The Role Of Girdin In Breast Cancer Progression

ObjectiveBreast cancer remains one of the most prevalent female malignant carcinoma and a leading cause of cancer-related mortality among females worldwide.Over last decades,survival of breast cancer patients has been improved obviously due to the advances in therapeutic methods and diagnostic tools.But the extremely biologically and clinically heterogeneous nature of breast cancer makes the clinical treatment of breast cancer still need to be improved.Moreover,it has been demonstrated that conventional clinco-pathological parameters(lymph node status,tumor size,histological grade)combined with a handful of markers(ER,PR,HER2 and Ki-67status)fall short in predicting relapse and prognosis.In view of these facts,there is an urgent need to better understand the underlying basis and discover novel therapeutic targets of this complex disease,which may help improved classification,risk stratification,and individualized treatments for breast cancer.Girdin,as an actin-binding protein,plays key roles in the development of various carcinomas.Recent studies have shown that the abnormal expression of Girdin is associated with the development of many human tumors.However,there are only few studies that focus on its subcellular distribution and prognostic value in breast cancer.The aim of this study was to discover the precise subcellular distribution of Girdin and the potential prognostic value corresponding to its cellular localization.MethodsThe subcellular distribution of Girdin was detected in human breast cancer cell line by immunofluorescence and Western blot analysis on nuclear/cytosol fractionation as well as in over 800 cases of human breast tissues by clinical pathological analysis.The subcellular expression of Girdin in invasive ductal carcinoma,ductal carcinoma in situ and benign lesions was detected by immunohistochemical method.The relationship between clinicopathological characteristics(lymph node status,tumor size,histological grade,ER,PR,HER2,Ki-67 status)and subcellular expression of Giridin was analyzed.And the prognostic value of its subcellular expression was evaluated using Kaplan-Meier survival analysis and univariate/multivariate Cox proportional hazard regression analysis.Results1.We found Girdin could localize in both cytoplasm and nucleus of cells in breast cancer cell line as well as in human breast tissue.Endogenous nuclear Girdin expression in MDA-MB-231 cells was observed in immunofluorescence analysis and validated by Western blot analysis on nuclear/cytosol fractionation.Moreover,nuclear expression of Girdin was elevated with Leptomycin B treatment.Giridin was discovered to attach to chormation or some other insoluble structure in nucleus by a small-scale biochemical fractionation.2.The score of cytoplasmic Girdin expression in mammal epithelial cells was gradually up-regulated from benign lesions to DCIS(P=0.026),and to IDC(P=0.034).Although nuclear Girdin score in IDC was much higher than that in DCIS and benign lesions,no statistic difference was observed between benign lesions and DCIS.Cytoplasmic Girdin expression in IDC tissues was higher than that in their corresponding adjacent non-neoplastic tissues.Cytoplasmic expression of Girdin was positively correlated with PR status,Ki-67 status,and recurrence or metastasis.And nuclear expression of Girdin was positively associated with ER status.3.The prognostic impact of cytoplasmic Girdin expression was evaluated in 398 IDC patients using Kaplan-Meier survival analysis and Cox proportional hazard regression analysis.(1)Patients with high cytoplasmic Girdin expression(irrespective of high or low nuclear Girdin expression)exhibited a shorter OS and PFS(OS:P=0.040;PFS:P=0.008).And the percentage of high cytoplasmic Girdin expression in patients who developed metastasis,recurrence or death within 5 years and those who were disease-free over 5 years were 48.8%(39/80)and 35.9%(88/245),respectively.Cytoplasmic expression of Girdin was positively correlated with bone metastasis(r_s=0.104,P=0.042).(2)High cytoplasmic Girdin expression led to a shorter OS and PFS in PR positive or Ki-67 negative subgroups or PR positive/Ki-67 negative subgroup.High cytoplasmic Girdin expression also indicated a worse prognosis in luminal subtype,especially in luminal A subtype.(3)In multivariate Cox proportional hazard regression analysis,cytoplasmic Girdin expression was an independent risk factor for PFS in breast cancer(hazard ratio[HR]1.714,P=0.011,95%confidence interval[CI]1.129-2.604).Moreover,cytoplasmic Girdin expression was also proved to be an independent risk factor for both OS and PFS in PR positive subgroup and luminal subtype.4.We also evaluated the prognostic impact of nuclear Girdin expression in 398 IDC patients using Kaplan-Meier survival analysis and Cox proportional hazard regression analysis.Patients with high nuclear Girdin expression exhibited a shorter OS and a similar PFS compared to patients with low nuclear Girdin expression.Nuclear Girdin expression was demonstrated to be an independent risk factor for OS in breast cancer.5.The prognostic value of combination analysis of cytoplasmic and nuclear Girdin expression was evaluated in 398 IDC patients using Kaplan-Meier survival analysis and Cox proportional hazard regression analysis.(1)Patients with high cytoplasmic/nuclear Girdin expression(C-high/N-high)exhibited a shorter OS and PFS.C-high/N-high was positively associated with ER status,PR status and Ki-67 status.(2)Girdin C-high/N-high indicated a worse OS and PFS in ER negative subtype,HER2 positive subtype and Ki-67 negative subtype.Girdin C-high/N-high was also associated with a shorter OS in HER2 negative subtype and Ki-67 positive subtype.Additionally,Girdin C-high/N-high led to a much shorter OS in both luminal and non-luminal subtype.Girdin C-high/N-high indicated a worse OS in luminal A subtype and triple negative subtype breast cancer patients.(3)In multivariate Cox proportional hazard regression analysis,Girdin C-high/N-high was an independent risk factor for both OS and PFS in IDC breast cancer patients.Conclusions1.Girdin localized in both cytoplasm and nucleus,and Girdin was discovered to attached to chromatin or some other insoluble structure in the nucleus.2.Cytoplasmic Girdin expression and nuclear Girdin expression exhibited different roles in prognosis prediction of breast cancer.Cytoplasmic Girdin expression was an independent prognostic factor for PFS(progression-free survival),whereas nuclear Girdin expression was an independent prognostic factor for OS(overall survival).3.Combination analysis of cytoplasmic and nuclear Girdin expression was an independent prognosis factor of both OS and PFS and it was a better survival predictor than either cytoplasmic or nuclear Girdin expression alone.