To investigate therapeutic strategies to overcome resistance to anastrozole (ANA) treatment, we have used a tumor model that simulates postmenopausal breast cancer patients with estrogen-dependent tumors. This model utilizes estrogen receptor (ERα) positive human breast cancer cells stably transfected with the aromatase gene (Ac1 cells), which are inoculated s.c. into ovariectomized SCID mice. The cells produce sufficient estrogen by aromatization of supplemented androstenedione (AD) to stimulate their proliferation and formation of tumors in the mice. In previous studies, increased expression of signaling proteins in the growth factor receptor/MAPK pathways was found in aromatase inhibitor resistant tumors and may be a mechanism whereby the estrogen receptor (ER) is phosphorylated in the absence of estrogen thus enabling initiation of transcription. We therefore investigated whether combining ANA treatment with the ER down-regulator fulvestrant (ICI) would prevent or delay development of resistance compared to ANA or ICI alone. The tumors treated with the combination of ANA and ICI had regressed by 35% during 18 weeks of treatment whereas the ANA and ICI treatments alone resulted in 750% and 100% increase in tumor size, respectively in the same time. Thus the combination of ANA plus ICI was more effective in maintaining suppression of tumor growth than either ANA or ICI alone. After tumors of ANA treated mice had doubled their initial size (week 8), mice were assigned to two groups. One continued on ANA treatment and the second group received ANA plus ICI. The tumors that remained in ANA alone showed an increase in tumor volume of 650%, while the tumors on the combination of ANA+ICI showed only a 250% increase. On week 18, mice were sacrificed and the tumors and uteri were removed, weighed and stored for further analysis. Treatment with ANA caused a reduction in uterine size that was further decreased by the addition of ICI. The tumors in all three groups were subjected to western blotting to examine the expression of ERα and pMAPK. ANA alone group showed overexpression of ERα compared to control. ANA+ICI group showed down-regulation of ERα as well as downregulation of pMAPK. ANA+ICI also caused a reduction of ERα transcriptional activity. On the other hand, ANA alone resulted in had higher ERα activation compared to control. A cell line was isolated from long term ANA treated tumors, designated as Ac1ANAR. This cell line was found to be refractory to growth stimulatory effects of E2 or AD and also to growth inhibitory effects of ANA, LET or TAM. The Ac1ANAR cells however, were responsive to ICI, suggesting the importance of ERα in growth of these cells. This suggests that the down-regulation of ER inhibits the crosstalk between ER and growth factor signaling and may prevent and inhibit resistance to aromatase inhibitors.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA