| Objective:Breast Carcinoma is the leading fetal cancer that affects the health of women now. With the development of economy and the improve- ment of peoples'life, the mortality of breast carcinoma is going up. Breast carcinoma recurrence and metastasis are the main death causes of these patients. However, the mechanism of occurrence and development remains obscure. Patients suffering from breast cancer have a poor prognosis because of deficiency in effective treatment measures in clinical practices.EGFR family encompasses members of erbB-1/EGFR, erbB-2/ HER2/ neu, erbB-3 and erbB-4. They belong to tyrosine kinase receptor which can be activated by tyrosine phosphorylation. HER2 is unactived oncogene in normal correlating with regulating to growth and differentiation. Amplification and overexpression of HER2 have been examined in a deep-going way and associated with invasion and diffusion. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, Nineteen percent of breast carcinoma which has no EGFR, ER and PR, is prone to recurrence and metastasis. Total survival rate is lower than that of other breast carcino ma.These patients have worse prognisis. But effective targeted therapy has not been emerged now. Studies have shown that overexpression of EGFR exists in this kind of breast carcinoma whose phenotype is significantly correlated with EGFR and lymphnode metastasis.EGFR distributes in the way of transmembrane controlling proliferation, differentiation, motility and survival. Appropriate amount expression in normal tissue is necessary for normal vital movement.Overexpression or strong activity of EGFR can promote proliferation system. The excessive proliferation and malignant phenotype will be emerged. Overexpression of EGFR has been in 35%～50 % breast carcinoma and correlates with prognosis. The expression of EGFR in metastasis is significantly higher than that of non-metastasis one. No recurrence and high survival rate result from negative expression of EGFR. Tyrosine phosphorylation is the key molecular event in the EGFR signaling pathway. The level of EGFR phosphorylation decides the prognosis of breast carcinoma. Higher phosphorylation of EGFR will lead to poor prognosis. However, the mechanisms accounting for the upregulation of EGFR activity are largely unknown. So searching the factors that affect EGFR activity is the key purpose and provides foundation for new target in tumor.Filamins A (Filamin-1, FLNa) are large actin-binding and scaffolding proteins expressed widely in cytoplasm. FLNa can distribute in the way of transmembrane or localize to the nucleus. Vertebrate filamins are elongated dimeric V-shaped proteins with two large polypeptide chains. Each monomeric chain of filamins consists of an N-terminal actin-binding domain (ABD), a long rod-like domain of twenty-four repeated and two hinges. FLNa is endued with powerful functions because of special structure. FLNa stabilizes cortical three-dimensional F-actin networks, anchors cytoskeleton to cellular membranes by binding to transmembrane receptors and integrate cell architecture and functions signaling, which is essential for cell motility correlating with proliferation, migration, organ development and potential role in oncogenesis;Acting as cross-linking proteins, Filamins not only link various signaling proteins to the cytoskeleton,but also collect costimulus molecule of cytomembrane. So they can take part in many important signaling transduction and make cell response to external stimulus in time;Overexpression of FLNa has been confirmed in many malignancy tumors. It correlates with migration, adhesion and invasion in lung and prostate carcinoma. High expression of FLNa exists in breast carcinoma, too. The expression of FLNa correlating with the malignancy degree and metastasis has been proved with immunohistochemistry and Flow cytometry. Fiori and Zhu et al have shown that FLNa can regulate the activity of EGFR in melanoma cells and the expressions of FLNa and EGFR in melanoma with highly metastatic potentiality are higher than that of lower one. Up to now, FLNa influencing EGFR phosphorylation or not has not been reported.Thus, the phosphorylation level of EGFR and correlated signaling molecules in breast carcinoma cells were observed after FLNa siRNA and full-lengh FLNa transfection, then the relationship between FLNa and EGFR phosphorylation as well as the effection of FLNa on proliferation were detected by Immunoprecipitation and MTT, respectively. Analysis was performed aggregately about the role of FLNa regulating to EGFR plosphorylation in breast carcinoma genesis from the levels of tissue, cell and molecule. In