A novel ESR2 frameshift mutation predisposes to medullary thyroid carcinoma and causes inappropriate RET expression

Introduction. Familial medullary thyroid cancer (MTC) and its precursor, C Cell hyperplasia (CCH) is associated with germline RET mutations causing multiple endocrine neoplasia type 2 (MEN2). The majority of germline RET mutations predisposing to MEN2 result in single amino acid substitutions causing inappropriate constitutive RET activation. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation (non-RET), suggesting that further predisposing gene alterations remain to be identified. Here, we investigated one kindred and isolated individuals with non-RET CCH/MTC and detected novel mutations in ESR2 encoding the beta subunit of the oestrogen receptor (ER). Functional studies were performed to determine the role of ESR2 germline mutations in MTC tumorigenesis.Material and Methods. We undertook exome sequencing studies in one family with apparent predisposition to CCH and MTC and identified a novel ESR2 frameshift mutation, c.948delT; p.G318Afs*22, which segregated with histological diagnosis following thyroid surgery. Sequencing of the ESR2 coding region in 19 individuals with apparently isolated MTC enabled the identification of a novel germline missense alteration, c.382G>C; p.V128L in a female who developed MTC age 36 years. This variant was also detected in a pair of siblings, one of whom had a papillary thyroid cancer age 60 years and the other a MTC age 57 years. In human cell lines (MCF-7, HCT116) the activity of ESR2 mutants was evaluated in the presence of ER agonists (E2, DPN, PPT) by luciferase and cell proliferation assays.Results. Luciferase reporter assays showed that ESR2-V128L retained transcriptional activity with a significant increase in luciferase activity in response to ESR2-agonist DPN in HCT116 (3.7-fold; P<0.01) and MCF-7 (1.8-fold; P<0.05) cells. In contrast, ESR2-G318Afs*22 was incapable of inducing luciferase activity in either cell line (P=NS). Furthermore, ESR2-G318Afs*22 failed to inhibit ESR1 driven luciferase activity in response to either 17-estradiol (E2) or ESR1-agonist PPT, or restrain ESR1-driven proliferation of MCF-7 cells (P=NS compared to ESR1 alone). In contrast, wild-type (WT) ESR2 and ESR2-V128L inhibited ESR1-driven luciferase activity (>60%. P<0.01) and cell proliferation (>30%, P<0.05). As RET expression is known to be stimulated by oestrogen, we then determined the influence of ESR2 mutants on RET in E2- and PPT-treated HCT116 cells. In contrast to WT ESR2, ESR2-G318Afs*22 was unable to oppose ESR1-stimulation of the RET proto-oncogene at both the mRNA and protein level (P=NS compared to ESR1 alone). Treatment with anti-oestrogen 4-hydroxytamoxifen was however capable of inhibiting E2-induced RET mRNA expression in cells with ESR2-G318Afs*22. Conclusion. Together these data indicate an emerging role for ESR2 as a novel susceptibility gene in non-RET MTC development, especially as ESR2 mutant G318Afs*22 was associated with elevated RET. These results also suggest that anti-oestrogens might represent a promising therapeutic strategy for MTC individuals with defective ESR2.