Estrogen receptor signaling and mechanism of resistance to endocrine therapy

We investigated mechanism of resistance to both faslodex and tamoxifen in a panel of 14 ER+ breast cancers cell lines. Growth inhibition assays indicated that some cell lines in the panel are significantly more sensitive to faslodex than to tamoxifen while other cell lines are either resistant or sensitive to both drugs. Tamoxifen did not show any inhibitory advantage over faslodex in any of the cell lines in the panel. In all the analyzed cell lines and regardless of their sensitivity or resistance to the drugs, faslodex reduced ER protein while tamoxifen caused accumulation of ER in the cells. Regardless of sensitivity or resistance of the cell lines to ER antagonists, an E2 responding-luciferase reporter assay showed several fold induction of the reporter by estradiol (E2) and reduction of the reporter by both drugs. However, faslodex was more effective in reducing the reporter level. Progesterone receptor (PR) is an E2 inducible gene. However in the panel of ER+ cell lines, PR expression was induced by E2 only in eight cell lines. Cell lines with higher sensitivity to both tamoxifen and faslodex showed relatively high or average expression of PR. Faslodex reduced PR level in all the PR+ cell lines. On the contrary, tamoxifen enhanced expression of PR in three of the cell lines and decreased PR levels in five other cell lines. Interestingly, in cell lines in which tamoxifen increased PR expression, the inhibitory effect of tamoxifen was significantly lower than faslodex. Treatment of these cell lines with combination of tamoxifen and PR inhibitor, ZK23021 1, increased the inhibitory effect of tamoxifen, while treatment of these cell lines with combination of faslodex and ZK230211 did not further enhance the inhibitory effect of faslodex. Therefore, induction of PR expression by tamoxifen in some cell lines contributes to their lower sensitivities to tamoxifen.;We also investigated the role of growth factor signaling in resistance to these ER antagonistic drugs. Activity of growth factor signaling was determined by measuring p-AKT and p-ERK1/2 levels in each cell line. The results indicated that ER+ cell lines with the highest combined levels of p-ERK1/2 and p-AKT were resistant to both faslodex and tamoxifen. ER+ cell lines with medium combined levels of p-ERK1/2 and p-AKT were significantly more sensitive to faslodex than to tamoxifen while those with the lowest levels of p-ERK1/2 and p-AKT were more sensitive to both drugs. In the panel of ER+ cell lines, resistance to faslodex was strongly (r = 0.8) and resistance to tamoxifen was weakly (r = 0.6) correlated with activity of growth factor signaling.;We also investigated mechanisms of acquired resistance to endocrine therapy by developing two resistant sub-lines from a highly sensitive cell line, MCF-7. Microarray analysis and other confirmatory assays indicated that E2 independent expression of AREG is highly increased in the resistant sub-lines, MCF-7R1 and MCF-7R2. Moreover in comparison to the parental cell line, higher levels of FGFR2, FGFR3, p-ERK1/2 and pAKT were detected in MCF-7R1 and MCF-7R2. Both resistant sub-lines showed higher sensitivities to the inhibitors of AREG function, cetuximab and lapatinib. Moreover, inhibition of AREG expression by SiRNA decreased the resistance of MCF-7R1 and MCF-7R2 cell lines to faslodex by several fold. In addition, inhibiting FGFR2 expression by SiRNA also enhanced the sensitivity of MCF-7R1 and MCF-7R2 cell lines to ER antagonistic drug faslodex. As parental MCF-7 and three other ER+ cell lines were stimulated by AREG, FGFs or AREG plus FGFs, we found that in MCF-7 and two other cell lines (T-47D and ZR-75-1) combination of these growth factors significantly enhanced the E2 independent growth and resistance to ER antagonists. Therefore, we conclude that ER+ tumor cells can acquire resistance to endocrine therapy by ER independent and autonomous production of AREG, overexpression of receptors for FGF growth factors and enhancing activity of growth factor signaling. (Abstract shortened by UMI.)...