this study, the putative connection between FLNa and EGFR plosphorylation was explored and would provide a new target for effective treatment of breast carcinoma.Part one The expression of filaminA in invasive breast carcinoma and its relationship with clinicopathological featuresMethods:Streptavidin-biotin-peroxidase (SP) Immunohistochemistry and Flow Cytometry were applied to detect the expression of FLNa and PCNA in 46 cases invasive breast cancer with different differentiation, using normal breast tissue or benign breast hyperplasia as negative control.Results:1. FLNa expression in breast cancer cells1.1 FLNa expression with immunohistochemical detection in breast cancer cells:FLNa expression was mainly distributed in the cytoplasm of mammary epithelial cells, staining shallow was regarded as negative in normal breast tissue (Fig.1-1); however, FLNa positive expression was gradually increased with the reducing of differentiation in invasive breast cancer. There were significant differences between the results of different differentiations (P <0.05); FLNa expression in the metastasis group was significantly stronger than that of non-metastasis one, the differences between them was statistically significant (P <0.05). FLNa has nothing to do with the histologic types in invasive breast cancer cells. (Fig.1-2～4 and Table1-1)1.2 FLNa expression with Flow cytometry in breast cancer cells:There was little expression of FLNa in normal breast tissue. It was gradually increasing with the reducing of differentiation. The highest expres- sion was in poorly differentiated breast cancer. The expression of FLNa in poorly differentiated breast carcinomas (1.22±0.13) was higher than that of well differentiated ones (1.10±0.11); Metastasis group (1.21±0.11) was higher than that of non-metastasis group (1.11±0.10), too. It has nothing to do with the histological types. There was a statistical difference between two groups (P <0.05). (Fig.1-6 and Table1-2)2. PCNA expression in breast cancer cells2.1 The results of PCNA with immunohistochemical detection in breast cancer cells:The positive expression of PCNA in nucleus of breast cancer cell was very similar to the expression tendency of FLNa. PCNA expression had also changed with the differentiation. PCNA expression showed strong positive expression in poorly differentiated invasive breast cancer. (Fig.1-5)2.2 The results of PCNA with Flow Cytometry in breast cancer cellsThe expression of PCNA increased with differentiation decreased. The expression of PCNA in poorly differentiated breast carcinoma (1.65±0.22) was significantly higher than that of well-differentiated ones (1.35±0.15); Metastasis group (1.56±0.16) was higher than that of non-metastasis group (1.42±0.15), too. There was a statistical difference between two groups (P <0.05). (Fig.1-7 and Table 1-3). The expression of PCNA was correlated positively with that of FLNa (P <0.05).Part two Effect of FLNa on the EGFR phosphorylation in breast carcinoma Methods:1. Effect of FLNa silence on EGFR phosphorylation in Human breast carcinoma.Human breast carcinoma MDA-MB-231 cells were cultured at 37℃, 5%CO2 in DMEM media containing 10% fetal calf serum and divided into two groups: FLNa-siRNA group and HK group. Each group was stimulated for 0min, 5min and 10min by 20nM EGF. The expression levels of FLNa, EGFR, p-EGFR, ERK and p-ERK were assessed by western-blot at the end.2. Effect of FLNa over expression on EGFR phosphorylation in Human breast carcinoma.MDA-MB-231 cells were cultured and divided into two groups: FLNa-full group and pcDNA3.1 group. Each group was stimulated for 0min, 5min and 10min by 20nM EGF. The expression levels of FLNa, EGFR, p-EGFR, ERK and p-ERK were examined by western-blot at last.3. FLNa regulating to EGFR phosphorylation was demonstrated by co- immunoprecipitation in human breast carcinoma.MDA-MB-231 cells were cultured and divided into two groups: FLNa-siRNA group and HK group.Each group was stimulated for 0min and 10min by 20nM EGF. Then 4G10 or EGFR protein immuneprecipitation was performed. The expressions levels of EGFR and p-EGFR were assessed by western-blot at the end.Results:1. Effect of FLNa silence on EGFR phosphorylation in Human breast carcinoma.(1)The expression of FLNaAfter FLNa-siRNA transfection,the expressions of FLNa in FLNa- siRNA group (1.40±0.10, 1.27±0.06) were lower than that of control group of HK. (3.97±0.29, 3.33±0.06). There was a statistical difference between two groups(P<0.01).