Regulation of epithelial-mesenchymal transition in tamoxifen-resistant human breast cancer by ERα66/ERα36 and Notch4/STAT3 pathways

Abstract

Breast cancer is one of the most common malignant tumors and leading causes of cancer-related death in women. Among all types of breast cancer, 70% are estrogen receptor alpha-positive (ERα (+)) breast cancer. Hormonal therapy to block the ERα pathway is high effective to ERα (+) breast cancer and tamoxifen (TAM) has emerged as the most effective drug. However, breast cancer cells continuous exposure to TAM recur acquired tamoxifen-resistant despite the initial responsive, subsequently stimulate cell proliferation, migration, invasion and metastasis. We previously demonstrated that tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells showed increased expression of mesenchymal marker proteins compared to the parent MCF-7 cells. ERα is a 66kDa, ligand-induced nuclear receptor transcription factor and it functionally plays important role in mediating many processes in human breast cancer. Several studies have reported the downregulation of ERα66 during the development of acquired TAM resistant breast cancer. It has been recently identified and cloned a 36kDa novel variant of ERα66, ERα36, which lacks both transcriptional activation domains (AF-1 and AF-2), but retains a truncated ligand-binding domain and an intact DNA-binding domain of the full-length ERα66. While ERα66 is mainly distributed in the nucleus, ERα36 predominantly localizes in the cytoplasm and plasma membrane. We observed the expression loss of ERα66 and elevation of ERα36 in TAM-resistant breast cancer (TAMR-MCF-7) compared to parental MCF-7 cells. In this study, we evaluated the role of ERα66 and ERα36 in the progression of acquired TAM resistance and EMT process in breast cancer. Our study revealed that ERα36 is a key signaling factor for estrogen-independent cell proliferation and tumorigenesis of tamoxifen-resistant breast cancer. Overexpression of ERα36 resulted in loss of ERα66, subsequently participating in acquisition of EMT. ERα36 seems to be involved in EMT process of TAMR-MCF7 cells by inhibiting ERα66 expression. However, the murine model of hepatic metastases performed via a hemispleen injection demonstrated that ERα36-overexpressing MCF-7 cells failed to cause metastatic tumor burden in liver though this cell type also displayed features of EMT-like phenotype similar to TAMR-MCF-7 cells. Moreover, overexpression of ERα66 in TAMR-MCF-7 cells could reverse the EMT to MET characterized by restoring the epithelial marker expression, E-cadherin. Likewise, overexpression of ERα66 in other ERα66-negative breast cancer cells such as MDA-MB-231, SKBR3 resulted in suppression of EMT and cell migration. However, spleen injection for liver metastases experiments revealed the same micrometastatic hepatic tumor burden between control TAMR-MCF-7 cells (TAMR-GFP) and ERα66-overexpressing TAMR-MCF-7 cells (TAMR-ER66). Thus, ERα66 function plays as a crucial factor in differentiation and maintenance of normal epithelial architecture. Notch is functionally important in the promotion of EMT during both development and progression of tumor. Notch1 and Notch4 have been reported as prognostic markers in human breast cancer. Here, we indicated that Notch4, but not Notch1, plays a critical role in the regulation of EMT signaling in TAMR-MCF-7 cells. Notch4 suppression by either Notch inhibitors or Notch4 siRNA attenuated EMT signaling. Tyrosine-phosphorylated STAT3 protein is known as a crucial signaling molecule in the regulation of tumorigenesis and metastasis. We found that TAMR-MCF-7 cells exhibited constitutive STAT3 phosphorylation, and Notch inhibition reduced the level of activated STAT3 in TAMR-MCF-7 cells. Our study also revealed that STAT3 bound physically with Notch4-intracellular domain (Notch4-ICD) but not the full-length of Notch4. Intrasplenic injection model of liver metastases was performed using TAMR-MCF-7 cells. Mice injected with N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butyl ester (DAPT

10 mg/kg

an inhibitor of γ-secretase) formed smaller splenic tumors and showed a reduced micrometastatic tumor burden in their livers compared with the control group treated with vehicle. This study reported for the first time the physical binding between STAT3 and Notch4-ICD and this interaction may play an important role in EMT progression during acquired tamoxifen-resistant breast cancer. Collectively, this study proposes two signaling pathways which are responsible for epithelial mesenchymal transition (EMT) phenotype during acquired tamoxifen-resistant human breast cancer.