Aromatase inhibitors and xenograft studies

Abstract

Aromatase inhibitors (AIs) have become the front-line choice for treatment of ER+ breast cancer. Nevertheless, although patients are responsive initially, they may acquire resistance and become unresponsive to further treatment. In addition, approximately 25% of breast cancers do not express the estrogen receptor (ERα) and consequently, are innately resistant to endocrine therapy. We have investigated the mechanisms associated with this lack of treatment response using xenograft models. We found that in cells and tumors that acquired resistance to the AI letrozole therapy, expression of the ER was reduced whereas growth factor signally was enhanced, including a marked increase in HER2 expression. Treatment with trastuzumab (HER2 antibody) resulted in a significant down-regulation of HER2 and p-MAPK as well as restoration of ERα expression. Thus, when trastuzumab was added to letrozole treatment at the time of tumor progression, there was significantly prolonged tumor suppression compared to trastuzumab or letrozole alone. This suggests that inhibition of both HER2 and ERα signaling pathways are required for overcoming resistance and restoring treatment sensitivity. ER negative tumors are innately resistant to endocrine therapy. Repression of the ERα has been found to be due to epigenetic modifications such as increased methylation and histone deacetylation. We found that entinostat (ENT), a histone deacetylase inhibitor (HDACi), activated not only expression of ERα but also aromatase in MDA-MB-231 ER-negative breast cancer cells, resulting in their ability to respond to estrogen and letrozole. Treatment with ENT in combination with letrozole significantly reduced tumor growth rate in xenografts compared to control tumors (p<0.001). ENT plus letrozole treatment also prevented the colonization and growth of MDA-MB-231 cells in the lung with a significant reduction (p<0.03) in both visible and microscopic foci. These results provide a strong indication for possible use of AIs in combination with HDAC inhibitors for the treatment of ER-negative breast cancer.

Figure 1

A. Effect of letrozole treatment on ERα, PGR, erB-2, and p-Shc expression in MCF-7Ca tumour xenografts. B. Effect of letrozole treatment on Grb2, p-MAPK, and p-ERα expression in MCF-7Ca xenografts. Letrozole-treated tumors were collected at 4 weeks (when they were responding to letrozole), 28 and 56 weeks (when they were growing on letrozole), analysed by Western immunoblotting, and were compared with vehicle-treated tumors collected at week 4 (control). Numbers below the blots represent fold change in protein expression compared with the control obtained by densitometric analysis.

Figure 2

A, Effect of trastuzumab alone or in combination with letrozole on the growth of MCF-7Ca xenografts: Tumor growth rate of the mice in the combination group was significantly lower than control (p=0.004), single agent ENT (p=0.009) or letrozole (p=0.049)

Figure 3

Effect of ENT alone or in Combination with Letrozole on the Growth of ER negative MDA-MB-231 Xenografts: The mice bearing MDA-MB-231 xenografts were treated with ENT alone or in combination with letrozole. Tumor volumes were measured twice a week. Tumor growth rate of the mice in the combination group was significantly lower than control (p=0.004), single agent ENT (p=0.009) or letrozole (p=0.049)

Figure 4

Effect of Treatment with ENT and Letrozole Alone or in Combination on Colonization of MDA-MB-231 Cells in the Lungs of Mice: MDA-MB-231 cells were injected into the tail vein. The mice were treated with after 3 weeks with vehicle, ENT+Δ⁴A, Δ⁴A+ letrozole or combination for 6 weeks. Visible or micrometastatic foci were quantitated at autopsy. (A) The combination of ENT (ENT) plus letrozole produced significantly fewer visible lung foci compared to control (*p=0.002) and ENT (†p=0.02). (B) Mice treated with a combination of ENT plus letrozole had significantly fewer micrometastases compared to control (*p=0.0269) and ENT (†p=0.038).