Abstract 2124: The HIV protease inhibitor Nelfinavir inhibits medullary thyroid cancer cell growth.
Authors: Kushchayeva Yevgeniya , Patel Aneeta , Costello John , Vasko Vasyl , Carroll Nancy , Jackson Rubie Sue , Burman Kenneth , Boyle Lisa 
Published: August 2014

Abstract
Background. Medullary thyroid cancer (MTC) is associated with RET mutations and activation of PI3K/AKT and MEK/ERK signaling pathways. Heat shock protein 90 (HSP90) contributes to functional stabilization of PI3K/AKT signaling and is required for growth factor inducible activation of the MEK/ERK pathway. A recent study showed that HSP90 is a molecular target for HIV protease inhibitor Nelfinavir (NFV). Treatment with NFV is associated with growth inhibition and induction of apoptosis in breast, ovarian and prostate cancer cell lines. Objectives. We hypothesized that NFV-inducible inhibition of HSP90 would lead to down-regulation of PI3K/AKT and MAPK/ERK signaling pathways and induction of apoptosis in MTC cells. Methods. MTC-derived cells (TT cells) harboring the RET C634W mutation were treated with NFV at concentrations from 1μM to 10μM. Activation of MAPK/ERK and AKT/ mTOR/p70S6K/pS6 signaling was assessed by Western blot using specific phospho-antibodies. Apoptosis was determined by Western Blot with antibody against cleaved caspase 3. Cell growth and viability were determined by Vi-Cell analyzer and Alamar blue assay. HSP90 expression was examined in 12 human MTC tumor samples by immunostaining. HSP90 expression was correlated with pAKT and pERK level. Results. Treatment with NFV (1μM to 10μM for 48 hours) did not affect expression of HSP90 or levels of total AKT and ERK. In contrast, pAKT and pERK were inhibited in TT cells after treatment with NFV (5μM and 10 μM). Since both AKT and ERK pathways contribute to regulation of p70S6K/pS6 activity, we examined activation of this signaling axis. NFV (10μM) decreased the level of p-p70S6K, p-pS6 and down-regulated Cyclin D1. Growth experiments demonstrated that the number of viable cells decreased by 30% and 85% after treatment with NFV at concentration 5μM and 10μM, respectively. Microscopy showed that exposure to NFV was associated with cell rounding and nuclear fragmentation, suggesting induction of apoptosis. Western blot analysis revealed that NFV induced caspase 3 cleavage in TT cells. In addition, treatment with NFV was associated with induction of γH2AX, indicating DNA damage. Immunostaining of human MTC showed increased expression of HSP 90 in 5/12 of examined MTCs compared to corresponding normal thyroid tissue. The highest level of cytoplasmic HSP90 was detected in 3 cases of MTC harboring RET mutation, and co-localization staining showed that cells with high level of HSP90 expression also exhibited prominent pAKT and pERK. Conclusion. Treatment of MTC cell with clinically achievable concentrations of NFV results in down-regulation of AKT and ERK signaling and inhibition of MTC cell growth. Expression of HSP90 in human tumors suggests that it has a role in maintenance of anti-apoptotic signaling in MTC cells. Together these data support the potential utility of NFV for the treatment of patients with MTC.
Citation Format: Yevgeniya Kushchayeva, Aneeta Patel, John Costello, Vasyl Vasko, Nancy Carroll, Rubie Sue Jackson, Kenneth Burman, Lisa Boyle. The HIV protease inhibitor Nelfinavir inhibits medullary thyroid cancer cell growth. [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 2124. doi:10.1158/1538-7445.AM2013-2124