Synergistic Signaling of KRAS and Thyroid Hormone Receptor β Mutants Promotes Undifferentiated Thyroid Cancer through MYC Up-Regulation

Abstract

Undifferentiated thyroid carcinoma is one of the most aggressive human cancers with frequent RAS mutations. How mutations of the RAS gene contribute to undifferentiated thyroid cancer remains largely unknown. Mice harboring a potent dominant negative mutant thyroid hormone receptor β, TRβPV (ThrbPV/PV), spontaneously develop well-differentiated follicular thyroid cancer similar to human cancer. We genetically targeted the KrasG12D mutation to thyroid epithelial cells of ThrbPV/PV mice to understand how KrasG12D mutation could induce undifferentiated thyroid cancer in ThrbPV/PVKrasG12D mice. ThrbPV/PVKrasG12D mice exhibited poorer survival due to more aggressive thyroid tumors with capsular invasion, vascular invasion, and distant metastases to the lung occurring at an earlier age and at a higher frequency than ThrbPV/PV mice did. Importantly, ThrbPV/PVKrasG12D mice developed frequent anaplastic foci with complete loss of normal thyroid follicular morphology. Within the anaplastic foci, the thyroid-specific transcription factor paired box gene 8 (PAX8) expression was virtually lost and the loss of PAX8 expression was inversely correlated with elevated MYC expression. Consistently, co-expression of KRASG12D with TRβPV upregulated MYC levels in rat thyroid pccl3 cells, and MYC acted to enhance the TRβPV-mediated repression of the Pax8 promoter activity of a distant upstream enhancer, critical for thyroid-specific Pax8 expression. Our findings indicated that synergistic signaling of KRASG12D and TRβPV led to increased MYC expression. Upregulated MYC contributes to the initiation of undifferentiated thyroid cancer, in part, through enhancing TRβPV-mediated repression of the Pax8 expression. Thus, MYC might serve as a potential target for therapeutic intervention.

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“…Immunohistochemistry (IHC) was conducted as previously described with some modifications [50]. For the antigen retrieval step, slides were heated in 0.05% citraconic anhydride solution (pH 7.4; Sigma-Aldrich, St. Louis, MO, USA) at 98°C for 60 minutes followed by treatment with rabbit anti-p-STAT3 antibody (1:100 dilution, Cell Signaling, Denver, MA, USA) and anti-cleaved caspase 3 antibody (dilution 1:300, cat.…”

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“…Immunohistochemistry (IHC) was conducted as previously described with some modifications (Zhu et al 2014). For the antigen retrieval step, slides were heated in 0.05% citraconic anhydride solution (Sigma-Aldrich; pH 7.4) at 98 8C for 60 min followed by treatment with antiKi-67 antibody (dilution 1:300, cat.…”

“…Preparation of whole-cell lysates from thyroid glands has been described previously (Zhu et al 2014). The protein sample (30 mg) was loaded and separated by SDS-PAGE.…”

SAHA-induced loss of tumor suppressor Pten gene promotes thyroid carcinogenesis in a mouse model

“…The Western blot analysis was carried out as described by Zhu et al (Zhu, et al 2014). Primary antibodies for p-AKT (#9271), total-AKT (#9272), PTEN (#9552), CDK4 (#2906), CDK6 (#3136), p-RB (#9307), MMP7 (#3801), and GAPDH (#2118) were purchased from Cell Signaling Technology (Danvers, MA).…”