P3-17-08: Macroautophagy Protects Breast Cancer MCF-7 Cells from TAM-Induced Apoptosis Via Mitogen-Activated Protein Kinase (MAPK) Pathway.
Authors: Hou YF , Ma XY , Liu ZB , Yu SJ , Shao ZM 
Published: June 2012

Abstract
Tamoxifen (TAM) has been used ubiquitously for endocrine therapy for the hormone-sensitive breast cancer. Several studies have revealed that tamoxifen treatment induced apoptosis and at the same time tamoxifen increased autophagic levels in MCF-7 cells. The previous studies attempt to elaborate the significance and mechanism of autophagy induced by TAM in breast cancer cells, however, there are contradictions among their conclusions, it is still not clear that autophagy protects MCF-7 from apoptosis or promotes apoptosis. Better understanding of the effect of autophagy induced by TAM in breast cancer cells on apoptosis will be of the important clinical significance in endocrine therapy for breast cancer.
The present study shows that tamoxifen treatment significantly increased autophagic levels by inducing autophagic vacuoles formation in MCF-7 cells observed by means of transmission electron microscopy and enhancing the expression of autophagy marker, icrotubule-associated protein light chain 3 measured by Western blot. Our research shows tamoxifen enhanced the phosphorylation of MAPKs when inducing autophagy and autophagy decreased significantly when kinase inhibitors were separately used to inhibit the phosphorylation of ERK, JNK, p38 MAPK. MAPK signal transduction pathway was involved in the process of autophagy in MCF-7 cells.
To investigate whether Estrogen receptor-α participated in autophagy caused by tamoxifen, we constructed Estrogen receptor-α gene ESR/1 shRNA expression vector and it could effectively inhibit the expression of ER-α in MCF-7 cells. Our research shows that autophagy was decreased with the downregulation of ER-α, so we conclude that Estrogen receptor-a also involved in autophagy induced by tamoxifen in MCF-7 cells.
To find out the specifical role of autophagy in tamoxifen treated breast cancer cell MCF-7, we inhibited autophagy producing after tamoxifen treatment by pretreating the cells with chloroquine and 3-methyladenine, both commonly used as autophagy inhibitors. Another method for autophagy inhibition was Becline-1 siRNA transfection into MCF-7 cells. Than we stained MCF-7 cells with anti-Annexin V FITC and PI and examinated apoptotic rate by flow cytometer and we also detected activity of caspase7 in MCF-7 cells. The results indicate that inhibition of autophagy by the methods mentioned induced higher apoptotic level, therefore, autophagy protected MCF-7 from apoptosis and inhibiting autophagy may be a new strategy to augment the theraputic effect of tamoxifen treatment. In our study, we induced breast cancer cell MCF-7 resistant to TAM in vitro and we found much higher autophagic level in TAM resisitant cells compared with TAM not resisitant MCF-7 cells. We draw the conclusion that inhibition of autophagy induced by tamoxifen may be a new therapy for tamoxifen resisitant breast cancer.
Key words: tamoxifen, breast cancer, autophagy, MCF-7 cells, MAPKs, estrogen receptor, inhibition, apoptosis
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-08.