1. Reactivation of the silenced thyroid hormone receptor beta gene expression delays thyroid tumor progression: Epigenetic silencing of the THRB gene is common in human cancers. We determined how DNA methylation affected the expression of the THRB gene in differentiated thyroid cancer (DTC) and how re-expression of the THRB gene attenuated the cancer phenotypes. We used methylation-specific PCR to examine the expression and promoter methylation of the THRB gene in DTC tissues. Thyroid cancer cells with hypermethylated THRB were treated with the demethylating agents 5'-aza-2'-deoxycytidine (5'aza-CdR) and zebularine to evaluate their impact on the cancer cell phenotypes. THRB mRNA expression in DTC was 90% lower than in normal controls, and this decrease was associated with a higher tumor/lymph node staging. The promoter methylation level of the THRB gene had a significant negative correlation with the expression level of the THRB gene. Treatment of FTC-236 cells with 5'aza-CdR or zebularine induced re-expression of the THRB gene and inhibited cell proliferation, and migration. FTC-236 cells stably expressing TRbeta exhibited lower cell proliferation, and migration through inhibition of beta-catenin signaling pathways compared with FTC-236 without TRbeta. 5'aza-CdR also led to suppression of tumor growth in vivo xenograft model using FTC-236 cells consistent with the cell-based studies. These finding indicate that TRbeta is a tumor suppressor and could be tested as a potential therapeutic target. 2. Diet-induced obesity increases tumor growth and promotes anaplastic change in thyroid cancer in a mouse model: Recent epidemiologic studies provide strong evidence suggesting obesity is a risk factor in several cancers, including thyroid cancer. However, the molecular mechanisms by which obesity increases the risk of thyroid cancer are poorly understood. Accordingly, we evaluated the effect of diet-induced obesity on thyroid carcinogenesis in our mouse model of TRbetaPV/PVPTEN+/- mice. A high fat diet (HFD) efficiently induced the obese phenotype in TRbetaPV/PVPTEN+/- mice after 15 weeks. Thyroid tumor growth was markedly greater and survival was significantly lower in TRbetaPV/PVPTEN+/- mice fed an HFD than in controls fed a low fat diet (LFD). HFD increased thyroid tumor cell proliferation by increasing protein levels of cyclin D1 and phosphorylated retinoblastoma protein to propel cell cycle progression. Histopathological analysis showed that the frequency of anaplasia of thyroid cancer was significantly greater (2.6 fold) in the HFD group than the LFD group. HFD treatment led to an increase in parametrial/epididymal fat pad and elevated serum leptin levels in TRbetaPV/PVPTEN+/- mice. Further molecular analyses indicated that the HFD induced more aggressive pathological changes that were mediated by increased activation of the JAK2-STAT3 signaling pathway and induction of STAT3 target gene expression. Our findings demonstrate that diet-induced obesity exacerbates thyroid cancer progression in TRbetaPV/PVPTEN+/- mice and suggest that the STAT3 signaling pathway could be tested as a potential target for the treatment of thyroid cancer. 3. Complex temporal changes in TGFbeta oncogenic signaling drive thyroid carcinogenesis in a mouse model: One key question in the understanding thyroid carcinogenesis is how dynamic temporal changes in global genomic expression alter as the disease progresses. Using cDNA microarrays, we compared global gene expression profiles of thyroid tumors of TRbetaPV/PV mice with the age- and gender-matched thyroids of wild-type mice at 3 weeks, 2 months, 4 months, 6 months, and 14 months. These time points covered the pathological progression from early hyperplasia to capsular invasion, vascular invasion and eventual metastasis. Microarray data indicated that 462 genes were up-regulated (Up-cluster genes) and 110 genes were down-regulated (Down-cluster genes). Three major expression patterns (trending up, cyclical, and spiking up and then down) and two (trending down and cyclical) were apparent in the Up-cluster and Down-cluster genes, respectively. Functional clustering of tumor-related genes followed by Ingenuity Pathways Analysis identified the TGFbeta-mediated network as key signaling pathways. Further functional analyses showed sustained activation of TGFbeta receptor-pSMAD2/3 signaling, leading to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, collagens, and laminins. These TGFbeta-induced changes facilitated epithelial-to-mesenchymal transition (EMT), which promotes cancer invasion and migration. Thus, complex temporal changes in gene expression patterns drive thyroid cancer progression, and persistent activation of TGFbeta-TGFRbetaII-pSMAD2/3 signaling leads to EMT, thus promoting metastasis. For the first time, our studies provide new understanding on the temporal changes at the genomics level during progression and metastatic spread of human thyroid cancer.
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