Multistep tumorigenesis of medullary thyroid carcinoma in man and transgenic mouse models

Abstract

Medullary thyroid carcinoma (MTC) originates from the thyroid gland C-cells. MTC is found as a sporadic tumor and MTC is the most important clinical manifestation of the Multiple Endocrine Neoplasia type 2 (MEN 2) syndrome. MEN 2 is associated with germline mutations in the RET gene. The RET proto-oncogene encodes the RET receptor tyrosine kinase. There are two subtypes of MEN 2. In MEN 2A the mutations affect cysteine residues in the extracellular domain of the protein and in MEN 2B the most common mutation results in a M918T substitution in the intracellular domain of the protein. This mutation is also found as a somatic mutation in 30-40% of sporadic MTC cases. To study the tumorigenesis of MTC we generated transgenic mice expressing either the human non-mutated RET proto-oncogene (CALC-WT-RET) or the human RET oncogene with the M918T mutation (CALC-MEN2B-RET) in the C-cells. Thyroid abnormalities were never observed in CALC-WT-RET transgenic mice or control non-transgenic mice analyzed up to an age of more than two years. In CALC-MEN2B-RET mice a thyroid phenotype was observed. Three out of eight CALC-MEN2B-RET transgenic founders presented with macroscopic bilateral MTC and MTC was detected in 13% of mice from an established CALC-MEN2B-RET line. These result showed that the MEN 2B mutation in the RET oncogene predisposes mice for MTC. The CALC-MEN2B-RET mice developed MTC with low incidence and a variable latency period, suggesting the involvement of additional oncogenic events. The nature of the additional oncogenic events involved in MTC progression was unknown. We studied a potential role for the P53 tumor suppressor in human and mouse RET-induced MTC development. In a panel of human MEN 2 and sporadic MTC samples and CALC-MEN2B-RET derived MTCs immunohistochemistry revealed P53 staining, which is indicative for mutated P53. A possible causal relation between P53 gene inactivation and MTC development was studied by monitoring CALC-MEN2B-RET transgenic mice lacking one or both alleles of the P53 gene. A significantly increased incidence of MTC was observed in composite transgenic mice compared to controls, which indicates that inactivation of P53 contributes to Ret-induced MTC development. Deregulation of the Wnt/beta-Catenin/Tcf signal transduction pathway was associated with several forms of cancer. Potential involvement of this pathway in RET-induced MTC development was investigated. Tumor-specific deregulation of the Wnt/beta-Catenin/Tcf signal transduction pathway was detected in man and transgenic mice. To study effects of deregulated Wnt/beta-Catenin/Tcf signaling on RET-induced MTC development in vivo, we generated CALC-MEN2B-RET transgenic mice also carrying the APC(min) mutation. Monitoring of these mice revealed that deregulation of Wnt/beta-Catenin/Tcf signaling contributes to RET-induced MTC development. A frequent finding in MTC is loss of heterozygosity at chromosome 1p, indicating a potential tumor suppressor gene of MTC development on this chromosome arm. Therefore, we performed mutation analysis of the P18 gene, located on 1p32 and involved in cell cycle regulation. In one MEN 2 and two sporadic MTC cases mutations in the P18 gene were detected. Both sporadic MTCs had also acquired a somatic RET mutation. These data strongly indicate, that P18 can act as a tumor suppressor of human MTC and suggest collaboration of loss of P18 function with activation of RET during MTC development. Identification of these three pathways as additional oncogenic events in RET-induced MTC development provides us with important novel targets for therapy, as there are currently no effective treatment modalities available for metastatic MTC.