Recent studies have revealed a role for platelets and the platelet-adhesive proteins, fibronectin and von Willebrand factor (vWF) in platelet-tumor cell interaction in vitro and metastasis in vivo. The present report documents the effect of thrombin treatment of platelets on this interaction in vitro and in vivo. In vitro, thrombin at 100-1,000 mU/ml maximally stimulated the adhesion of six different tumor cell lines from three different species two-to fivefold. As little as 1-10 mU/ml was effective. The effect of thrombin was specific (inhibitable by hirudin, dansyl-arginine N43-ethyl-1,5 pentanediyl) amide and unreactive with the inactive thrombin analogue N-P-tosyl-L-phenylchloromethylketone-thrombin and D-phenylalanyl-L-propyl-L-arginine chloromethylketone-thrombin (PPACK-thrombin), and required high-affinity thrombin receptors (competition with PPACK-thrombin but not with N-P-tosyl-L-lysine-chloromethyl-ketone-thrombin). Functionally active thrombin was required on the platelet surface. Binding of tumor cells to thrombin-activated platelets was inhibitable by agents known to interfere with the platelet GPIIb-GPIIIa integrin: monoclonal antibody 10E5, tetrapeptide RGDS and 'y chain fibrinogen decapeptide LGGAKQAGDV, as well as polyclonal antibodies against the platelet adhesive ligands, fibronectin and vWF. In vivo, thrombin at 250-500 mU per animal increased murine pulmonary metastases fourfold with CT26 colon carcinoma cells and 68-413-fold with B16 amelanotic melanoma cells. Thus, thrombin amplifies tumor-platelet adhesion in vitro twoto fivefold via occupancy of high-affinity platelet thrombin receptors, and modulation of GPIIb-GPIIIa adhesion via an RGD-dependent mechanism. In vivo, thrombin enhances tumor metastases 4-413-fold with two different tumor cells lines. (J. Clin. Invest. 1991. 87:229-236.).

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Anti-estrogen therapies are not effective in ER- breast cancers, thus identifying mechanisms underlying lack of ER expression in ER- breast cancers is imperative. We have previously 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. This study was performed to identify mechanisms of hMAPK-induced transcriptional repression of ER. We found that ER promoter activity is significantly reduced in the presence of hMAPK signaling, yet did not identify specific promoter sequences responsible for this repression. We performed an epigenetic compound screen in an ER- breast cancer cell line that expresses hMAPK yet does not exhibit ER promoter hypermethylation. A number of HDAC inhibitors were identified and confirmed to modulate ER expression and estrogen signaling in multiple ER- cell lines and tumor samples lacking ER promoter methylation. siRNA-mediated knockdown of HDACs 1, 2, and 3 reversed the mRNA repression in multiple breast cancer cell lines and primary cultures and ER promoter-associated histone acetylation increased following MAPK inhibition. These data implicate histone deacetylation downstream of hMAPK in the observed ER mRNA repression associated with hMAPK. Importantly, histone deacetylation appears to be a common mechanism in the transcriptional repression of ER between ER- breast cancers with or without ER promoter hypermethylation.

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