Abstract PD05-11: Development, Characterization, and Effective In Vitro Treatment of an Endoxifen Resistant Breast Cancer Cell Line

Abstract

Background: A major issue surrounding the use of therapeutic drugs for the treatment of breast cancer is the eventual development of resistance. Endoxifen, the most potent tamoxifen metabolite, is being developed as a novel endocrine therapy for the treatment of endocrine responsive breast cancer patients. While numerous studies have investigated the process of tamoxifen resistance, no such data exist regarding the mechanism by which cells develop resistance to endoxifen. Here, we describe the development and characterization of a novel endoxifen resistant MCF7 breast cancer cell line and the identification of a specific treatment to effectively target these resistant cells. Methods: MCF7 cells were chronically exposed to concentrations of endoxifen previously demonstrated to be associated with the greatest reductions in estrogen stimulated proliferation and transcription (1000 nM). Changes in the physiological and molecular properties of these cells were monitored during the course of resistance using a wide range of techniques. Results: Following 15 months of endoxifen exposure, an epithelial to mesenchymal transition (EMT) was induced in MCF7 cells, characterized by loss of E-cadherin expression, up-regulation of fibronectin and vimentin expression and increased responsiveness to TGFβ. Resistant cells exhibit a 7-fold increase in their proliferation rates relative to parental cells and display basal like properties (triple negative) due to silencing of ERα , progesterone receptor and CD24 expression. Resistant cells were confirmed to be estrogen insensitive through the use of cell proliferation and gene expression studies. Wound healing and cell migration assays revealed that resistant cells are highly aggressive with a significant level of metastatic potential. Microarray analysis revealed over 7500 genes to be differentially expressed in the resistant cell line relative to parental cells using a 2-fold cutoff, of which only 52 genes (0.7%) were determined to be up-regulated. Interestingly, a number of the most highly altered genes have previously been implicated in the development of EMT and/or resistance including Twist2, IGF binding protein 5, GATA and β-tubulin. Based on the identification of P-tubulin as the most up-regulated gene in resistant cells, 2-methoxyestradiol (2ME2) was recognized as a candidate drug to specifically target this resistant cell line due to it known roles in blocking tubulin polymerization. Indeed, 2ME2 exposure resulted in the induction of apoptosis and significant cell death in vitro. Conclusions: Chronic exposure of MCF7 cells to high concentrations of endoxifen led to induction of EMT with cells that are basal like, estrogen and SERM insensitive, highly TGFP responsive with significantly increased proliferation rates and metastatic potential relative to parental cells. These findings suggest that the mechanisms of resistance to endoxifen may differ from those observed with long term exposure to tamoxifen and have identified 2ME2 as a potentially successful alternative therapy for endoxifen resistant breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-11.

The Modulation of Drug Efflux Transporter by Curcumin in McF7 Breast Cancer Cells After Repeated Exposure of Endoxifen and Estradiol

“…However, treatment effectiveness is limited by high rates of de novo and acquired resistance during treatment [8]. E, an active metabolite of TMX, can also fail because of acquired resistance [6,7]. Hence, the aim of the present study was to determine the efficacy of CM administered for the prevention of decreased sensitivity of BC cells to E by coadministration with β-E2.…”

“…E exerts its anticancer effects by degrading ER alpha, which leads to the inhibition of estrogen-induced BC cell proliferation [4]. However, BC cell resistance to endocrine treatment, including E, is likely to develop overtime [5][6][7].…”

“…Numerous mechanisms are responsible for the development of endocrine resistance, including downregulation of components in the ER signaling pathway, alterations to the cell cycle, crosstalk with growth factor signaling pathways, BC stem cells, and modulation of drug transporters [6,8]. A known mechanism of BC cell resistance to TMX is the overexpression of the drug efflux transporters P-glycoprotein, BCRP, MRP1, and MRP2 [9,10].…”

Increased vimentin mRNA expression in MCF-7 breast cancer cell line after repeated endoxifen-treatment

“…1 Currently, endoxifen is being developed as a novel drug for the treatment of endocrine responsive breast cancer patients. 3 Hawse et al 2 showed that endoxifen had similar actions to selective estrogen receptor antagonist (fulvestrant) with regard to its ability to degrade ERα through proteasome degradation system, to block ERα-mediated transcriptional activation and to prevent estrogeninduced breast cancer cell proliferation. 2 A study was conducted by Wu et al 1 and showed that cells treated with high endoxifen (E) concentrations, i.e.…”