(Fig. 2-1, Table 2-1)(2)The expression of phosphorylated EGFRAfter stimulation of EGF at 5min and 10min, the expressions of phosphorylated EGFR in FLNa-siRNA group (0.73±0.01, 0.37±0.05) were lower than that of control group of HK (1.08±0.01, 1.30±0.06). There was statistical difference between two groups(P<0.01). (Fig. 2-2, Table 2-2)(3) The expression of phosphorylated ERK After stimulation of EGF at 5min and 10min, the expressions of phosphorylated ERK in FLNa-siRNA group (0.17±0.00, 0.12±0.04) were lower than that of control group of HK (0.32±0.02, 0.54±0.04). There was a statistical difference between two groups(P<0.01). (Fig. 2-3, Table 2-3).2. Effect of FLNa over expression on EGFR phosphorylation in Human breast carcinoma.(1)The expression of FLNaAfter FLNa-full transfection, the expressions of FLNa in FLNa-full group (5.93±0.38, 6.07±0.32) were higher than that of control group of pcDNA3.1 (4.50±0.20, 4.60±0.17). There was a statistical difference between two groups (P<0.01). (Fig. 2-4, Table 2-4).(2)The expression of phosphorylated EGFRAfter stimulation of EGF at 5min and 10min, the expressions of phosphorylated EGFR in FLNa-full group ( 1.53±0.11,0.86±0.14) were higher than that of control group of pcDNA3.1(0.57±0.01, 0.44±0.02).There was statistical difference between two groups(P<0.01, P<0.05). (Fig. 2-5, Table 2-5).(3) The expression of phosphorylated ERK after FLNa-full transfectionAfter stimulation of EGF at 5min and 10min, the expressions of phosphorylated ERK in FLNa-full group (1.83±0.07, 1.06±0.07) were higher than that of control group of pcDNA3.1 (0.74±0.01, 0.59±0.03). There was statistical difference between two groups(P<0.01). (Fig. 2-6, Table 2-6). 3. FLNa regulating to EGFR phosphorylation was proved by Immunoprecipi- tation in human breast carcinoma.3.1The expressions of FLNa with Western blot detection after FLNa-siRNA transfectionAfter FLNa-siRNA transfection,the expressions of FLNa in FLNa- siRNA group (3.53±1.08,4.33±1.33) were lower than that of control group of HK (7.23±2.20,7.53±1.86). There was statistical difference between two groups(P<0.01). (Fig. 2-7, Table 2-7)3.2 The expression of phosphorylated EGFR after EGFR IP: After stimulation of EGF at 10min,the expression of phosphorylated EGFR in FLNa-siRNA group (1.09±0.09) was lower than that of control group of HK (2.32±0.22).There was statistical difference between two groups(P<0.01). (Fig. 2-8, Table 2-8)3.3 The expression of EGFR after 4G10 IP:After stimulation of EGF at 10min,the expression of EGFR in FLNa- siRNA group(0.42±0.01) was lower than that of control group of HK (1.07±0.01). There was statistical difference between two groups(P<0.01). (Fig. 2-9, Table 2-9)Part Three Effect of FLNa on proliferation of breast carcinoma Methods:MDA-MB-231 cells were cultured and transferred cells into 96 holes plate. MDA-MB-231 cells were divided into two groups: FLNa-siRNA and HK-siRNA transfection group;FLNa-full and pcDNA3.1 plasmid transfection group.EGF stimulating was done with concentration of 4nM, 20nM and 100nM on the two groups overnight. The MDA-MB-231 cells proliferation was examined by MTT after FLNa-siRNA and FLNa-full plasmid trans- fection.Results:1. MTT detection after FLNa-siRNA transfectionUnder the EGF stimulation with concentration of 4nM, 20nM and 100nM, the proliferation level of FLNa-siRNA group (1.28±0.09, 1.46±0.02, 1.58±0.06 was lower than that of control group of HK(1.55±0.12, 1.62±0.06, 1.68±0.12).There was statistical difference between two groups (P<0.01).The proliferation level of two groups was increasing following concentration of EGF.2. MTT detection after FLNa-full transfectionUnder the EGF stimulation with concentration of 4nM, 20nM and 100nM, the proliferation level of FLNa-full group (1.14±0.08, 1.28±0.11,1.51±0.03) were higher than that of control group of pcDNA3.1 (1.09±0.17, 1.21±0.17, 1.25±0.05). There was statistical difference between two groups(P<0.01). The proliferation level of two groups was rising following concentration of EGF.Conclusion:1. The expression of FLNa is little in normal breast tissue. It is gradually increasing with the reducing of differentiation and correlated with lymphnode metastasis in breast carcinoma. The expression of FLNa is correlated positive- ly with proliferation of breast carcinoma.2. The expression of FLNa can regulate EGFR phosphorylation which active the molecule of ERK through MAPK signaling pathway and effect the proliferation of breast carcinoma.3. FLNa silence can lead to the proliferation level of breast carcinoma decreasing, while the overexpression of FLNa can lift the the proliferation level of breast carcinoma.The results show that FLNa can regulate phos- phorylation of EGFR and effect genesis and development of breast carcinoma. |
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