Role of MEN2A-derived RET in maintenance and proliferation of medullary thyroid carcinoma

Background: Dominant-activating mutations in the RET protooncogene, a receptor tyrosine kinase, have been identified as a cause of medullary thyroid carcinoma. Such oncogenic RET mutations induce its ligand-independent constitutive trans-autophosphorylation. We investigated the role of endogenous oncogenic RET autophosphorylation in maintaining the neoplastic phenotype in medullary thyroid carcinoma cells and orthotopic medullary thyroid carcinomas in RET transgenic mice. Methods: We constructed adenoviral vectors expressing a dominant-negative truncated form of RET, termed RE[T.sup.[DELTA]TK], and analyzed its effect on cell viability, apoptosis, and proliferation of TT medullary thyroid carcinoma cells. We investigated the effect of RE[T.sup.[DELTA]TK] on downsteam signaling by assessing alterations in phosphorylation or in gene expression. The effect of RE[T.sup.[DELTA]TK] in primary medullary thyroid carcinomas in transgenic mice was assessed by monitoring tumor growth. All statistical tests were two-sided. Results: Cell viability was reduced. Phosphorylation of Akt and extracellular signal-regulated kinase (ERK), components of downstream signal transduction pathways, was abolished, and cell cycle progression was reduced. Expression of cell cycle regulator cyclin D1 was decreased, and expression of cell cyle regulators [p21.sup.CIP1/WAF1] and [p27.sup.KIP1] was increased. Apoptosis was stimulated and concurrently the expression of BCL-2 was decreased. All in vitro experiments compared TT cells expressing RE[T.sup.[DELTA]TK] with untreated control cells or control vector-treated cells. Furthermore, 2 weeks after injecting adenovirus-carrying RE[T.sup.[DELTA]TK] into thyroid glands of transgenic mice with orthotopic medullary thyroid carcinoma, tumors were statistically significantly smaller than their initial size in mice treated with RE[T.sup.[DELTA]TK] (43.6%, 95% confidence interval [CI] = 30.7% to 56.5% ; P = .010; two-sided unpaired Student's t test), whereas tumors in mice treated with a control vector were larger than their initial size (139.8%, 95% CI = 120.3% to 159.3%; P