Expression Of Extracellular Matrix Metalloproteinase Inducer (CD147) And Phosphorylated Activated Extracellular Signal-regulated Kinase (p-ERK) In Invasive Ductal Carcinoma Of Human Breast

[Background] Breast carcinoma is one of the most common female malignancies. Invasion and metastasis of tumor cells is a multi-step process, during which malignant cells present pathophysiological transformations such as basilemma degradation, diosmosis, tumor move through vascellum and metastasis of target tissue. This induces remodeling of basilemma and extracellular amatrix mediated by proteolytic enzymes .CD147 is extracellular matrix metalloproteinase inducer (EMMPRIN), it sitimulates fibroblast to secrete matrix metalloproteinase to degrade extracellular matrix and promote invasion and metastasis of tumor cells. CD 147 is involved in three steps such as adherency, degradation and movement proposed by Liotta. Thus, more researchers pay attention to its role of tumour cells invasion and metastasis.MMP-9 is one of MMPs family, belonging to gelatinase.secrets as proenzyme in vivo. When it is activated by many factors, MMP-9 may degradate collagen IV,V,VII,X and gelatin. So MMP-9 is closely correlated to invasion and metastasis of tumor cells .p-ERK is, phosphor-ERK, an activated form. The contribution of ERK in cell proliferation mediated through growth factor have been generally accepted. Growth factor cytokine hormone signal mediated by caryocinesia and differentiation can activate ERK. p-ERK regulates generation cycle, cell proliferation and differentiation, but it resists apoptosis. Phosphor-ERK in active form, is transfered to cellular nucleus and initiates oncogene transcription up-regulation and leads to cellular malignant transformation. In vitro and animal experiment, over activated p-ERK is significantly correlated to tumorigenesis and metastasis in mouse. Several investigations indicate that GD147 may stimulates fibroblast to secret MMPs via MAPK(including of ERK P38 JNK/SAPK, BMK) signal transduction pathway,the relationship of CD 147 to MAPK molecule level in human breast carcinoma is unclear yet.[objects] The expression of CD147 ,MMP-9 and p-ERK protein in IDC and DCIS of breast cancer wasinvestigatedto expore the relationship of invasion and metastatic of breast cancer to CD147 ,MMP-9 and p-ERK expression.[Materials] All samples were selected from the pathology department of the Second Affiliated Hospital, College of Medical Science in Zhejiang University.96 cases of invasive ductal carcinoma (DCIS) and 34 cases of ductal carcinoma in situ (DCIS) were obtained from excision biopsy procedures or mastectomies. None of the patients with invasive breast cancer had received any preoperative therapy. They were female, aging from 28 to 84 years.mean age being 53.5 years. All specimens were fixed in neutral 4% buffered formaldehyde. We reexamined ductal carcinoma in situ and invasive ductal carcinoma by two pathologists. Grading of invasive cancer was done according to the procedure of Elston and Ellis.[Immunohistochemistry]Immunohistochemistry Envision two-step method was used to investigate the expression of CD147.MMP-9 and p-ERK protein in 96 cases of invasive ductal carcinoma and 34 cases of ductal carcinoma in situ of breast. Tissue sections were 4 um in thickness and were deparaffined in xylene. Antigen unmasking were performed by boiling tissue sections in 0.01 M citrate buffer, pH 6.0 for 20 min followed by cooling at room temperature 20 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min. CD147 (ZA-0455, Beijing ZhongShan Biotechnology CO.LTD), MMP-9(M-0393,Maixin Biotechnology) and p-ERK (Sc7383 , Santa Cruz Biotechnology) were incubated overnight at 4^Cin a dilution of 1:100 ,l:50and 1:200 . Between each steps washed in PBS three times for 5 minutes. Antibody binding was visualized by the DAB method. The primary antibody was replaced by PBS as a negative control.[Quantification] CD 147 was mostly located in the membrane and cytoplasm of breast carcinoma cell, some staining in fibroblast;MMP-9 staining in cytoplasm;p-ERK staining showing in nuclear or/and cytoplasm. The immunohistochemical results for CD147and MMP-9 proteins were classified as follows: -, no staining;+, positive cells percentage less than 10%;++, positive cells percentage in 10-50%;+++, positive cells percentage more than 50%. Tissue sections stained with p-ERK were quantitated subjectively using the folowing scale 0:none;+:positive cells percentage less thanl0%;++:positive cells percentage more than 10%.(Statistical analysis] The data analyses were performed using SPSS for Windows 10.0 with a significant level of P < 0 .05 .we performed X2 test and Spearman correlate analysis.[Results] The positive rates of CD147, MMP-9 and p-ERK were 59.4% ,54.2% and 52.l%in IDC of breast, respectively,which were significantly higher than those in DCIS of breast (35.3% ,32.6% ,29.4%)and breast adenosis tissue(0%). CD 147 and MMP-9 protein expressions were closely related to tumor size (r=0.336;0.306, PO.05) ,TNM stage (r=0.384;0.308, PO.01) , histological grading (r=0.272;0.323, P<0.05) , lymph node metastasis (r=0.276;0.251, PO.05) and 5-year survival rate (r=r0.225;0.251, PO.05), unrelated to patients' age. p-ERK protein expressions was only significently related to histological grading(r=0.301, PO.05). CD147expression was correlated with MMP-9(r=0.261, PO.05), but p-ERK expression showed no relationship of CD147 to MMP-9 (r=0.013;0.122, P>0.05) .{Conclusions] (1) The positive rates of CD 147and MMP-9 protein in IDC were significantly higher than those in DCIS, and they were also higher than those in breast adenosis tissue. The difference of expressive rates between IDC and DCIS was statistically significant. (2) The enhanced expression of CD 147 and MMP-9 in IDC was associated with TNM stage, the tumor size, histological grading, lymph node metastasis and 5-year survival rate. (3) The enhanced expression of p-ERK was associated with the increase of the grade of breast cancer. The difference of expressive rates p-ERK between in IDC and DCIS was statistically significant. (4) CD 147 expression was correlated with MMP-9, but p-ERK expression showed no relationship to CD147 or MMP-9. Thus, our data suggested that tumor-derived CD147 stimulates breast carcinoma cells to secret MMP-9 , it doesn't stimulate fibroblast to secret MMP-9 via ERK signaling.