Hyperactive ERK and persistent mTOR signaling characterize vemurafenib resistance in papillary thyroid cancer cells
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Elyse K. Hanly1, Neha Y. Tuli1, Robert B. Bednarczyk1, Robert Suriano1,2, Jan Geliebter1, Augustine L. Moscatello3, Zbigniew Darzynkiewicz4, Raj K. Tiwari1
1Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA
2Division of Natural Sciences, College of Mount Saint Vincent, Bronx, NY 10471, USA
3Department of Otolaryngology/Head and Neck Surgery, New York Medical College, Valhalla, NY 10595, USA
4Department of Pathology, New York Medical College, Valhalla, New York 10595, USA
Raj K. Tiwari, e-mail: [email protected]
Keywords: thyroid cancer, vemurafenib, BRAFV600E, drug resistance, cell signaling
Received: August 31, 2015 Accepted: November 25, 2015 Published: December 28, 2015
Clinical studies evaluating targeted BRAFV600E inhibitors in advanced thyroid cancer patients are currently underway. Vemurafenib (BRAFV600E inhibitor) monotherapy has shown promising results thus far, although development of resistance is a clinical challenge. The objective of this study was to characterize development of resistance to BRAFV600E inhibition and to identify targets for effective combination therapy. We created a line of BCPAP papillary thyroid cancer cells resistant to vemurafenib by treating with increasing concentrations of the drug. The resistant BCPAP line was characterized and compared to its sensitive counterpart with respect to signaling molecules thought to be directly related to resistance. Expression and phosphorylation of several critical proteins were analyzed by Western blotting and dimerization was evaluated by immunoprecipitation. Resistance to vemurafenib in BCPAP appeared to be mediated by constitutive overexpression of phospho-ERK and by resistance to inhibition of both phospho-mTOR and phospho-S6 ribosomal protein after vemurafenib treatment. Expression of potential alternative signaling molecule, CRAF, was not increased in the resistant line, although formation of CRAF dimers appeared increased. Expression of membrane receptors HER2 and HER3 was greatly amplified in the resistant cancer cells. Papillary thyroid cancer cells were capable of overcoming targeted BRAFV600E inhibition by rewiring of cell signal pathways in response to prolonged vemurafenib therapy. Our study suggests that in vitro culture of cancer cells may be useful in assessing molecular resistance pathways. Potential therapies in advanced thyroid cancer patients may combine vemurafenib with inhibitors of CRAF, HER2/HER3, ERK, and/or mTOR to delay or abort development of resistance.
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