Functional activation of the estrogen receptor-α and aromatase by the HDAC inhibitor entinostat sensitizes ER-negative tumors to letrozole

Abstract

Approximately 25% of breast cancers do not express the estrogen receptor-α (ERα) and consequently do not respond to endocrine therapy. In these tumors, ERα repression is often due to epigenetic modifications such as methylation and histone deacetylation. For this reason, we investigated the ability of the histone deacetylase inhibitor entinostat (ENT) to trigger reexpression of ERα and aromatase in breast cancer cells, with the notion that this treatment would restore sensitivity to the aromatase inhibitor (AI) letrozole. ENT treatment of tumor cells increased expression of ERα and aromatase, along with the enzymatic activity of aromatase, in a dose-dependent manner both in vitro and in vivo. Notably, ERα and aromatase upregulation resulted in sensitization of breast cancer cells to estrogen and letrozole. Tumor growth rate was significantly lower in tumor xenografts following treatment with ENT alone and in combination with letrozole than in control tumors (P > 0.001). ENT plus letrozole also prevented lung colonization and growth of tumor cells, with a significant reduction (P > 0.03) in both visible and microscopic foci. Our results show that ENT treatment can be used to restore the letrozole responsiveness of ER-negative tumors. More generally, they provide a strong rationale for immediate clinical evaluation of combinations of histone deacetylase and aromatase inhibitors to treat ER-negative and endocrine-resistant breast cancers.

Figure-1

(A) Time Course of ENT Effect on mRNA Expression of ERα and Aromatase: Expression of mRNA was examined in MDA-MB-231 cells by RT-PCR at different time points (0-72h). Image shows ERα, aromatase (CYP-19) and 18s ribosomal RNA (rRNA) as loading control. (B) Effect of ENT in Presence or Absence of Estrogen or Δ⁴A and Letrozole on the mRNA Expression of pS2, PgR and Aromatase in MDA-MB-231 Cells: RT-PCR analysis shows pS2, PgR and aromatase (CYP-19) with 18s ribosomal RNA (rRNA) as loading control. (C) Effect of ENT on Aromatase Activity: MDA-MB-231 cells were treated with ENT with or without fulvestrant for 18h and then assayed for aromatase activity with (ENT⇒ Letrozole) or without letrozole (*p=0.006 versus control). (D) Effect of Combining Letrozole with ENT or SAHA in MDA-MB-231 Cells. Cell viability was measured by MTT assay after 6-day treatment with increasing concentrations of letrozole alone (IC₅₀>10μM) or in presence of ENT (100nM). IC₅₀ value for letrozole was 6.17nM when combined with ENT (100nM). (E) Effect of Δ⁴A on Response of MDA-MB-231 Cells to ENT. MTT assay was performed after 6-day treatment with ENT alone, or in the presence of aromatizable Δ⁴A (10nM) (F) Response of ER-positive or ER-negative cells to Δ⁴A with or without ENT Pre-treatment. MDA-MB-231 cells were pre-treated with ENT (1nM) or vehicle for 3 days and then with Δ⁴A for 3 days. MCF-7Ca cells were treated with Δ⁴A for 6 days. Cell viability was measured after 6 days using MTT assay.

Figure-2. ChIP Assay

Association of (a) acetyl histone H₃ (b) ERα (c) total histone H₃ or (d) negative control (no antibody) with the aromatase PI.3/II promoter. MDA-MB-231 cells were treated with indicated agents. Lane-1) control, 2) E₂ (10nM), 3) ENT (1μM), 4) ENT+E₂ 5) Δ⁴ A (100nM), 6) ENT plus Δ⁴A, 7) Δ⁴A plus letrozole (1μM) and 8) ENT plus Δ⁴A plus letrozole.

Figure-3

(A) Effect of ENT on the Growth of MDA-MB-231 Xenografts: MDA-MB-231 xenografts were grown in OVX athymic nude mice. Mice were treated with increasing doses of ENT and tumor volumes were plotted versus time (B) Effect of Doses of ENT on Tumor and Uterine Weights of Mice with MDA-MB-231 Xenografts: Tumor (left y-axis) and uterine (right y-axis) weights were measured at autopsy of above mice in 3A (C) Effect of Doses of ENT on ERα and Aromatase Protein Expression in MDA-MB-231 Xenografts: Western analysis of lysates of tumors from above mice in Fig 3A; from: Lane-1) vehicle treated control, 2) ENT (1mg/kg/day), 3) ENT (2.5mg/kg/day), 4) ENT (5mg/kg/day) and 5) ENT (10mg/kg/day). Blots show ERα, aromatase (CYP-19) and β-actin (D) Effect of Doses of ENT on Aromatase Activity of Mice with MDA-MB-231 Xenografts: Aromatase activity was measured by ³H₂O release assay and corrected for total protein concentration (*p<0.05 versus control) (E) Effect of Doses of ENT on the mRNA Expression of ERα, pS2 and Aromatase in MDA-MB-231 xenografts: Expression of mRNA was analyzed by RT-PCR. Lane-1) control, 2) ENT (1mg/kg/day), 3) ENT (2.5mg/kg/day), 4) ENT (5mg/kg/day), and 5) ENT (10mg/kg/day). A representative gel image shows ERα and aromatase (CYP-19) and 18s ribosomal RNA (rRNA) as loading control.

Figure-4

(A) Effect of ENT Alone or with Δ⁴A on the Growth of MDA-MB-231 Xenografts: MDA-MB-231 xenografts were grown in OVX nude mice and treated with vehicle, Δ⁴A (100μg/day), ENT (2.5mg/kg/day) or ENT plus Δ⁴A. The growth rates were not significantly different across the four treatment groups (B) Effect of ENT Alone or with Δ⁴A on the Tumor and Uterine Weights of Mice Bearing MDA-MB-231 Xenografts: Tumor (left y-axis) and uterine (right y-axis) weights were measured at autopsy of above mice in 4A (C) Effect of ENT Alone or with Δ⁴A Supplement on the mRNA Expression of PgR and pS2 in MDA-MB-231 Xenografts: RT-PCR analysis. Lane-1) control, 2) Δ⁴A (100 μg/day), 3) ENT (2.5 mg/kg/day), 4) ENT plus Δ⁴A, 5) Δ⁴A plus letrozole (10μg/day) and 6) ENT plus Δ⁴A plus letrozole. A representative gel image shows ERα and aromatase (CYP-19) and 18s ribosomal RNA (rRNA) as loading control.

Figure-5

(A) Effect of ENT alone or in Combination with Letrozole on the Growth of 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) (B) Effect of ENT Alone or in Combination with Letrozole on the Tumor and Uterine Weights: The tumors and uteri were collected and weighed at autopsy on day 53 of above mice in 5A. Tumor (left y-axis) and uterine (right y-axis) weights (C) Effect of ENT and Letrozole Alone or in Combination on the Protein Expression in MDA-MB-231 Xenografts: Tumors from of above mice in 5A were analyzed by western blotting. Blots show ERα, aromatase (CYP-19) and β-actin (D) Effect of ENT alone or in Combination with Letrozole on the Aromatase activity of MDA-MB-231 Xenografts: The aromatase activity in the tumors from mice above in 5A, treated with ENT is significantly higher (*^a p<0.001) than control, letrozole or ENT plus letrozole. The aromatase activity was significantly lower in letrozole or ENT plus letrozole group than ENT alone († p<0.001).

Figure-6. Effect of ENT with or without Letrozole on the Growth of Hs578T Xenografts

Xenografts of Hs578T cells were grown in ovariectomized female nude mice. Mice were treated as indicated. Each data point represents volume of one tumor. Tumor growth was significantly slower in the tumors of mice treated with ENT + letrozole (**p<0.01) compared to control, ENT+Δ⁴A and Δ⁴A+letrozole.

Figure-7. 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 quantitiated 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).