Abstract
Constitutive MAPK activation silences genes required for iodide uptake and thyroid hormone biosynthesis in thyroid follicular cells. Accordingly, most BRAFV600E papillary thyroid cancers (PTC) are refractory to radioiodide (RAI) therapy. MAPK pathway inhibitors rescue thyroid-differentiated properties and RAI responsiveness in mice and patient subsets with BRAFV600E-mutant PTC. TGFB1 also impairs thyroid differentiation and has been proposed to mediate the effects of mutant BRAF. We generated a mouse model of BRAFV600E-PTC with thyroid-specific knockout of the Tgfbr1 gene to investigate the role of TGFB1 on thyroid-differentiated gene expression and RAI uptake in vivo. Despite appropriate loss of Tgfbr1, pSMAD levels remained high, indicating that ligands other than TGFB1 were engaging in this pathway. The activin ligand subunits Inhba and Inhbb were found to be overexpressed in BRAFV600E-mutant thyroid cancers. Treatment with follistatin, a potent inhibitor of activin, or vactosertib, which inhibits both TGFBR1 and the activin type I receptor ALK4, induced a profound inhibition of pSMAD in BRAFV600E-PTCs. Blocking SMAD signaling alone was insufficient to enhance iodide uptake in the setting of constitutive MAPK activation. However, combination treatment with either follistatin or vactosertib and the MEK inhibitor CKI increased 124I uptake compared to CKI alone. In summary, activin family ligands converge to induce pSMAD in Braf-mutant PTCs. Dedifferentiation of BRAFV600E-PTCs cannot be ascribed primarily to activation of SMAD. However, targeting TGFβ/activin-induced pSMAD augmented MAPK inhibitor effects on iodine incorporation into BRAF tumor cells, indicating that these two pathways exert interdependent effects on the differentiation state of thyroid cancer cells.
Highlights
Radioiodine is a key treatment modality for patients with recurrent or metastatic-differentiated thyroid cancer and is widely applied in the post-operative adjuvant setting
To determine whether the TGFβ/SMAD pathway is activated in BRAFV600E-driven papillary thyroid cancers (PTC) in vivo, we performed pSMAD Western blots in tumor lysates from TPO-Cre/LSL-BrafV600E mice, which develop thyroid cancers with high penetrance by 5 weeks of age (Franco et al 2011)
Treatment of mice with 1.5 mg/kg of the MEK inhibitor CH5126766 (CKI), which binds to MEK and places it in an inactive conformation bound to RAF (Ishii et al 2013), potently blocked MAPK signaling and reduced pSMAD levels as well as expression of the TGFβ downstream target NOX4 (Fig. 1B) (Azouzi et al 2017)
Summary
Radioiodine is a key treatment modality for patients with recurrent or metastatic-differentiated thyroid cancer and is widely applied in the post-operative adjuvant setting. BRAFV600E-mutant thyroid cancers have a high MAPK output because this class 1 BRAF-mutant signals as a monomer and is insensitive to the negative feedback effects of ERK on activated RAF dimers (Yao et al 2015) They have the most profoundly decreased thyroid differentiation score (TDS) (Cancer Genome Atlas Research Network 2014), a quantitative integrated readout of a set of thyroid differentiation markers, and tend to be more refractory to RAI therapy (Xing et al 2005). BRAFV600E-induced suppression of Nis expression was shown to be mediated by a TGFB1driven autocrine loop in PCCL3 (Riesco-Eizaguirre et al 2009) This group found that the inhibition of Nis transcription was MEK-independent, implying that redifferentiation is attainable in the setting of high constitutive MAPK activation. BRAFV600E clearly increases TGFB1 expression and pSMAD in cell lines and mouse PTCs (Riesco-Eizaguirre et al 2009, Knauf et al 2011), prompting us to investigate the functional role of this pathway in a genetically accurate in vivo context
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