HSP90 INHIBITION AS A NOVEL THERAPEUTIC APPROACH FOR THE TREATMENT OF AGGRESSIVE THYROID CARCINOMAS

McMillin W.D.¹, Kotoula V.², Negri J.¹, Hayden P.¹, Koletsa T.², Mitsiades S.C.¹, Mitsiades N.^{1, 3}

¹Department of Medical Oncology, Dana Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston MA, USA, ²Department of Pathology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece, ³Memorial Sloan Kettering Cancer Center, New York, USA

Abstract

Context: Hsp90 is a molecular chaperone that interacts with several intracellular proteins, such as Akt, IGF1R, Raf, etc, to facilitate their proper folding and preserve their correct 3-dimensional conformation and functional status. Hsp90 inhibitors (e.g.17- allylamino- 17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG)) have shown promising anticancer activity and are undergoing clinical evaluation.
Objective – Methods: We investigated the effect of 17-AAG and 17-DMAG (Calbiochem, San Diego, CA) on a panel of 6 thyroid carcinoma cell lines with MTT assay, flow cytometry, gene expression profiling, RT-PCR and immunoblotting. The DNA binding activity of p53 was quantified by enzyme linked immunosorbent assay (ELISA) using the Trans-AMT p53 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA). PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis), a cell-permeable small molecule that can restore the activity of mutant p53, was from Calbiochem.
Results: Both 17-DMAG and 17-AAG induced growth arrest and apoptosis in vitro in thyroid carcinoma cells, including those of anaplastic and medullary origin, suppressed Akt, IGF1R and B-Raf expression, upregulated Hsp90 expression and induced caspase-3 and PARP cleavage. Apoptosis induced by 17-DMAG was partially abrogated by the caspase inhibitor ZVAD-FMK, as well as by transfection of myristoylated (constitutively active) Akt orBcl-2 cDNA. 17-DMAG suppressed p53 expression and DNA-binding activity, as well as expression of the p53-dependent pro-apoptotic molecules Bax, Noxa and PUMA. Concurrent treatment with the p53 activator PRIMA-1 or the DNA-damaging chemotherapeutic doxorubicin enhanced 17-DMAG-induced apoptosis.
Conclusions: Hsp90 inhibitors suppress the expression of several proliferative/anti-apoptotic mediators and induce growth arrest and caspase-dependent apoptosis in thyroid carcinomas. Hsp90 chaperones p53, and Hsp90 inhibition suppresses p53 expression and activity. Reactivation of p53, such as with the small molecule PRIMA-1 or with DNA-damaging chemotherapy may sensitize 17-DMAG-treated thyroid carcinoma cells to apoptosis. These findings have direct implications for the development of novel combination therapies for aggressive thyroid carcinomas.