P4-01-14: Fulvestrant Regulates Epidermal Growth Factor (EGF) Ligands and Induces EGF Receptor Activation in MCF-7 Breast Cancer Cells.

Abstract

Abstract Selective Estrogen Receptor Modulators (SERMs) 4 Hydroxy Tamoxifen (4-HT) and fulvestrant (fulv) inhibit estrogen receptor a (ER) positive breast cancer growth. We have observed that treatment (24 to 48 hours) of fulv in MCF-7 cells induced a 190kDa tyrosine phosphorylated band, which was blocked by EGFR and HER-2 tyrosine kinase inhibitors erlotinib and lapatinib. Immunoprecipitation showed EGFR, HER-2, and HER-3 were all phosphorylated after fulv treatment. 4-HT and estradiol did not cause this phosphorylation. No changes in receptor level after fulv treatment were noted. Downstream MAPK signaling was also blocked by erlotinib and lapatinib. Fulv induced activation of EGFR was ER dependent, since fulv treatment in C4-12, an ER negative cell line derived from MCF-7 cells, did not induce EGFR activation. Co-treatment with estradiol and fulv prevented EGFR activation. To explore the possibility that fulv enhanced ligand expression, we collected conditioned media (CM) from MCF-7 cells after 48 hours of treatment. pEGFR was lost when CM was removed, but recurred within 30 minutes. Cycloheximide abolished the ability of fulv to activate EGFR suggesting autorcine production of EGFR ligands was induced by fulv. To detect specific EGFR ligands, we used qPCR to measure various EGFR ligand mRNA levels in MCF-7 and C4-12 cell at 1, 4, 24 and 48 hours after fulv treatment. TGF-α and HB-EGF mRNAs were upregulated over 48 hours, which correlated well with pEGFR activation. 4-HT did not affect mRNA levels of these ligands. In contrast, amphiregulin (AREG) mRNA levels were substantially reduced 48 hours after fulv treatment. A similar trend was seen for AREG mRNA levels in 4HT treated cells but to a much lesser extent. There was no change in any EGFR ligand mRNA levels in ER negative C4-12 cells. These qPCR data suggested differential regulation of EGFR ligands by fulv treatment contributed to EGFR family member activation and was dependent on ER expression. Upon fulv treatment, levels of ER were diminished with decreased detection of S167 and S118 ER phosphorylation sites. Monolayer cell growth analysis showed that while fulv treatment in SFM reduced MCF-7 cell numbers compared to control treatment, erlotinib plus fulv significantly reduced cell numbers below the level of cells maintained in fulv alone. In conclusion, we show that fulv, but not E2 or 4-HT, activates EGFR family members accompanied by upregulation of ligands. Since SERM resistance has been associated with EGFR family member activation, differential control of EGFR ligand gene expression by ER may contribute to the development of fulvestrant resistance in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-14.