The Prompting Effects of MT1-MMP on EMT in Breast Cancer

Objective Epithelial to mesenchymal transition (EMT) was the initial process of invasion and metastasis leading to a relapse of breast cancer following resection and chemo-radiotherapy. Membrane type-1 Matrix Metalloproteinase (MT1-MMP) was conrmed to play an important role in EMT in various cancers. However, the MT1-MMP effects on EMT in breast cancer had not yet been studied. Here,We investigated the MT1-MMP effects on breast cancer EMTonset, invasion, and migration abilities in MCF-7 cells. Methods Expressions of MT1-MMP and EMT-associated proteins including E-cadherin, N-cadherin, and Vimentin were detected by immunohistochemistry in 71 breast cancer resection samples. The relationships of MT1-MMP with clinic- pathological parameters were statistically analyzed, as well as EMT-associated proteins. Western blot tests were performed to test MT1-MMP and EMT-associated proteins expression levels in MCF-7 cells transfected by MT1-MMP plasmid. Wound-healing and transwell experiments were used to estimate MT1-MMP-induced invasion and migration. The proteins including and levels were higher both MT1-MMP positive the results of and


Introduction
Breast cancer (BC), a common life-threatening malignancy, has become the leading cause of cancerrelated deaths in women worldwide. Globally, breast cancer accounted for approximately 1.67 million new cases and 0.5 million deaths in 2012 [1]. Despite the early detection and treatment of breast cancer, it remains the second leading cause of cancer-related death in America [2].
Cancer relapse and metastasis are primary reasons for poor survival and prognosis among patients receiving successful resection or chemo-radiotherapy. Epithelial to mesenchymal transition (EMT) is the initial process of cancer progression, during which tumor cells dissociate from the primary tumor to invade into the neighboring tissue, traveling through the blood vessels, and nally forming colonies at a secondary site [3].
EMT has been de ned as a critical step in metastasis progression. Cancer cells can use EMT to initiate invasion and metastasis [4]. Tumor cells, when combined with immuno uorescence staining for mesenchymal markers, have identi ed EMT as a very early event in a pancreatic ductal adenocarcinoma mouse model [5]. EMT activation in human breast cancer cells could also enhance metastatic dissemination [6].
EMT is characterized by the decrease of E-cadherin expression(which is an epithelial protein marker) and the increasement of N-cadherin (the cadherin switch, which is one of the hallmarks of EMT) [7], as well as vimentin (the mesenchymal proteins in the main extracellular matrix) [8].
Membrane type1 Matrix metalloproteinase (MT1-MMP) is the rst discovered membrane-associated Matrix metalloproteinase, which has been con rmed to be extensively associated with many kinds of tumor growths and negative prognosis [9]. Its indirect effect on the EMT and tumorigenesis process transformation was also displayed in cultured endocardial cushion cells and transgenic animals by activation of MMP-2,-3,-8,-13 [10].
Whereas, further research of MT1-MMP on EMT of breast cancer samples was rarely reported, thus MT1-MMP-affected breast cancer progression is still unknown.
In this study, the expressions of MT1-MMP in breast cancer tissues veri ed that MT1-MMP was associated with some breast cancer clinic-pathological parameters. Furthermore, based on the above analysis, MT1-MMP plasmid-transfected breast cancer cells were used to verify that MT1-MMP was involved with the EMT procedure, the progression of breast cancer cell migration, and invasion.

