Studies On Aurora A As A Novel Subgroup Biomarker For Moleculer Calssicifation And A Potential Therapeutic Target For Luminal A Breast Cancer

BackgroundAccording to the existed breast cancer molecular markers ER, PR, Her-2, Ki67, breast cancer can be divided into better prognostic Luminal A subtype and Luminal B subtype, poor prognostic triple-negative breast cancer and Her-2 over expression subtype. As a kind of highly heterogeneous tumor, there are huge difference between the prognostic of breast cancer subtypes. Even among the Luminal A subtype breast cancer patients, there are part of them have the same prognosis as to triple-negative ones, thus the present molecular subtype is not perfect. Therefore, to identify the prognostic risk markers out of various subtypes of breast cancer has important clinical significance. Aur-A, as the key kinase of mitosis, has a close relationship with the tumorigenesis of many kinds of tumors, but whether it could be an independent prognostic factor of breast cancer or not is still controversial. Furthermore, the specific mechanism of Aur-A in the prognostic of breast cancer remains unclear and needs to explore.ObjectiveTo explore whether Aur-A can be used as a more accurate marker for different kinds of breast cancer molecular subtype to distinguish prognosis. Study of the molecular signaling pathway, to better understanding of the biological function of Aur-A and its mechanism of action.Methods(1) Immunohistochemistry staining was performed to analyze the expression of Aur-A in breast cancer tissue and the relationship between the expression and prognosis of breast cancer patients on tissue microarray which contained 310 breast cancer patients with 12 years follow-up data. Through further stratified analysis to understand whether Aur-A can successfully distinguish poor prognosis subgroup of patients from different subtypes of breast cancer patients. (2) On behalf of the luminal A subtype of breast cancer, MCF-7 cell line was set as the experimental object in vitro. Using siRNA to knock down Aur-A in MCF-7 cells or use Amg900 which is the selective inhibitor of Aur-A to inhibit the activation of Aur-A in MCF-7 cells. Then, colony formation, CCK-8 assay, Scratch assay and Transwell were performed to study the effect of Aur-A on the proliferation and invasion and metastasis of Luminal A breast cancer, Western blot and RT-PCR was used to study molecular mechanism of Aur-A. (3) Establish Nude Mice Xenografts with MCF-7 cells respectively after lentivirus knock down Aur-A or after treated with Amg900 inhibitor drug, corresponding negative and blank controls were set up to this two models. Observate the growth rate of this two kinds of tumor model and immunohistochemical Aur-A, Ki67, Cofilin, P-Cofilin, PHH3 and Src expression of the tumor tissue.ResultsPart 1:Aur-A is highly expressed in infiltration breast cancer, among 310 patients positive expression rate reached 80.65%(250/310), the distribution of positive and negative expression of Aur-A had no correlation with age(P=0.451), pathological grade(P=0.318), axillary lymph node metastasis(P=0.777), tumor location(P=0.387), clinical staging(P=0.902), CK5/6(P=0.099), AR(P=0.512), but has significant correlation with tumor size(P=0.0.030), ER(P=0.044), PR(P=0.027), Her-2(P=0.000), Ki-67(P=0.013), p53(P=0.029), EGFR(P=0.036),Cofilin(P=0.000) and molecular subtype (P=0.000). Kaplan Meier survival function analysis found that overexpression of Aur-A is an independent risk factor for poor prognosis of breast cancer. Further stratified analysis found that expression of Aur-A had statistical significance to the prognosis in Luminal A subtype breast cancer(P=0.03). Univariate COX analysis results showed that Aur-A, ER, PR, EGFR and AR, FAK, Cofilin, Aur-B, molecular subtype, tumor stage have statistical significance, however, multivariate analysis shows that only Aur-A and Cofilin could be used as independent prognostic factors. The results suggested that Aur-A could be used as a more accurate risk marker for Luminal A subtype breast cancer.Part 2:On the basis of the first part of the study, the biological function and mechanism of Aur-A were studied. Aur-A is high expressed in luminal A subtype breast cancer cell line MCF-7, after knockdown Aur-A by siRNA in MCF-7 cells or after effected by Aur-A selective inhibitors Amg900 in MCF-7 cell line, cell proliferation, migration, invasion ability, scratch healing and clone formation rate was significantly lower than that in the control group. In siRNA knock downgroup phosphorylation of Cofilin-F-actin pathway increased, thereby reducing the non phosphorylated part related with Cofilin activity, lead to the reduced activity of Cofilin-F-actin signal pathway, then after knock down of Aur-A theexpression levelof epithelial mesenchymalrelated molecules TGFβ,Snail, Vimentin were decreased, the expression level of E-cadherin protein increased; while invasion metastasis related molecules,Src and FAK, were changed at the same time. Alterations in these pathways are likely to be the molecular mechanisms that are associated with poor prognosis of breast cancer caused by Aur-A.Part 3:Nude mice xenografts and inhibited intervention experiment results show that, tumor volume and weight of virus knock down Aur-A group was significantly less than that of blank group and virus negative control group; selective inhibition of Amg900 by Aur-A can effectively control the growth of the nude mice xenografts tumor with MCF-7 cells. Tumors of nude mice by immunohistochemical results show that in Aur-A virus treatment group there were no obvious differences of Cofilin, PHH3, but the expression of Src, Aur-A, Ki67 were significantly lower than that of control group, while P-Cofilin was significantly higher expression.ConclusionAur-A can be used as a independent prognostic factor for breast cancer, especially in Luminal A subtype of breast cancer.Aur-A, as upstream gene could regulate Cofilin-F-Actin, Src, FAK and EMT related protein TGFβ,Snail, Vimentin, E-cadherin and other signaling proteins, then achieve the regulation of invasion and transfer of tumor cell in the interaction with molecules from variety of invasion and metastasis related signaling pathway.Aur-A can be used as a marker for evaluating the prognosis of Lumianl A subtype breast cancer and a potential therapeutic target.