Endocrine Abstracts

Thyroid cancer is the most common type of endocrine tumor and reaches the peak of incidence between the age of twenty and fifty years. It has 4-fold higher prevalence in females than males, suggesting that estrogens and their receptors could be involved in thyroid cancer pathogenesis. Previous studies demonstrated allosteric interference operated by G protein-coupled estrogen receptor (GPER) to molecules structurally similar to the thyroid-stimulating hormone (TSH) receptor (TSHR). We hypothesize that G protein-coupled estrogen receptor (GPER) may interact with TSHR, differently modulating proliferative signals in healthy versus cancer thyroid cells. Mechanistic experiments evaluating TSHR/GPER heteromers and signaling were performed on papillary thyroid carcinoma (K1), follicular thyroid epithelial (Nthy-ori 3-1) and COS7 (control) cell lines, and confirmed in papillary, follicular, and anaplastic tumors versus healthy primary thyroid tissues by immunofluorescence (IF) and proximity ligation assay (PLA). Cell lines were co-transfected with specific plasmids to evaluate effects on the TSH-induced activation of Gαs and Gq protein-associated transduction pathways, under TSHR/GPER co-expression. Cell lines were treated with 300 nM TSH and 730 pM estradiol. Intracellular levels of cAMP and calcium ion (Ca²⁺) increase were measured by bioluminescence resonance energy transfer (BRET), while IP1 by homogeneous time resolved fluorescence (HTRF). Results were compared by Kruskall-Wallis test (n=6; P<0.05) and corrected by Dunn’s post-hoc test. We found TSHR/GPER co-expression and physical interaction in healthy thyroid follicles using two different methods (IF and PLA), and confirmed these results in cell lines by BRET. Surprisingly, no GPER expression was found in histological sections of papillary, follicular and anaplastic thyroid cancer, as confirmed by the absence of IF signal and heteromers. In TSHR expressing cell lines, TSH activated Gαq-mediated signals, i.e. IP1 (n=6; P<0.05) and Ca²⁺ (n=3; P<0.05), and cell proliferation, while it was not under TSHR/GPER co-expression. Control experiments with Gq and PLC inhibitors, i.e. YM-254890 and U73122, confirmed GPER-like inhibition of TSH-induced IP1 production. Instead, the presence of GPER unaffected TSH/GαS-mediated cAMP production (n=6; p≥0.05). Cell treatment with estradiol and GPER antagonist (5 µM; G15) had no effects (n=6; p≥0.05), revealing that GPER inhibits TSHR/Gq regardless of ligands. GPER/TSHR heteromers drive potentially protective effects in thyroid cells, while the lack of GPER unlock TSH/Gq-dependent proliferative intracellular pathways in tumor cells. This data suggests that lack of GPER may be related to thyroid tumor pathogenesis.