title: 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.affiliation: Preclinical Chemotherapy and Pharmacology Unit, and Nuclear Medicine Unit, Istituto Nazionale Tumori, Milan, Italy.
authors.forename: Giuditta
authors.initials: G
authors.lastname: Cuccuru
authors.forename: Cinzia
authors.initials: C
authors.lastname: Lanzi
authors.forename: Giuliana
authors.initials: G
authors.lastname: Cassinelli
authors.forename: Graziella
authors.initials: G
authors.lastname: Pratesi
authors.forename: Monica
authors.initials: M
authors.lastname: Tortoreto
authors.forename: Giovanna
authors.initials: G
authors.lastname: Petrangolini
authors.forename: Ettore
authors.initials: E
authors.lastname: Seregni
authors.forename: Antonia
authors.initials: A
authors.lastname: Martinetti
authors.forename: Diletta
authors.initials: D
authors.lastname: Laccabue
authors.forename: Chiara
authors.initials: C
authors.lastname: Zanchi
authors.forename: Franco
authors.initials: F
authors.lastname: Zunino
mesh_terms: D000284:Administration, Oral; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D015153:Blotting, Western; D018276:Carcinoma, Medullary / Q000188:drug therapy* / Q000209:etiology / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D007211:Indoles / Q000008:administration & dosage / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018813:Multiple Endocrine Neoplasia Type 2a / Q000150:complications*; D015513:Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051966:Phospholipase C gamma; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013964:Thyroid Neoplasms / Q000188:drug therapy* / Q000209:etiology / Q000378:metabolism; D014183:Transplantation, Heterologous; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D007211:Indoles; C000715470:MAS1 protein, human; D015513:Oncogene Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; C477359:RPI-1 compound; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma