Cellular effects and antitumor activity of RET inhibitor RPI-1 on MEN2A-associated medullary thyroid carcinoma

Giuditta Cuccuru, Cinzia Lanzi, Giuliana Cassinelli, Graziella Pratesi, Monica Tortoreto, Giovanna Petrangolini, Ettore Seregni, Antonia Martinetti, Diletta Laccabue, Chiara Zanchi, Franco Zunino
Journal of the National Cancer Institute 2004 July 7, 96 (13): 1006-14

BACKGROUND: The RET proto-oncogene encodes a receptor tyrosine kinase. RET oncogenes arise through sporadic and inherited gene mutations and are involved in the etiopathogenesis of medullary thyroid carcinoma, a cancer that responds poorly to conventional chemotherapy. Medullary thyroid carcinoma is a component of multiple endocrine neoplasia type 2 or MEN2 syndromes.

METHODS: We investigated the cellular effects of RPI-1, a novel 2-indolinone Ret tyrosine kinase inhibitor on cells that express RET C634 oncogenic mutants common in the MEN2A syndrome: NIH3T3 fibroblasts transfected with RET(C634R) and human medullary thyroid carcinoma TT cells that express endogenous RET(C634W). RPI-1 antiproliferative activity was determined by cell proliferation and anchorage-independent growth assays. Expression and phosphorylation of Ret and of proteins involved in downstream signaling pathways were examined by immunoblotting. Antitumor activity of oral RPI-1 treatment was tested by using two dosing levels in nude mice bearing subcutaneous TT xenograft tumors. All statistical tests were two-sided.

RESULTS: The RPI-1 IC50 value for cell proliferation was 3.6 microM (95% confidence interval [CI] = 1.8 to 5.4 microM) in NIH3T3 cells expressing the Ret mutant compared with 16 microM (95% CI = 12.3 to 19.7 microM) in non-transfected NIH3T3 cells, and that for colony formation in soft agar was 2.4 microM (95% CI = 0.8 to 4.0 microM) and 26 microM (95% CI = 17 to 35 microM) in RET mutant-transfected and H-RAS-transfected NIH3T3 cells, respectively. In NIH3T3 cells expressing the Ret mutant, Ret protein and tyrosine phosphorylation were undetectable after 24 hours of RPI-1 treatment. In TT cells, RPI-1 inhibited proliferation, Ret tyrosine phosphorylation, Ret protein expression, and the activation of PLCgamma, ERKs and AKT. In mice, oral daily RPI-1 treatment inhibited the tumor growth of TT xenografts by 81% (P<.001 versus control mice) and reduced the plasma levels of the specific biomarker calcitonin (P =.01 versus control mice). Twenty-five percent of RPI-1-treated mice were tumor-free.

CONCLUSIONS: Ret oncoproteins represent exploitable targets for therapeutic intervention in MEN2A-associated medullary thyroid carcinoma. The antitumor efficacy and oral bioavailability of RPI-1 support its therapeutic potential.

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