Anti-estrogen therapy and breast cancer: Molecular mechanisms of efficacy and resistance

Anti-estrogen therapies like tamoxifen are the most successful breast cancer treatments to date. However, molecular mechanisms responsible for decreased efficacy and the evolution of resistance are poorly characterized. 4-hydroxytamoxifen (OHT) is a tamoxifen metabolite with much higher binding affinity to the estrogen receptor (ER). For this reason, many of the anti-estrogenic effects of tamoxifen are attributed to OHT and not the parent drug. Others have shown that additional metabolites of tamoxifen are present in sera at much higher levels than OHT. The concentration of at least one of these metabolites is lowered by genetic variations in cytochrome P450 CYP2D6 and coadministration of anti-depression drugs that are potent inhibitors of the same. We report here a facile three step synthetic method through which 4-hydroxylated tamoxifen metabolites can be synthesized. Through these synthetic means, we confirm that N-desmethyl-4-hydroxytamoxifen (Endoxifen) is the unknown metabolite affected by variations in and inhibitors of CYP2D6. Endoxifen binds to ER with similar affinity as OHT and inhibits estrogen-dependent ERE-driven transcription as well as cell proliferation with similar potency and efficacy as OHT. We conclude that endoxifen is not only present at higher concentrations in sera than OHT but also equally effective as an anti-estrogen. Therefore, endoxifen likely plays an important role in the anti-estrogen effects of tamoxifen, and genetic variations or drug interactions may be responsible for decreased efficacy of tamoxifen therapy. Phases of anti-estrogen resistance are defined by a tumor\u27s response to ligands. One type of resistance is identified by a tumor response profile where anti-estrogens like tamoxifen stimulate growth whereas estradiol represses the same. Serum response factor is a growth promoting transcription factor activated by growth factor signaling pathways partially responsible for tamoxifen resistance and known to cross-talk with ER. Here we report that in MCF-7 cells estradiol activates SRF-dependent transcription whereas all anti-estrogens have little to no effect. However, in SKBR3 cells transiently transfected with ER, raloxifene, tamoxifen, and fulvestrant activate SRF activity. That activity is repressed by both estradiol and anti-estrogen GW-7604. These data suggest that ER-modulation of SRF activity could play a role in changes in ligand response in different cellular contexts