Abstract PD05-03: HDAC Inhibitor Entinostat Restores Responsiveness of Letrozole Resistant MCF-7Ca Xenografts to AIs through Modulation of Her-2
Authors: Sabnis GJ , Kazi A , Goloubeva O , Brodie AMH. 
Published: April 2011

Abstract
Development of aromatase inhibitors (AIs) has significantly improved the treatment outcome of hormone responsive post-menopausal breast cancer. However, not all tumors respond and some eventually acquire resistance. We have developed a xenograft model that mimics post-menopausal hormone responsive breast cancer (MCF-7Ca). Results obtained using this model have been confirmed by numerous clinical trials. Using this model, we have established that single agent AI is better than tamoxifen in controlling tumor growth. We also observed that although, AI letrozole provides a longer control over tumor growth, tumors eventually began to grow. A cell line was isolated from these Long-Term Letrozole Treated tumors and designated as LTLT-Ca. These cells and the tumors had lower expression of ER and aromatase compared to parental MCF-7Ca cells. On the other hand, growth factor receptor Her-2 and downstream kinases such as MAPK, Akt were upregulated. Inhibition of Her-2 using trastuzumab resulted in reversal of resistance and restoration of sensitivity to estrogens, antiestrogens and AIs.
In order to restore sensitivity to AIs, we treated letrozole resistant MCF-7Ca tumors to HDAC inhibitor entinostat (ENT). In studies performed with ER negative MDA-MB-231 cells, ENT has upregulated ERα and restored sensitivity to AIs. To examine the effect of this combination on tumors that have acquired resistance to letrozole, we utilized MCF-7Ca xenografts model. We inoculated ovariectomized athymic nude mice MCF-7Ca cells and then tumors were allowed to form in the presence of androstenedione (≥4A), aromatizable substrate for estrogen. When the tumors reached ∼300mm3, the mice were randomized such that the mean tumor volume was not significantly different across the groups (p=0.13). 50 mice were treated with letrozole for total 16 weeks, during this time the tumors regressed, but eventually began to grow. When the tumors had reached double the initial volume, they were randomized again into 5 groups; letrozole continued, exemestane (250µg/day), ENT (50µg/day) or the combination of ENT with letrozole or exemestane. The mice were treated till week 26. The growth rates of tumors of mice treated with the combination of ENT with letrozole or exemestane was significantly better than single agent alone (P&lt;0.05). Tumors were collected at autopsy and analyzed. ENT was able to increase ERα expression and aromatase activity in the letrozole resistant tumors. In addition, ENT also downregulated Her-2, p-Her-2, p-MAPK and p-Akt.
We have shown that upregulation of Her-2 in LTLT-Ca cells is a result of longer half-life of Her-2 protein and not gene amplification. To determine the mechanism of effect of ENT on Her-2 expression, we examined the interaction of Her-2 with HSP-90 in LTLT-Ca cells and effect of ENT on this interaction. We observed that ENT reduced the association of Her-2 protein with HSP-90, possibly reducing the stability of Her-2 protein the LTLT-Ca cells. This reduced interaction was also seen the tumors treated with the combination of ENT plus letrozole or exemestane. Our results suggest that HDAC inhibitor entinostat may modulate Her-2 and result in reverse the acquired resistance of letrozole resistant cells and tumors.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-03.