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Multiple endocrine neoplasia type 2 (MEN2) is a hereditary cancer syndrome. MEN2 patients develop endocrine tumors like MTC and pheochromocytoma (PC). MEN2 is caused by activating germline mutations in the proto-oncogen RET, which are also detected as somatic mutations in a subset of sporadic MTCs and PCs. Tumorigenesis is known to be a multistep process in which several genes are involved. Here we investigated the role of cell cycle inhibitor P18(INK4C) in the multistep tumorigenesis of medullary thyroid carcinomas (MTC). Upon screening of human MTC and PC, we detected inactivating somatic mutations in the P18 gene. Because all these P18 mutations coincided with RET mutations, transgenic MEN2B-RET mice were crossed with P18 knockout mice to study the involvement of RET and P18 in the multistep tumorigenesis of MTC. Plasma calcitonin (CT) levels were measured to monitor MTC development. Up to nine months of age, RET;P18 mice showed a much higher frequency of MTC development (26%) as compared to the single mouse strains RET (0%) and P18 (2%), indicating a synergistic effect of loss of P18 and activation of RET in MTC development. Furthermore, MTC incidence was higher in homozygous P18 knockout groups compared to heterozygous P18 knockouts. Some of the MTCs of heterozygous P18 knockout mice displayed loss of P18 expression, suggesting LOH of P18 in these tumors. Loss of P27 is known to contribute to the phenotype of P18 knockout mice: 42% of P18;P27 double knockout mice developed MTC up to nine months of age. Activation of RET in this background did not result in higher MTC incidence (44%), however, it resulted in earlier onset and probably larger MTCs, as indicated by significantly increased CT levels. Taken together, our results indicate that P18 classifies as a classical tumor suppressor gene in MTC, which functionally cooperates with RET in the multistep tumorigenesis of these tumors.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA