Abstract 2098: Histone deacetylation underlying hMAPK-induced ER mRNA repression
Authors: Plotkin Amy J. , Volmar Claude-Henry , Ayad Nagi , El-Ashry Dorraya 
Published: May 2015

Estrogen receptor (ER)-negative breast cancer is more aggressive and associated with both shorter disease-free and overall survival than ER+ breast cancer. While anti-estrogen therapies have greatly improved treatment for ER+ breast cancer, both de-novo and acquired resistance occurs. Anti-estrogen therapies are not effective in ER- breast cancers, thus identifying mechanisms underlying lack of ER expression in ER- breast cancers is imperative. Hypermethylation of the ER promoter has been demonstrated to repress ER, and is found in ∼ 25% of ER- breast cancers. Previously we demonstrated that hyperactivation of MAPK (hMAPK) downstream of overexpressed EGFR or overexpression/amplification of Her2 represses ER protein and mRNA expression. Abrogation of hMAPK in ER- breast cancer cell lines and primary cultures causes re-expression of ER and restoration of anti-estrogen responses. hMAPK-mediated ER mRNA repression involves repression of transcription. We found that the ER mRNA synthesis rate and ER promoter activity, assessed with run-on assays and ER promoter reporter constructs, respectively, were significantly reduced in hMAPK-MCF-7 cell line models, with little to no ER expression, compared to control-transfected MCF-7 (coMCF-7) cells with high ER expression. Co-transfection of dnERK1/2 abrogated this repression restoring promoter activity to levels comparable with coMCF-7s but promoter bashing did not identify specific sequences responsible for this hMAPK repression. Thus we hypothesized that epigenetic mechanisms may be involved in hMAPK mediated repression of ER transcription; however, we could not detect ER promoter methylation in our hMAPK-MCF-7 models, nor in several breast cancer cell lines with hMAPK.

In this study, we performed an epigenetic compound screen in the ER- SUM 149 breast cancer cell line, which presents hMAPK, and had been previously demonstrated to re-express ER with MAPK inhibition. Three candidate compounds, all HDAC inhibitors, were identified, validated in individual luciferase assays, and evaluated with rtPCR and Western blotting to verify their ability to modulate ER expression and estrogen signaling. They have been validated in additional ER- cell lines and clinical tumor samples lacking ER promoter methylation, as well. Finally, they have been used in our hMAPK-MCF-7 cells to demonstrate the role of hMAPK in histone deacetylation of the ER promoter.

While HDAC inhibitors have been evaluated previously for their ability to sensitize anti-estrogen resistant ER+ breast cancers and re-express ER in ER- breast cancer cell lines exhibiting ER promoter methylation, histone deacetylation of ER in ER- breast cancers lacking ER promoter methylation has not been previously identified. Collectively, these data link histone deacetylation as an underlying mechanism in hMAPK-repression of ER mRNA suggesting this is may be a common epigenetic mechanism in the generation of ER-negative breast cancer.

Citation Format: Amy J. Plotkin, Claude-Henry Volmar, Nagi Ayad, Dorraya El-Ashry. Histone deacetylation underlying hMAPK-induced ER mRNA repression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2098. doi:10.1158/1538-7445.AM2014-2098