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 a bout 30 per cent of hyperfunctioning human thyroid adenomas and in a minori ty of differentiated thyroid carcinomas. The mutations activating the TSHR gene(s) in the thyroid carcinomas, were located at the codon 623 changing a n Ala to a Ser (GCC-->TCC) or in codon 632 changing a Thr to Ala or Ile (AC C-->GCC or ACC-->ATC). In order to study if the constitutively activated TS HR gene(s) has played a role in the determination of the malignant phenotyp e 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 responsable(s) for the malignant phenotype of the ce lls transformed by these constitutively activated TSHR cDNAs, Our results s how that (1) the TSHRM623 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) s uggest that the fully transformed phenotype of our 3T3 cells, may be the co nsequence of the additive effect of the activation of at least two differen t 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 p laying a key role as an effector in the MAPK pathway activation in our 3T3- transformed cells is PI3K gamma. Signaling from PI3K gamma to MAPK appears to require in our murine cellular system a tyrosine kinase (still not chara cterized), Shc, Grb2, Sos, Ras and Raf. It is proposed that the constitutiv ely activated TSHR genes detected in the thyroid carcinomas, may have playe d an oncogenic role, participating in their development through these two p athways.