The Role Of DAB2Downregulation In The Breast Cancer

Object:Dab2is a pluripotent adaptor protein, which has been showed the down-regulation and absence in a variety of human cancer types, has a wide influences on cell functions. This study has been focused on the relation between the down-regulation or the absence of Dab2in breast cancer and the tumor cell function, analyzed the functions of tumor associated gene Dab2in occurrence and development of breast cancer, emphasized on discussion about the influences of Dab2on endocytic and trafficking itinerary of TGF-β receptor subunits. Moreover, we also investigated the role of Dab2in the TGF-β depletion, by which the existence of cross-talk between the tumor and immune cells might regulate anti-tumor immune responses. The study has been fully elaborated the significance of Dab2in pathogenesis of breast cancer and its application value in clinical diagnosis treatment of breast cancer.Method:1) We collected76cases with breast cancer in our hospital from January2011to November2011. The expressions of Dab2in breast cancer were tested by IHC, compared with which in normal breast tissues, and analyzed the relationship with various clinical pathological features.2) This study analyzed Dab2protein expression of common cell lines of breast cancer through Western-blot, made SK-BR-3cell line re-express Dab2(SK-BR-3Dab2) through plasmid transfection, observed the influences of Dab2on cell capacity of proliferation, migration and invasion, and the changes of MAPK signal pathway protein phosphorylation levels, compared to the control cells transfected with empty vector SK-BR-3V cells(SK-BR-3V).3) We used mink lung epithelial cells which stably infected with a luciferase reporter gene driven by a PAI-1-sensitive promote to measure the TGF-β concentration in the cell culture supernatant, observed the impacts of Dab2on TGF-beta depletion of SK-BR-3cell line, and the influences on the classic Smad signal pathway.4)We collected the culture supernatant of SK-BR-3Dab2cells and control SK-BR-3V cells after TGF-β stimulation, then co-culture system was used to simulate tumor microenvironment in vitro, in order to observe whether the differences of TGF-β concentration in supernatant could account for the different effects of Treg induction. Neutralizing anti-TGF-β antibody was added to the co-culture system for neutralization experiments. The carboxyfluorescein diacetatesuccinimidyl ester(CFSE) dilution assay was performed to evaluate the suppression characteristics of induced Tregs.Results:1) In76cases of patients with primary breast cancer, only18carcinoma tissues (24%) were positive in Dab2immunohistochemical staining, the others58were negative, including the specimens of ductal in situ carcinoma with microinvasion also has been showed the absences of expression. In10cases of patients with normal breast tissues and1breast cysts specimen, Dab2immunohistochemistry results have been showed intermediate to high level positive staining of cytoplasm. No significant correlation was observed between the expression of Dab2protein and clinicopathological parameters, such as ages tumor sizes, lymphatic metastasis stage(P>0.05).2) The testing results of western blot in breast cancer cell lines showed the non-malignant breast cell line MCF-10A had the Dab2expression of molecular weight105kDa, and the MDA-MB-231and SK-BR-3cells had only a little weak expressions of endogenous Dab2, but MCF-7cells was completely undetectable. Compared with the control group SK-BR-3V, the cells proliferation inhibition rate of SK-BR-3Dab2reached28%after72hours. Significant difference of the number of the migrated cells was observed between SK-BR-3Dab2and SK-BR-3V cells, in the migration and invasion assay (P<0.05, the inhibition rate of32%). Dab2still had been showed the inhibitor of migration ability under the TGF-β stimulation of concentration5ng/ml (P<0.05, the inhibition rate reached37%). Similar effects of Dab2expression in SK-BR-3on invasion assay were found (P<0.05. the inhibition rate reached78%with TGF-β, the inhibition rate reached63%without TGF-β). The expression of c-FOS of SK-BR-3Dab2cells was much weaker under the stimulation of serum, compared with the control group. And the ERK1/2activities were unchanged upon serum stimulation in two group cells.3) SK-BR-3cells to the certain extent retained TGF-β receptor with function, being able to sense the ligand and pass down signals. When the TGF-β initial concentration is60pM in the cell culture supernatant, the concentration of SK-BR-3Dab2was reduced12pM compared with the control group, which was showed the enhancement of depletion capacity at4hours. After half an hour TGF-β stimulation between SK-BR-3Dab2cells and SK-BR-3V cells, the former expression of p-Samd2was obviously much weaker. Two groups of Samd2phosphorylation are maintained at the similar levels after that. Tfn recycling assay proved that the block of TGF-β receptors recycling via a clathrin-and Rab11-dependent pathways was got improved after re-expression of Dab2. Dab2expression improved diffused Tfn staining by55%4) The breast cancer microenvironment was modeled by establishing the co-culture of breast cancer cell line, SK-BR-3Dab2or SK-BR-3V, with sorting naive CD4+T cells. Our results showed that SK-BR-3Dab2cell supernatant can induce a lower population of CD4+CD25+Foxp3+Tregs than SK-BR-3V (P<0.05). The CFSE dilution assay further confirmed that the induced Treg was functional and able to suppress the proliferation of effector T cells.Conclusion:The absent or down-regulated of Dab2in breast cancer cells, not only made cells lose control of various fundamental biological activities such as proliferation, migration and invasion, but also blocked Rab11-dependent pathway in the trafficking of TGF-β receptors. Moreover, the TGF-β depletion of the cells was impaired, which would therefore contribute to the accumulation of TGF-β in the tumor microenvironment. The enhanced levels of TGF-β would induce the conversion of naive CD4+T cells to CD4+CD25+Foxp3+Tregs, which were functional and able to suppress the proliferation of effector T cells. Thus it showed the absent or down regulation of Dab2could finally impact on the immune suppression in the tumor microenvironment.