Beclin1Downregulation Enhances Tamoxifen Sensitivity And Predicts A Favorabie Outcome For ER Positive Breast Cancer

Background:Breast cancer is the most common female malignant tumor and about70%breast cancer were estrogen receptor positive. Endocrine therapy represented by tamoxifen is one of the most effective treatment for estrogen receptor(ER)-positive breast cancer, however drug resistance greatly prevents patients to get benefit from it. Further understanding of tamoxifen resistance is badly in need to improve therapeutic outcome of patients.Objectives:To uncover the role of beclin1in tamoxifen resistance and in prognosis of ER positive breast cancer.Methods and results:In this study, by immunohistochemistry assessment, we found that beclin1level was not only positively correlated with level of human epidermal growth factor receptor2(HER2) but also negatively correlated with overall survival of patients with ER-positive breast cancer. No association was found between beclin1expression and other clinicopathological characteristics including pathological grade, tumor size, lymph node status, or tumor stage. Using bioinformatic methods, we further found that level of beclin1was negative correlated with overall survival in ER-positive breast cancer patients receiving tamoxifen (TAM) treatment. Then we established a TAM resistant cell subline (MCF-7R) from ER-positive breast cancer MCF-7cells, in which beclin1and HER2protein levels were discovered higher than MCF-7. Beclin1was silenced using RNA interference in these two cell lines (MCF-7/Beclin1-siRNA and MCF-7R/Beclin1-siRNA). We found that MCF-7or MCF-7R cells with lower beclin1exhibit decreased proliferation rate together with enhanced apoptosis. Moreover, silencing beclin1significantly promoted TAM sensitivity of MCF-7and MCF-7R in vitro manifested as decreased proliferation and clone formation ability, increased cell apoptosis, G0/G1cell cycle arrest, decreased invasion and migration ability. Real-time PCR and Western blotting analysis revealed that cells with downregulated beclin1expression possessed decreased HER2expression both at mRNA level and protein level. After TAM treatment, MCF-7/Beclin1-siRNA and MCF-7R/Beclin1-siRNA showed decreased HER2protein levels, alone with decreased phosphorylated Akt and ERKl/2level and Bcl-2level, increased cleaved caspase-3and PARP levels, compare with control cells. Although autophagy protein LC3B expression level was increased after TAM treatment, it did not show significant differences between MCF-7/Beclin1-siRNA and MCF-7/Con-siRNA. In the subsequent studies, we established stable beclin1knockdown cell line MCF-7/Beclin1and control cell line MCF-7/V by shRNA expression lentiviral vectors. MCF-7/Beclin1also exhibited increased sensitivity to TAM and decreased HER2level compare with MCF-7/V. We overexpressed HER2in MCF-7/Beclin1and MCF-7/V cells by stable transfection. After overexpression of HER2, formerly increased TAM sensitivity was reversed in MCF-7/Beclin1. In addition, with the presence of TAM, HER2upregulation could partially restore the phosphorylation levels of Akt, ERK1/2and Bcl-2and could decreased the level of cleaved caspase-3and PARP in MCF-7/Beclin1.Conclusion:This study indicated beclin1level was not only positively correlated with level of human epidermal growth factor receptor2(HER2) but also negatively correlated with overall survival of patients with ER-positive breast cancer. Downregulation of beclin1may contribute to enhanced tamoxifen sensitivity through lower HER2expression.Therefore, beclin1could be a potential prognosis predictor and novel therapeutic target for TAM resistance in ER-positive breast cancer patients.