cadherin、Vi mentin、Snail mRNA的表达。结果TGF-β1处理72h后的MCF-7细胞穿透能力明显增强。E-cadherin蛋白及mRNA表达减少,Vi mentin、Snail蛋白及mRNA表达增加。结论E-cadherin、Vi mentin是细胞发生EMT的重要生物学标志,Snail可能在转录水平上调控E-cadherin、Vi mentin蛋白的表达,Snail在EMT和乳腺癌的发生发展中起着重要的作用。     

Alternatively spliced protein arginine methyltransferase 1 isoform PRMT1v2 promotes the survival and invasiveness of breast cancer cells

Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several common cancer types; however, their precise contribution to the cell transformation process is not well understood. We previously reported that the PRMT1 gene generates several alternatively spliced isoforms, and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and subcellular localization. We focus here on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform, and we have found that its relative expression is increased in breast cancer cell lines and tumors. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in an aggressive cancer cell line significantly decreased cell invasion. We also demonstrate that PRMT1v2 overexpression in a non-aggressive cancer cell line was sufficient to render them more invasive. Importantly, this novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. Moreover, this activity requires both proper subcellular localization and methylase activity. Lastly, PRMT1v2 overexpression altered cell morphology and reduced cell-cell adhesion, a phenomenon that we convincingly linked with reduced β-catenin protein expression. Overall, we demonstrate a specific role for PRMT1v2 in breast cancer cell survival and invasion, underscoring the importance of identifying and characterizing the distinct functional differences between PRMT1 isoforms.     

In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy

Animal imaging requires the use of reliable long-term fluorescence methods and technology. The application of confocal imaging to in vivo monitoring of transgene expression within internal organs and tissues has been limited by the accessibility to these sites. We aimed to test the feasibility of fibred confocal fluorescence microscopy (FCFM) to image in situ green fluorescent protein (GFP) in cells of living animals. We used transgenic rabbits expressing the enhanced GFP (eGFP) gene. Detailed tissue architecture and cell morphology were visualised and identified in situ by FCFM. Imaging of vasculature by using FCFM revealed a single blood vessel or vasculature network. We also used non-transgenic female rabbits mated with transgenic males to visualise eGFP expression in extra-foetal membranes and the placenta. Expression of the eGFP gene was confirmed by FCFM. This new imaging technology offers specific characteristics: a way to gain access to organs and tissues in vivo, sensitive detection of fluorescent signals, and cellular observations with rapid acquisition at near real time. It allows an accurate visualisation of tissue anatomical structure and cell morphology. FCFM is a promising technology to study biological processes in the natural physiological environment of living animals.     

Fascin is a key regulator of breast cancer invasion that acts via the modification of metastasis-associated molecules

The actin-bundling protein, fascin, is a member of the cytoskeletal protein family that has restricted expression in specialized normal cells. However, many studies have reported the induction of this protein in various transformed cells including breast cancer cells. While the role of fascin in the regulation of breast cancer cell migration has been previously shown, the underlying molecular mechanism remained poorly defined. We have used variety of immunological and functional assays to study whether fascin regulates breast cancer metastasis-associated molecules. In this report we found a direct relationship between fascin expression in breast cancer patients and; metastasis and shorter disease-free survival. Most importantly, in vitro interference with fascin expression by loss or gain of function demonstrates a central role for this protein in regulating the cell morphology, migration and invasion potential. Our results show that fascin regulation of invasion is mediated via modulating several metastasis-associated genes. We show for the first time that fascin down-regulates the expression and nuclear translocation of a key metastasis suppressor protein known as breast cancer metastasis suppressor-1 (BRMS1). In addition, fascin up-regulates NF-kappa B activity, which is essential for metastasis. Importantly, fascin up-regulates other proteins that are known to be critical for the execution of metastasis such as urokinase-type plasminogen activator (uPA) and the matrix metalloproteases (MMP)-2 and MMP-9. This study demonstrates that fascin expression in breast cancer cells establishes a gene expression profile consistent with metastatic tumors and offers a potential therapeutic intervention in metastatic breast cancer treatment through fascin targeting.     

Automated whole animal bio-imaging assay for human cancer dissemination

A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.     

Mammosphere Formation in Breast Carcinoma Cell Lines Depends upon Expression of E-cadherin

Tumors are heterogeneous at the cellular level where the ability to maintain tumor growth resides in discrete cell populations. Floating sphere-forming assays are broadly used to test stem cell activity in tissues, tumors and cell lines. Spheroids are originated from a small population of cells with stem cell features able to grow in suspension culture and behaving as tumorigenic in mice. We tested the ability of eleven common breast cancer cell lines representing the major breast cancer subtypes to grow as mammospheres, measuring the ability to maintain cell viability upon serial non-adherent passage. Only MCF7, T47D, BT474, MDA-MB-436 and JIMT1 were successfully propagated as long-term mammosphere cultures, measured as the increase in the number of viable cells upon serial non-adherent passages. Other cell lines tested (SKBR3, MDA-MB-231, MDA-MB-468 and MDA-MB-435) formed cell clumps that can be disaggregated mechanically, but cell viability drops dramatically on their second passage. HCC1937 and HCC1569 cells formed typical mammospheres, although they could not be propagated as long-term mammosphere cultures. All the sphere forming lines but MDA-MB-436 express E-cadherin on their surface. Knock down of E-cadherin expression in MCF-7 cells abrogated its ability to grow as mammospheres, while re-expression of E-cadherin in SKBR3 cells allow them to form mammospheres. Therefore, the mammosphere assay is suitable to reveal stem like features in breast cancer cell lines that express E-cadherin.