The Study Of IMP3Inducing Epithelial-mesenchymal Transition And Multidrug Resistance In Breast Cancer

[Background]Breast cancer is one of the most common cancers in women with greater than1,300,000cases and450,000deaths each year worldwide. Clinically, breast cancer is a heterogeneous disease, and is composed of a variety of different types, each type has its special shape, immunohistochemical characteristics and the corresponding clinical behavior. The occurrence and development of breast cancer is a complex process, involving a variety of transcription factors, proto-oncogenes activation and inactivation of tumor suppressor genes.Distant metastasis is the main reason for the high mortality rate of breast cancer, the5-year survival rate for patients with metastasis breast cancer is below30%. Although malignant progression has been the subject of scrutiny for decades, it remains poorly understood owing to the complexity of the process and the difficulty of modeling it in the laboratory.Accumulating evidence from both animal tumor models and human cancers has established the notion that the progression of an epithelium-derived tumor into an invasive phenotype involves fibroblast-like transition, the event referred to as epithelial-mesenchymal transition (EMT). During this transition, tumor cells lose epithelial characteristics that include cell apical-basal polarity, membrane-associated adherents, and the cell-cell adhesion protein E-cadherin. Concurrently, they undergo a dramatic remodeling of the cytoskeleton to facilitate cell mortality and invasion, and also transform to obtain a spindle-like phenotype. Through EMT, tumor cells exhibit the invasive ability to detach from their origin, invade host stroma, escape via vasculature and/or lymphatics, and eventually survive and regrow in a distant region, a process termed tumor metastasis.Slug, also known as SNAI2, is a member of the Snail-related family. As a zinc-finger transcription factor, it can recognize the CAGGTG sequence. The Snail family has been linked with mesoderm and neural-crest formation, cell division and endoreduplication, cell adhesion, invasion and the regulation of cell cycle and apoptosis. In addition to participate in the normal development process, they were also found to participate in the epithelial mesenchymal transition in multiple tumors. As a main regulatory factor, Slug can influence the expression of a series of genes, including in the cell adhesion process. It can repress the expression of E-cadherin and play an important role in the process of tumor growth and metastasis.Drug resistance is an important reason for limiting the effectiveness of chemotherapy in breast cancer. Research found that many tumors can not only resistant to one drug, but also resistant to a wide spectrum of anti-cancer agents with different structures or different target sites, this phenomenon is called "multidrug resistant". P-glycoprotein (P-gp), which is encoded by the multidrug resistance gene1(MDR1), plays an important role in the primary drug resistance in breast cancer. And studies have found patients undergoing drug treatment could have the upregulation of the expression of MDRl/P-gp, which inducing higher risk of failure of chemotherapy. Thus regulating the expression of MDRl/P-gp is the key to reverse multidrug resistance and improve the effect of treatment of breast cancerIMP3is a member of the human insulin-like growth factor Ⅱ (IGF-Ⅱ) mRNA binding protein (IMP) family. They have important roles in RNA trafficking and stabilization, cell growth, and cell migration during the early stages of embryogenesis. IMP3expressed in a variety of malignant tumors, but in adjacent benign or normal tissues showed lower expression or no expression. Recent study has found that IMP3protein promotes chemoresistance in breast cancer cells by regulating breast cancer resistance protein (BCRP) expression. IMP3is an oncofetal protein, and plays an important role in tumor proliferation, invasion, metastasis and drug resistance. The study found IMP3associated with tumor invasion and metastasis, but the relationship between the IMP3expression and EMT leaves unclear. Therefore, in this study, we designed IMP3overexpression or interference, to detect the expression of E-cadherin, Vimentin and Slug, explore its role in the regulation of epithelial-mesenchymal transition thereby affecting the invasion and metastasis of breast cancer. We preliminarily analyzed the relationship and possible regulatory mechanism between IMP3and MDR/P-gp.[Methods]1.180paraffin-embedded breast tissue samples were diagnosed as invasive ductal carcinoma at the Department of Pathology of Qilu Hospital, Shandong University, from2007to2009. We used a tissue microarray punching instrument to punch out the selected areas, which were placed into the recipient block side by side. The immunohistochemical method to study altered protein expression was performed. All the markers were incubated with the sections overnight at4℃, including IMP3, E-cadherin, Vimentin and Slug. For negative controls, the Ab were replaced with PBS.2. The siRNA sequence targeted to IMP3or Slug were designed. IMP3expression vector pEGFP-C1-IMP3was a gift from Dr. Yung-Ming Jeng, Department of Pathology, National Taiwan University Hospital. The human Slug cloning vector was obtained and the target gene was subcloned into a mammalian expression vector pcDNA3.1(+), after comfirmed by enzyme digestion and DNA sequencing, the plasmids were amplified.3. MDA-MB-231cells were transfected with IMP3-specific siRNA (231-siRNA-IMP3) with