Oncogenic potential of a mutant human thyrotropin receptor expressed in FRTL-5 cells

Abstract

An abnormal stimulation of the cAMP pathway has been recognized as the primary event in various pathological situations that lead to goitrogenesis or thyroid tumors. Thyroid adenomas are monoclonal neoplasms that become independent of thyroid stimulating hormone (TSH) in their secretory function and growth. Mutated forms of the TSH receptor (TSHR) and the adenylyl cyclase-activating Gsa protein, which confer a constitutive activity on these proteins, have been observed in human adenomas. The FRTL-5 rat thyroid cell line is a permanent but untransformed line; the growth of which depends on the presence of TSH, and at least in part, on the stimulation of the cAMP pathway. In order to compare the oncogenic potential of the activated mutant Gsa protein and the constitutively activated TSHR, we have transfected FRTL-5 cells with an expression vector bearing either the cDNA of the Gsa gene carrying the A201S mutation or the cDNA of the TSH receptor carrying the M453T mutation recently identi®ed in a case of congenital hyperthyroidism. The expression of these two cDNAs was driven by the bovine thyroglobulin gene promoter. We show that, although the expression of both the Gsa or TSHR mutant proteins leads to TSHindependent proliferation and to constitutive cAMP accumulation in FRTL-5 cells, only the mutant TSHR is able to induce neoplastic transformation, as demonstrated by growth in semi-solid medium and tumorigenesis in nude mice.

Role of the cAMP and MAPK pathways in the transformation of mouse 3T3 fibroblasts by a TSHR gene constitutively activated by point mutation

“…The development of hyperfunctioning adenomas in transgenic mice has also been observed after expression of a G as mutated transgene (Michiels et al, 1994). On the contrary, until now there has been no report of the induction of a fully neoplastic transformed phenotype in vivo or in vitro by either a mutationally activated G as or by a TSHR wild type genes (Fourne s et al, 1998;Russo et al, 1995a;SaõÈ d et al, 1994).…”

Contrasting effects of activating mutations of GαS and the thyrotropin receptor on proliferation and differentiation of thyroid follicular cells

“…Similar TSH-independent stimulation of cAMP and proliferation have been reported separately both for GSP (Muca and Vallar, 1994) and a range of TSHR mutants (Porcellini et al, 1997) but few functional parameters were examined. The only previous study to attempt a direct comparison between GSP and TSHR clones obtained from the same parent population (Fournes et al, 1998) concluded that GSP exerted a weaker e ect than mTSHR (M453T) in terms of cAMP stimulation and doubling time in monolayer. However the former result was obtained in the presence of IBMX, and the latter may have been in¯uenced by the method of clonal selection (TSH-independent proliferation rather than`neutral' selection in G418).…”

“…Intriguingly though, di erential sensitivity of ligand-dependent and -independent stimulation to downregulatory control could provide an explanation for the observation (Fournes et al, 1998) that anchorage-independent opposed to monolayer growth of FRTL5 was strikingly stimulated by mTSHR, but not by TSHstimulation of the wt receptor (nor by GSP).…”

“…TSH-independent proliferation has also been seen with mTSHR, the extent varying in one study with the nature of the mutant (Porcellini et al, 1997), although it was not clear whether even the most potent construct (T632I) reached the level of TSH stimulated controls. In another study, comparing the M453T mutant with GSP, only the mutant receptor was able to induce clones with anchorage-independence and tumorigenicity in nude mice (Fournes et al, 1998), providing a ®rst hint that mTSHR may be a more potent oncogene.…”

“…Some were performed on pools of clones (Porcellini et al, 1997) which, given the heterogeneity of the response expected in gene transfer experiments, can lead to information being lost due either to overgrowth of more vigorously growing clones or by a dilution e ect from clones with low expression of the transgene. Others involved selection of individual clones based on their ability to overgrow the background culture in the absence of TSH (Fournes et al, 1998), which introduces a strong bias for the most proliferative phenotype and risks masking di erences in potency between GSP and TSHR. Furthermore, most previous studies have examined only e ects on proliferation rather than di erentiated thyrocyte functions.…”