HSP90 inhibition is a potential therapeutic strategy in treatment of adenocarcinoma of the esophagus

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The HSP90 dependent proteins are termed as client-proteins (CP), including many oncogenic signaling proteins, receptors, kinases and more. Functional inhibition of HSP90 by Geldanamycin (17-AAG) leads to down regulation of CP and represents a promising therapeutic strategy in malignant diseases. Esophageal carcinoma is a deadly disease. Molecular profiling of adenocarcinoma of the esophagus uncovered Insulin-like-Growth-Factor I Receptor (IGF-IR), Epidermal-Growth-Factor-Receptor (EGF-R1) and HER2 being involved in tumor progression. Efforts are made to counteract tumor progression by targeting these receptors by specific antibodies. However IGF-IR, EGF-R1 and HER2 represent CP of HSP90 hence HSP90 represents a therapeutic target in adenocarcinoma of the esophagus.
 Aim of our study was to evaluate the efficacy of different HSP90 inhibitors (17-AAG, 17-AEPGA and 17-DMAG) on several esophageal adenocarcinoma cell lines in vitro and in vivo.
 Primary human esophagus carcinoma cells PT1590, OE19, OE33 and lymph node metastasis cell line (LN1590) were used for in vitro experiments. Antiproliferative effect of 17-AAG, 17-AEPGA and 17-DMAG was evaluated by the MTT reduction assay. HSP90 functional inhibition and down regulation of IGF-IR, EGF-R1 and HER2 were evaluated by immuno-cytochemistry. For verification and quantification of down regulation of IGF-IR, EGF-R1 and HER2 western blot (WB) analysis were performed. Apoptosis was proven by caspase-3 and poly (ADP-ribose) polymerase (PARP) staining and WB analysis. Furthermore in an orthotopic nude-mice model efficacy of 17-DMAG was tested in vivo. Nude-mice were implanted previously subcutaneously grown OE19 cells orthotopically. After 10 days magnetic resonance imaging (MR) was performed. In all animals successful tumor implantation was confirmed. Animals were randomized by a third person into treatment and sham group. At time of randomization the tumor volume did not differ between both groups. Treatment group received 17-DMAG intraperitoneally three times a week for total 30 days. Tumor growth was traced by regular MR scanning.
 17-AAG, 17-AEPGA and 17-DMAG inhibited cell proliferation in all three tested esophagus carcinoma cell lines highly significantly (p < 0.001). Also the metastasis cell line (LN1590) was significantly inhibited (p < 0.001). The calculated IC50 for all tested cell lines was under 1 µM. All three tested inhibitors were highly effective. Western blot analysis performed with protein extracts of the treated and non-treated cells revealed highly significant down regulation of IGF-IR, EGF-R1 and HER2 in all tested cell lines including metastasis (p < 0.0001). Down regulation was confirmed by immuno-cytochemistry. Apoptosis was confirmed as the underlying mechanism by caspase-3 and PARP detection. The nude mice experiment with intra-peritoneal treatment with 17-DMAG resulted in significant reduced tumor volume in treated animals compared to sham group.
 Client-proteins like IGF-IR, EGF-R1 and HER2 are essential for esophageal adenocarcinoma progression and represent therapeutic targets. Our data suggest HSP90 inhibition is a promising therapy modality in adenocarcinoma of the esophagus and takes advantage of simultaneous down regulation of multiple CP. Ongoing experiments will disclose new CP involved in tumor progression of adenocarcinoma of the esophagus.