Materials And Methods
Immunohistochemistry: 71 breast cancer cases were observed in the rst a liated hospital of the Shihezi University, school of medicine, Xinjiang, China. The median ages were 43 . All cases were diagnosed as Ductal Breast Cancer by the pathologist. 13 cases were grouped into stage I, 41 into stage II, and 17 into the advanced stage. All cases were grouped according to the UICC 2009 TNM clinical staging standard.
Paired normal breast tissue samples were taken from the sites at more than 3 centimeters (cm) away from the cancerous lesions, and were used as controls. Informed consent was taken from all patients in this study, which was authorized by the institutional ethics committee of the First A liated Hospital of Shihezi University, School of Medicine.
5-micrometer (μm) thick sections of formalin-xed, para n-embedded tissue were used to perform the immunohistochemical staining. Mouse anti-human monoclonal primary antibody (Epitomics, America) at a dilution of 1:300 was used to detect MT1-MMP. Mouse anti-human E-cadherin (1:500; Abcam America), rabbit anti-human monoclonal antibody vimentin (1:400; Abcam, America), and anti-N-cadherin (1:800; Abcam, America) primary antibodies were incubated with slides overnight at 4°C. All positives (in the kit) and negative-controls (PBS instead of the rst antibody) in the experiments were performed to verify the speci city of the immunostaining reaction. The second antibody EnVision kit (Dako, America) (Cat:201503, Lot:10051369) was used for immunohistochemical staining, which was assessed by a semi-quantitative scoring system [11] as follows: In terms of the staining intensity, no staining was recorded as 0 (negative), and positivity required that at least 10% of the tumor cells would be stained. Pale yellow was recorded as 1 (weakly positive), brownishyellow was recorded as 2 (positive), and brown was recorded as 3 (intensive positive). No signi cant age difference was found between the elementary stage group ( + ) and the advanced stage group ( + ). Wound-healing assay: MCF-7 cells transfected with control plasmid and MT1-MMP plasmid were seeded at a density of 1×10 6 /well in 6-well tissue culture plates overnight. Once con uence was reached, 3 parallel lines were drawn on the bottom of the 6-well plates before seeding the cells. Using a sterilizing 100 uL pipette tip, an area vertical to the lines, approximately 5 mm in width, was cleared to create a scratch. Then the cells were maintained in DMEM without fetal bovine serum (FBS) medium for 24 and 48 hours, in which repopulation and closure were subsequently monitored daily under phase contrast microscope 4×objective lens. The closure was quanti ed using Image J 2.0.0 software by measuring the width of the scratch each day and normalizing it to the initial size of the scratch.
Transwell assay in vitro: MT1-MMP and pcDNA 3.1 control vector were transiently transfected into MCF-7 cells (5×10 4 ), which were then allowed to migrate for 24h through Transwell ® cell culture inserts (8-μm pore size, 6.5-mm diameter; Costar, Cambridge, MA). Transwell ® inserts were coated with Matrigel ® , and the lower compartment was lled with 600 μL of DMEM supplemented with 20% fetal bovine serum. After incubation at 37 °C in an incubator for 24 h, lters were rinsed with phosphate-buffered saline, xed with 4% paraformaldehyde (15min at -4 °C), and stained with crystal violet (0.1%; Thermo Fisher Scienti c) for 15min. Cells on the upper surface of the lters were removed with a cotton swab. Cells that migrated through the lters were counted under the microscope at a magni cation of 400×. Each clone was tested in triplicate in at least three independent assays. Data was expressed as mean ± standard error.
Western blot: Cellular proteins were prepared using cell lysis buffer (50 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 2mM ethylenediaminetetraacetic acid, 10mM NaCl, 2mg/mL aprotinin, 5mg/mL leupeptin, 2mg/ml pepstatin, 1mM dithiothreitol, 0.1% sodium dodecyl sulfate, and 1mM phenylmethylsulfonyl uoride). Equal amounts of protein samples (8μg) were separated by polypropylene gel electrophoresis with 5% concentrated gel and 12% separating gel under a stable voltage of 80 for 60 minutes , and then β -actin immunoblots served as loading controls. The membrane was photographic, developed and xed, and an ECL reagent kit (Thermo Systems) was used to detect the bands on the membrane. Finally, the data and images were collected by the Gel Imaging System (Bio-Rad, America) and X-ray exposure. specimens. Strong expression of MT1-MMP was also observed in the nest of in ltrating stroma. (Fig 1 A  and B).
The enhanced expression level of MT1-MMP (positive and strong positive expressions) in the lymph node metastasis group was signi cantly higher than that in the non-lymph node metastasis group (p<0.05, Table 1). It could be seen that the bigger size of tumors were more positively expressed in Breast Cancer. Compared with the clinical stages I and II, the signi cantly higher expressions of MT1-MMP could be seen in the advanced stages of Breast cancer III and IV (p<0.05, Table 1).
Other clinic-pathological factors such as age, estrogen receptor, and progestogen receptor showed no signi cant correlation with the expressions of MT1-MMP in Breast cancer tissues.  Table 2). The membrane expression of Vimentin in breast cancer tissue was more than that in the cytoplasm. A higher positive rate of the Vimentin expression was observed in cancer with a signi cant difference (60.6% in the cancer group, while 38.2% in paracancerous tissue, p<0.05). The level of Vimentin immunohistochemical expressions was strongly related to that of MT1-MMP (χ 2 =6.459, p =0.011).
2. MCF-7 breast cancer cell lines transfected with MT1-MMP plasmid exhibited promoting BC cancer cell migration and invasion in vitro. (Fig. 2). The results showed that the morphology of the cells changed distinctly with controls, presenting elongated, broblast-like cells with less cell-to-cell contact (panel 3).
2.2 Next, MT1-MMP protein expression was estimated by Western blot analyses, which revealed that the protein of MT1-MMP band (66-kDa) was seen in transient transfection MCF-7 cell lines, which was not observed in a control cell line with pcDNA3.1 vector-transfected (see Fig. 3). However, the β -actin band (42-kDa) was detected in both groups of cells. The results showed that MCF-7 with overexpression of MT1-MMP was established successfully, furthermore, the association of EMT and MT1-MMP could be testi ed effectively in vitro.