target sequence GCUGGAGCUUCAAUUAAGATT. We also established negative control (231-nc) and mock (231-mock) transfected cells. T47D cells were transfected with the pEGFP-C1-IMP3vector (T47D-IMP3). The empty pEGFP-C1vector served as a control (T47D-vector). After another48h culture, qRT-PCR was adopted to detected the expression of IMP3, E-cadherin, Vimentin and Slug. Western blot was used to analysis protein expression. Selected clones produced by the transient transfections of IMP3-siRNA to the MDA-MB-231line and IMP3expression vector in the T47D line were studied morphologically by phase contrast microscopy. Assays were performed using transwell chambers for migration and invasion, respectively. The interaction between IMP3protein and Slug mRNA was determined using a ribo-immunoprecipitation qPCR assay.4.231-siRNA-IMP3cells transfected with Slug vectors are referred as231-siRNA-IMP3/vector and231-siRNA-IMP3/Slug, and T47D-IMP3cells transfected with Slug-specific siRNA are referred as T47D-IMP3/nc and T47D-IMP3/siRNA-Slug. After another48h culture, qRT-PCR and western blot were adopted to detected the expression of IMP3, E-cadherin, Vimentin and Slug. Morphology changes were studied by phase contrast microscopy. Migration and invasion assays were performed using transwell chambers.5. The ADM cells were transfected with siRNA-IMP3and negative control respectively. After another48h culture, qRT-PCR was adopted to detected the expression of IMP3and MDR1mRNA, Western blot was used to analysis the IMP3and P-gp protein expression. Rhl23efflux assays were performed to detecte the function of P-gp. MTS assay was used to assess the effect of IMP3silencing on the chemosensitivity of ADM cells to4anticancer drugs, doxorubicin, paclitaxel, cisplatin, and5-Fu.[Results]1. In the analysis of a180-member tissue microarray (TMA), IMP3expression was seen in23cases (12.8%);157tumors (87.2%) did not express IMP3. To explore the correlation, we stained for the expression of three genes (E-cadherin, vimentin, and Slug) which have tight relation with EMT. E-cadherin belonged to markers of epithelial tissues. Vimentin and Slug were always treated as markers of mesenchymal tissues. The immunohistochemical expression of IMP3inversely correlated with E-cadherin (P=0.042), which was further confirmed by Spearman correlation analysis (r=-0.163, P=0.029). IMP3expression directly correlated with both Slug (P=0.004) and vimentin (P<0.001), and its Spearman correlations were0.27(P<0.001) and0.366(P<0.001), respectively.2. With overexpression of IMP3, the expression of E-cadherin was reduced (P<0.05) accompanied with the up-regulation of Slug and vimentin (P<0.05) in transfected T47D cells; in contrast, with knockdown of IMP3, a dramatic decrease came up in Slug and vimentin expression (P<0.05) and turned up a significant increase in E-cadherin expression (P<0.05). T47D-IMP3cells exhibited a slight alteration in morphology, growing in elongated individual cells rather than tight aggregates. The231-siRNA-IMP3cells grew in more tightly-organized rather than irregular sheets. In Transwell assay, the MDA-MB-231line was highly invasive, but it exhibited reduced invasion and migration after transfection than that in the control group (P<0.05). T47D cells were poorly invasive, but it showed moderately high invasion and migration after transfection (P<0.05)3. Ribo-immunoprecipitation qPCR revealed that IMP3binds Slug mRNA directly. To further determine whether IMP3exerts its function through regulation of Slug, we performed gain-of-function analyses by overexpressing Slug in231-siRNA-IMP3cells and loss-of-function analyses by knocking down Slug in T47D-IMP3cells. As expected, overexpression of Slug rescued the morphological change, cell migration and invasion caused by silencing IMP3in MDA-MB-231cells. The upregulation of vimentin and the downregulation of E-cadherin were also observed. On the other hand, knockdown of Slug in T47D-IMP3cells could also have the opposite change in EMT, morphology, migration and invasion. Taken together, these results suggest that Slug is a functional target of IMP3.4. The expression of MDR1and IMP3mRNA was down-regulated remarkably in comparison to the control. Western blot analyses showed that IMP3and MDRl/P-gp were down-regulated in response to IMP3interference. The P-gp efflux function was measured by flow cytometry. The fluorescent retention in IMP3-silenced ADM cells obviously exceeded that of the control, indicating that the efflux function of biologically active MDR1/P-gp decreased after IMP3knock-down. MTS assay showed that IC50values of the four anti-cancer drugs all decreased remarkably after IMP3interference.[Conclusions]1. There was an inverse correlation of IMP3with E-cadherin protein expression and directly correlated with both Slug and vimentin. The overexpression of IMP3therefore contributed to the EMT in breast cancer progression.2. IMP3could bind to Slug mRNA directly. Overexpression or knockdown of Slug could rescue IMP3-induced alterations in morphology, migration, and invasion. In this study, we confirmed that Slug was a novel, direct and functional target of IMP3.3. IMP3was overexpressed in ADM cells, consistent with MDRl/P-gp expression. The silencing of IMP3decreased MDR1expression and reversed the multidrug resistance in ADM cells.4. IMP3appeared to mediate migration and invasion by regulating the Slug-E-cadherin-EMT pathway, and is possible to be identified as a new molecular marker to predict metastasis and target for multidrug resistance reversing in breast cancer cells.