Abstract 2283: Endoxifen exhibits potent in vitro and in vivo antitumor activity in ER+/HER2+ breast cancer and tamoxifen refractory tumors
Authors: Reinicke Kathryn E. , Hou Xiaonan , Goetz Matthew P. , Suman Vera J. , Kuffel Mary J. , Haluska Paul , Reid Joel M. , Ames Matthew M. 
Published: June 2012

Abstract
Background: In estrogen receptor positive (ER+) breast cancer human epidermal growth factor receptor 2 (HER2) expression is associated with resistance to tamoxifen (Tam). Endoxifen is the most abundant active metabolite of Tam, resulting in part from cytochrome P450 2D6 (CYP2D6) metabolism. While Tam has been extensively used to model proliferative ER/HER2 cross-talk, there are no published data regarding endoxifen's effect on ER/HER2 nongenomic nor ER genomic signaling.
Methods: Using ER+ cells (MCF7) and ER+/HER2+ cells (MCF7-HER2, BT474) we compared the effects of Tam and endoxifen on proliferation using crystal violet assays, Erk1/2 phosphorylation by western blotting, and E2-stimulated ER-regulated gene expression of amphiregulin (AREG), c-Myc (MYC) and progesterone receptor (PGR) by real-time RT-PCR assays. To examine the anti-proliferative affects of endoxifen compared to Tam in vivo we performed E2-stimulated MCF7-HER2 xenograft studies in athymic nude mice. Endoxifen doses of 25 (low) and 75 mg/kg (high) were used based on our prior pharmacokinetic studies showing achievable steady state levels of 100 nM and 1 uM, respectively. Z-Endoxifen HCl was synthesized by NCI.
Results: Endoxifen, unlike Tam, potently inhibits growth in both ER+ and ER+/HER2+ breast cancer cell lines at concentrations observed in human CYP2D6 intermediate and extensive metabolizers (&gt;40 nM). Tam (10-1000 nM) stimulates phosphorylation of Erk1/2 in stark contrast to endoxifen at similar doses. Endoxifen (100-1000 nM) significantly decreases E2-stimulated transcription of ER-regulated genes in MCF7-HER2 cells; AREG 2-3 fold, MYC 1-2 fold, and PGR 2-4 fold. Conversely, Tam (10-1000 nM) maintains or stimulates transcription; AREG 2-3 fold, MYC 0.5-2.5 fold, and PGR 0-3 fold. After 7 weeks of treatment of MCF7-HER2 xenografts, both high (p=0.001) and low dose endoxifen (p=0.01) resulted in greater antitumor activity than Tam, with high dose superior to low dose endoxifen (p=0.024). MCF7-HER2 xenografts that progressed on Tam to 200% growth at 7 weeks were randomized to either continued Tam or endoxifen. Four weeks after randomization endoxifen was superior to Tam (p&lt;0.0001) with tumor size decreased by ≥ 20%. No significant toxicity was observed.Conclusions: We have demonstrated in both ER+ and ER+/HER2+ breast cancer cell lines that in contrast to Tam, endoxifen potently inhibits growth (both in vitro and in vivo), does not activate Erk1/2 signaling, and inhibits E2-stimulated transcription of ER-regulated genes. Endoxifen exhibited potent antitumor activity in MCF7-HER2 xenografts refractory to Tam. These data support the ongoing NCI sponsored phase I studies of Z-endoxifen at Mayo Clinic and NCI.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2283. doi:10.1158/1538-7445.AM2011-2283