Discussion
To nd effective diagnostic strategies and targeted therapies, it was essential to understand well the MT1-MMP biology and molecular expressions in breast cancer and to identify the relationship between MT1-MMP and Breast cancer EMT. In this study, we rst examined the MT1-MMP protein expression level through immunohistochemistry in 71 surgically resected breast cancer cases and their paired adjacent normal breast tissue samples, respectively. The results showed that MT1-MMP was strongly expressed in breast cancer tissues compared with their paracancerous tissues, which suggested that the overexpression of MT1-MMP in breast cancer tissues was positively related to the incidences of breast cancer disease. Similar results were found in a microarray analysis in nasopharyngeal carcinoma [12], and a high expression of MT1-MMP was also observed in human lung cancer [13], prostate cancer [14], and glioblastomas [15]. Gigantic strides in animal experiments were also identi ed and related to abnormal MT1-MMP over-expression induced mammary gland abnormalities and adenocarcinoma in transgenic mice [10].
Furthermore, in the analysis of clinical statistical data, it was found that the MT1-MMP expression level was positively correlated with the lymph node metastasis and the breast cancer clinical stages, while no signi cant expression differences were found on other clinical parameters such as age, PR, and ER in breast cancer cases, which indicated that MT1-MMP played a promotional role in breast cancer clinical metastasis and progression.
EMT has been considered as a favored explanation of how tumor cells leave the primary tumor site, disseminate through the body, and eventually form distant metastases. In the multiple-step metastasis, the function of EMT was attributed to the initial events [16]. Similarly, clinical statistics data con rmed that EMT had a strong association with distant metastasis in patients younger than 40 years old and in lymphovascular invasion patients [17]. Previous studies also revealed that over-expression of MT1-MMP in carcinoma tissues up-regulated expression of EMT-associated genes including N-cadherin, vimentin, snail, and slug in nasopharyngeal carcinoma [12], gastric cancer [18], and esophageal squamous cell carcinoma [19].
Many studies signi ed that EMT was popularly accepted as an initial process of cancer procession, i.e. gaining the migratory and invasive properties and allowing cancer cells to leave the primary tumor, invading into blood vessels [20] [21]. The process was characterized by epithelial cells losing their apicobasal polarity with delocalization of tight and adherens junction proteins (decreasing epithelial proteins of E-cadherin [22]) but acquiring a spindle-shaped mesenchymal-like morphology with an upregulated expression of mesenchymal markers, N-cadherin [23] and Vimentin [24].
To identify that MT1-MMP promotes the invasion and metastasis of breast cancer, we did further experiments in vitro to explore the role of MT1-MMP in breast cancer cell migration and invasion. Compared with the empty vector-transfected group ( g.3, panel3), MCF-7 breast cancer cells transfected with MT1-MMP-GFP pcDNA3.1 plasmid were found to obtain morphological mesenchymal changes characterized by the spindle-shaped mesenchymal-like structure. Wound healing assay and transwell experiment results ( g.4 and g.5) also showed that constitutive activation of MT1-MMP enhanced the breast cancer cell migration and invasion potential in vitro. It seemed that the MT1-MMP expressed in breast cancer cell membrane had positive effects on cancer EMT and invasion ability in this experimental data.
Interestingly Collectively, our experimental data demonstrated that MT1-MMP was over-expressed in breast cancer tissue, and MT1-MMP played a critically positive role in breast cancer EMT through the mechanism of repressing E-cadherin and enhancing N-cadherin and Vimentin expressions, whilst MT1-MMP also prompted breast cancer metastasis and migration. Therefore, MT1-MMP was promised to become a potential candidate as the diagnostic or therapeutic measure in breast cancer disease.

Declarations Funding
The study was supported by the researh project from Shiheizi Universitiy in China, project number: ZZZC201717A.

Con ict of interest/Competing interests
All authors declare no con ict of interests, including employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding except the funding declared.
Availability of data and material (data transparency) All authors declare that the data transparency were con rmed.
Code availability (software application or custom code) software application were available.

Authors' contributions
All authors contributed to the study conception and design, more contributions from Cao weiwei. Material preparation, data collection and analysis were performed by He Jianwei, Zhang Hongmei and Zhang Licui.The rst draft of the manuscript was written by Cao weiwei and Inayat Azeem. All authors commented on previous versions of the manuscript.and all authors read and approved the nal manuscript.

Ethics approval
The retrospective protocol of this study was approved by the