To study recurrence-free survival (RFS), the Kaplan-Meier method was used; to compare RFS among different treatment groups, the log-rank test was used. A Cox proportional hazards regression model was used for relative risk estimation in multivariate analysis. All hazard ratios have been adjusted for age (continuous), Nottingham histological grade (NHG; I/II versus III), lymph node status (negative versus positive), and for cohort II also for Ki-67 nuclear fraction (0% to1%, 2% to 10%, 11% to 25%, 26% to 50%, and 51% to 100%); data for Ki-67 were not available for cohort I. We evaluated whether the effect of tamoxifen treatment was modified by stromal ��-arrestin-1 by adding an interaction term to ALK inhibitor
the Cox regression model. All P values corresponded to two-sided tests and a P value of <0.05 was considered statistically significant. This study was conducted according to the guidelines provided by the REMARK study. 35 ��-Arrestin-1 antibody reactivity was first tested by siRNA silencing of ��-arrestin-1 in MDA-MB-468 and MDA-MB-231 breast cancer cells. Significantly decreased levels of the protein were detected by immunocytochemistry for ��-arrestin-1 siRNA transfected cells, compared with control, 48 hours after transfection (Figure <a href="http://www.selleckchem.com/products/AG-014699.html
">selleck 1B). In parallel with the immunohistochemical validation of the antibody, we tested the migratory capacity of MDA-MB-468 and MDA-MB-231 cells after overexpression and silencing of ��-arrestin-1. Overexpression was detected with anti-GFP antibody using Western blot analysis (Figure 1A). Of note, ��-arrestin-1 overexpression significantly reduced the migratory propensity of both cell lines 48 hours after transfection (Figure 1C), whereas silencing resulted in prominently increased migration (Figure 1D). To exclude the possibility that decreased migration was an effect of reduced cell viability due to vector transfection, we used Western blot analysis to monitor proliferation and apoptosis in both cell lines 48 hours after vector transfection. No change in proliferation or increased apoptosis was observed in cells overexpressing ��-arrestin-1, compared with control transfected cells, Fluconazole
indicating that the decreased migration observed was a true effect of ��-arrestin-1 overexpression (see Supplemental Figure S1 at http://jmd.amjpathol.org
). Based on the discovery that overexpression of ��-arrestin-1 expression was restricted exclusively to breast cancers in the Human Protein Atlas, we further investigated its protein expression in two independent cohorts of breast cancer cases. ��-Arrestin-1 protein expression was first evaluated in cohort I, consisting of a TMA of 179 breast tumors.