The Effect Of Integrin β1-Rac1 On The Polarity Reversal Of Breast Invasive Micropapillary Carcinoma

Objective Invasive micropapillary carcinoma(IMPC)is a type of high metastatic malignant tumor.Polarity reversal growth pattern with its cluster growth,invasive and metastasis may be the key factor of its highly invasion and metastasis.But the mechanism of polarity reversal was still unclear.Our previous studies have shown that IMPC tumor cells exhibit cluster growth,invasive and metastasis pattern.Fu et al reported that IMPC tumor cells have a large number of microvilli on their cell memberanes,lining the outer surfaces of the cell clusters.In addition,abundant filaments were identified in the cytoplasm.Meanwhile tight junctions were identified between cells.Immunohistochemistry test showed that E-cadherin(E-cad)was present on the intercellular contact surface of tumor cells but not on the outer membranous surfaces of IMPC cells.It is reported that Integrin β1 plays an important role in the formation of the lumen and maintains the polarity of epithelial cell.Rac1 is an integrin regulated switch that has a central role in a variety of actin dependent processe.Rac1 has been linked to the induction of epithelial polarity in cells adhering to extracellular matrix.But the expression and regulatory relationship of Integrin β1 and Rac1 in IMPC have not been reported.Here we examined the expression of Integrin β1,Rac1 and their regulation effect on polarity at the level of cytology.And we further verified the results by clinical data.In order to discuss the possible mechanism of polarity reversal of IMPC.Methods 1)We scilenced Integrin β1 and Rac1 expression in MCF-10 A cells using si RNA-Integrin β1 and si RNA-Rac1 respectively.We use si RNA-ctrl as negative control.We examined the interference effect of si RNA-Integrin β1 and si RNA-Rac1 in MCF-10 A cells at both the m RNA and protein levels by Realtime PCR and Western blot.2)To verify whether polarity changes were specifically induced by Integrin β1,we planted MCF-10 A cells in collagen gel for 3D culture.And then down-regulated Integrin β1 expression using si RNA.3)Rhodamine-conjugated affinipure goat anti-rabbit Ig G was used to mark MUC-1(red).Nuclei were counterstained with DAPI.We used si RNA-ctrl as negative control.We use immunofluorescence to detect cell polarity.4)To further explore whether there is a regulatory relationship between Integrin β1 and Rac1,we treated breast cancer cell lines and IMPC primary tumor cells with AIIB2,which is a kind of Integrin β1 inhibitor that has been well used in many studies.We treated MCF-7,MDA-MB-231 as well as IMPC and IDC-NOS primary tumor cells with AIIB2(7.5μg/ml)when 3D cell culture.The hole without AIIB2 was as control.After 72 hours,the expression of Rac1 in the two groups were tested by western blot respectively.5)We planted IMPC primary tumor cells in collagen gel for 3D culture.And then treated the cell clusters with AIIB2.Rhodamine-conjugated affinipure goat anti-rabbit Ig G was used to mark MUC-1(red),Fluorescein-conjugated affinipure goat anti-mouse Ig G was used to label E-cadherin(green).Nuclei were counterstained with DAPI.We use immunofluorescence to detect cell polarity.6)We performed immunohistochemical analysis of Integrin β1 and Rac1 in 102 cases of breast IMPC and 100 cases of IDC-NOS.The expression of these two proteins was analyzed by combining with clinicopathological characteristics and prognosis of the patients.Results 1)Integrin β1 m RNA was significantly decreased in MCF-10 A cells which were transfected with si RNA-Integrin β1 compared with control cells.And Rac1 m RNA was also significantly decreased in MCF-10 A cells which were transfected with si RNA-Rac1 compared with control cells.2)After treated with si RNA-Integrin β1,both of Integrin β1 and Rac1 were decreased.When transfected MCF-10 A cells with si RNA-Rac1,only Rac1 expression was downregulated,but there was no significant variation of Integrin β1 expression.3)The cell polarity was changed in MCF-10 A cell clusters after silenced Integrin β1 and Rac1.We further found that after treated with Integrin β1 inhibitor AIIB2,the expression of Rac1 was down regulated and the polarity was also changed in IMPC primary cell clusters.4)Clinical analysis of 102 cases of breast IMPC and 100 cases of IDC-NOS showed that the expression of Integrin β1 and Rac1 were both upregulated in IMPC comparing to that in IDC-NOS.And the high expression of these two proteins was positively correlated with polarity reversal and LNM.5)Even in the IDC-NOS group,the levels of LNM and lymph nodes grade in Integrin β1 and Rac1 weakly positive expressed groups were significantly higher than those in the negative expression group.6)Kaplan-Meier survival curves showed that the DFS of patients with high expression of Integrin β1,Rac1 and polarity reversal was significantly shorter than low expression of Integrin β1,Rac1 expression and no polarity reversal patients in IMPC group.The DFS of Integrin β1 and Rac1 double high expression group was shorter than that of the single high group.7)Univariate and correlation analysis showed that the expression of Integrin β1,Rac1 was correlated with tumor cell polarity reversal,lymph nodal metastasis and the disease-free survival of IMPC patients.Multivariate analysis showed that the polarity reversal was an independent predictor for poor disease-free survival of breast IMPC patients.Conclusion In conclusion,IMPC is a high metastatic malignant tumor that occurs in multiple organs of the body.Our results indicate that high metastatic characteristic of IMPC is closely related to the polarity reversal clusters structure of tumor cells.Overexpression of Integrin β1 may upregulate Rac1,which is the key factor of polarity reversal clusters structure in IMPC.Inhibition of the expression of the two protein is a new idea for the treatment of this high metastatic tumor.In view of polarity reversal is the precursor of metastasis,detection the expression of these two proteins in tumor tissue to predict the prognosis,which can provide evidence for the plan of individual treatment.