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

Abstract

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.

• Authors: Giuditta Cuccuru, Cinzia Lanzi, Giuliana Cassinelli, Graziella Pratesi, Monica Tortoreto, Giovanna Petrangolini, Ettore Seregni, Antonia Martinetti, Diletta Laccabue, Chiara Zanchi, Franco Zunino
• Journal: Journal of the National Cancer Institute (J Natl Cancer Inst) Vol. 96 Issue 13 Pg. 1006-14 (Jul 07 2004) ISSN: 1460-2105 [Electronic] United States
• PMID: 15240784 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antineoplastic Agents
• Indoles
• MAS1 protein, human
• Oncogene Proteins
• Proto-Oncogene Mas
• Proto-Oncogene Proteins
• RPI-1 compound
• Protein-Tyrosine Kinases
• Proto-Oncogene Proteins c-ret
• RET protein, human
• Receptor Protein-Tyrosine Kinases
• Protein Serine-Threonine Kinases
• Proto-Oncogene Proteins c-akt
• Mitogen-Activated Protein Kinases
• Type C Phospholipases
• Phospholipase C gamma

Topics

• Administration, Oral
• Animals
• Antineoplastic Agents (administration & dosage, pharmacology)
• Blotting, Western
• Carcinoma, Medullary (drug therapy, etiology, metabolism)
• Cell Division (drug effects)
• Female
• Gene Expression Regulation, Neoplastic (drug effects)
• Indoles (administration & dosage, pharmacology)
• Mice
• Mice, Nude
• Mitogen-Activated Protein Kinases (metabolism)
• Multiple Endocrine Neoplasia Type 2a (complications)
• Oncogene Proteins (antagonists & inhibitors, metabolism)
• Phospholipase C gamma
• Phosphorylation (drug effects)
• Precipitin Tests
• Protein Serine-Threonine Kinases (metabolism)
• Protein-Tyrosine Kinases (antagonists & inhibitors)
• Proto-Oncogene Mas
• Proto-Oncogene Proteins (metabolism)
• Proto-Oncogene Proteins c-akt
• Proto-Oncogene Proteins c-ret
• Receptor Protein-Tyrosine Kinases (antagonists & inhibitors, metabolism)
• Signal Transduction (drug effects)
• Thyroid Neoplasms (drug therapy, etiology, metabolism)
• Transplantation, Heterologous
• Type C Phospholipases (metabolism)