Role of the cAMP and MAPK pathways in the transformation of mouse 3T3 fibroblasts by a TSHR gene constitutively activated by point mutation.
Constitutive activating mutations of the TSHR gene, have been detected in about 30 per cent of hyperfunctioning human thyroid adenomas and in a minority of differentiated thyroid carcinomas. The mutations activating the TSHR gene(s) in the thyroid carcinomas, were located at the codon 623 changing an Ala to a Ser (GCC-->TCC) or in codon 632 changing a Thr to Ala or Ile (ACC-->GCC or ACC-->ATC). In order to study if the constitutively activated TSHR gene(s) has played a role in the determination of the malignant phenotype presented by these tumors, we investigated: (1) the transforming capacity after transfection of mouse 3T3 cells, of a TSHR cDNA activated by an Ala-->Ser mutation in codon 623 or an Thr-->Ile mutation in codon 632 and (2) the pathway(s) eventually responsible(s) for the malignant phenotype of the cells transformed by these constitutively activated TSHR cDNAs. Our results show that (1) the TSHR(M623) or (M632) cDNAs give rise to 3T3 clones presenting a fully neoplastic phenotype (growth in agar and nude mouse tumorigenesis); this phenotype was weaker in the cells transformed by the 632 cDNA; (2) suggest that the fully transformed phenotype of our 3T3 cells, may be the consequence of the additive effect of the activation of at least two different pathways: the cAMP pathway through G(alpha)s and the Ras dependent MAPK pathway through G(beta)gamma and PI3K and (3) show that the PI3K isoform playing a key role as an effector in the MAPK pathway activation in our 3T3-transformed cells is PI3Kgamma. Signaling from PI3Kgamma to MAPK appears to require in our murine cellular system a tyrosine kinase (still not characterized), Shc, Grb2, Sos, Ras and Raf. It is proposed that the constitutively activated TSHR genes detected in the thyroid carcinomas, may have played an oncogenic role, participating in their development through these two pathways.
Du Villard
J A
JA
Laboratoire d'Instabilité Génétique et Cancer (UPR 2169), Institut de Recherches sur le Cancer, CNRS, Villejuif, France.
Wicker
R
R
Crespo
P
P
Russo
D
D
Filetti
S
S
Gutkind
J S
JS
Sarasin
A
A
Suárez
H G
HG
D016475:3T3 Cells / Q000378:metabolism / Q000637:transplantation; D019943:Amino Acid Substitution; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D058544:Class Ib Phosphatidylinositol 3-Kinase; D003062:Codon / Q000235:genetics; D000242:Cyclic AMP / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000502:physiology*; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D005786:Gene Expression Regulation; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009857:Oncogenes; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D017354:Point Mutation; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology*; D013964:Thyroid Neoplasms / Q000235:genetics; D014162:Transfection; D018909:src Homology Domains
D003062:Codon; D018076:DNA, Complementary; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D058544:Class Ib Phosphatidylinositol 3-Kinase; C546760:PIK3CG protein, human; C546764:Pik3cg protein, mouse; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins