Abstract 3432: HSP90 expression and antitumoral activity of geldanamycin and its analog 17-AAG in gallbladder cancer.

Abstract

Abstract Background. Gallbladder carcinoma (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. In Chile, it has an extremely high incidence and is the second cause of death by cancer among women. Heat shock protein 90 (HSP90) is a molecular chaperone and is one of the most abundant proteins expressed in cells. Many oncogenic proteins responsible for the transformation of cells to cancerous forms are client proteins of HSP90. Two molecules have shown to inhibit the action of HSP90, geldanamycin (GA) and its analog 17-allylamino-17-des-methoxygeldanamycin (17-AAG) and hence a potential target for adjuvant therapy. HSP90 expression and antitumoral activity of GA and 17-AAG have not been studied in GBC. Methods. Expression of HSP90 was analyzed in 47 selected GBC cases by immunohistochemical staining using a monoclonal mouse HSP90 antibody (clone JPB24, dilution 1:200, Novocastra, Newcastle upon Tyne, UK). A positive case was interpreted if at least 50% of the tumor showed cytoplasmic staining. HSP90 expression was also evaluated in 6 GBC cell lines by Western blot analysis. A viability assay was performed in GB-d1 cells to determine the median lethal dose (LD50) of GA and 17-AAG. After treatment with different concentrations of each inhibitor for 48 h, the percentage of viable cells was assessed with a colorimetric MTS cell proliferation assay (Promega, Madison, WI). Treatment with 0.1% of DMSO as solvent was used as control. We tested the change of migration capability of GB-d1 cells by Transwell chambers after 24h with GA (15μM) and 17-AAG (12μM) treatment. Western blotting of GB-d1 cells before and after treatment with each inhibitor was done to evaluate the expression of HSP90, EGFR, ERK1/2, Cyclin B1 and ACTB. Results. HSP90 was frequently overexpressed in GBC primary tumor and cell lines. The staining intensity ranged from weak to strong, with 35 cases (74%) showing moderate intensity. Average expression was 78%. The MTS cell viability demonstrated that both GA and 17-AAG elicited cytostatic and cytotoxic effects on GB-d1 cells. After 24h treatment with GA or 17-AAG, the migration rates of GB-d1 cells decreased drastically up to 5% together with the down-regulation of EGFR and Cyclin B1. Conclusions. Our results showed that HSP90 is highly expressed in GBC making it a good candidate for targeted therapy. HSP90 inhibition with GA or 17-AAG induces cytotoxicity and significantly reduces in vitro cell migration and the expression of oncogenic client proteins of HSP90. Our results suggest that the inhibition of HSP90 function by GA and 17-AAG may provide a promising therapeutic strategy for the treatment of GBC. However, these results must be complemented with in vivo assays and clinical trials. Grant Support: FONDECYT 1090171 Citation Format: Jose R. Valbuena, Juan C. Roa, Pamela A. Leal, Patricia A. Garcia, Alejandro Corvalan. HSP90 expression and antitumoral activity of geldanamycin and its analog 17-AAG in gallbladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3432. doi:10.1158/1538-7445.AM2013-3432