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 RET(DeltaTK), and analyzed its effect on cell viability, apoptosis, and proliferation of TT medullary thyroid carcinoma cells. We investigated the effect of RET(DeltaTK) on downsteam signaling by assessing alterations in phosphorylation or in gene expression. The effect of RET(DeltaTK) 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(CIP1/WAF1) and p27(KIP1) was increased. Apoptosis was stimulated and concurrently the expression of BCL-2 was decreased. All in vitro experiments compared TT cells expressing RET(DeltaTK) with untreated control cells or control vector-treated cells. Furthermore, 2 weeks after injecting adenovirus-carrying RET(DeltaTK) into thyroid glands of transgenic mice with orthotopic medullary thyroid carcinoma, tumors were statistically significantly smaller than their initial size in mice treated with RET(DeltaTK) (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<.001).


CONCLUSIONS
Selective disruption of oncogenic RET signaling in medullary thyroid carcinoma in vitro and in vivo is associated with loss of the neoplastic phenotype of medullary thyroid carcinoma and should be investigated further as the basis for new therapeutic approaches for this disease.
Drosten
Matthias
M
Department of Vectorology and Experimental Gene Therapy, University of Rostock, Schillingalle 70, D-18055 Rostock, Germany.
Hilken
Gero
G
Böckmann
Miriam
M
Rödicker
Florian
F
Mise
Nikica
N
Cranston
Aaron N
AN
Dahmen
Uta
U
Ponder
Bruce A J
BA
Pützer
Brigitte M
BM
D000256:Adenoviridae; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D018276:Carcinoma, Medullary / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D045744:Cell Line, Tumor; D002470:Cell Survival; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018813:Multiple Endocrine Neoplasia Type 2a / Q000378:metabolism*; D015513:Oncogene Proteins / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000235:genetics / Q000378:metabolism*
D015513:Oncogene Proteins; D015514:Oncogene Proteins, Fusion; D011518:Proto-Oncogene Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C022616:thiazolyl blue; D001973:Bromodeoxyuridine