Expression of ERα and Aromatase in MDA-MB-231 Tumors by HDAC Inhibitor Entinostat Leads to Growth Inhibition by Aromatase Inhibitor Letrozole.

Abstract

Abstract The treatment for hormone receptor-positive breast cancer has improved significantly since the development of aromatase inhibitors (AIs). Nevertheless, AIs are ineffective in estrogen receptor-negative (ER-) tumors, which comprise of approximately 25% of breast cancers and tend to be more aggressive. Studies have shown that repression of ER in these hormone receptor-negative tumors may be due to epigenetic modifications. The discovery of recruitment of histone deacetylase enzymes in gene silencing provides a rationale for inhibition of HDAC activity to release transcriptional repression as a potential therapeutic strategy. The objective of the present study was to express ERα and aromatase with HDACI treatment and thereby sensitize tumors to growth inhibition with aromatase inhibitors. In this study we used ER negative, hormone refractory MDA-MB-231 human breast cancer cells. Treatment with HDAC inhibitor entinostat led to upregulation of ERα, aromatase and its activity in a dose dependent manner in cells and xenografts. MDA-MB-231 xenografts were grown in ovariectomized female nude mice. Mice were inoculated with 2.5 X 106 cells per site subcutaneously. When the tumors reached 150 mm3, the mice were grouped into 4 groups (n=10), so that the mean tumor volume was not statistically different across groups (p=0.88). Tumor volumes were measured twice weekly. The mice in the letrozole group had a mean tumor growth rate (β = 0.023 ± 0.014) that was not statistically different (p=0.76) from that of the control group (β = 0.038 ± 0.007). Also, the growth rate of entinostat group (β = 0.034 ± 0.011) was not significantly lower than that of the control (p=0.33). However, the growth rate of entinostat plus letrozole group ((β = -0.003 ± 0.013) was significantly lower than that of the control (p=0.01), entinostat (p=0.03) and letrozole (p=0.049) groups. The combined treatment of entinostat plus letrozole was significantly more effective than either agent alone. In addition, the ability of this combination to inhibit migration in vitro was examined by wound healing assay. The combination of entinostat plus letrozole provides superior inhibition of migration (p<0.001) compared to control, entinostat and letrozole alone. To test the efficacy of this combination in preventing the outgrowth of tumor foci in the lung, mice received an inoculation of 3 X 106 cells intravenously via the tail vein. They were treated three weeks later with entinostat alone, letrozole alone, or the combination. Mice were treated for six weeks, and then euthanized. Treatment with entinostat alone or letrozole was not significantly effective, but the combination resulted in a significantly reduced number of both visible and micrometastases (p=0.03) compared to no treatment (control) and the entinostat alone group. Thus, up-regulation of ERα and aromatase resulted in sensitization of tumors to significant inhibition of growth, cell migration and formation of micro-metastases by the aromatase inhibitor letrozole.Our results provide the basis for possible use of AIs in combination with HDAC inhibitors for the treatment of hormone refractory ERα negative breast cancer. This could open a new avenue for the management of ER- breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 401.