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Purpose: The role of dual-specificity phosphatase-5 (DUSP5) in BRAF-mutant thyroid cancers remains unclear. The aims of this study are to investigate the role of DUSP5 in BRAF-mutant thyroid cancer cells, explore its value in the diagnosis and evaluate therapeutic potential of targeting DUSP5 combined with sorafenib for BRAF-mutant thyroid cancer patients., Methods: The role of DUSP5 in thyroid cancer cells was determined by a series of in vitro and in vivo experiments. Underlying mechanisms were explored by western blotting analysis. The diagnostic value of combination detection of DUSP5 expression and BRAF V600E mutation was evaluated using ROC curve., Results: Knocking down DUSP5 in BRAF-mutant thyroid cancer cells significantly inhibited colony formation, cell migration and invasion, meanwhile, induced cell cycle arrest and cell apoptosis. Moreover, inhibition of DUSP5 improved the anti-tumor efficacy of sorafenib both in vitro and in vivo. Besides, combination detection of DUSP5 expression and BRAF V600E mutation showed much more accuracy in preoperative diagnosis of thyroid cancer., Conclusions: Our data demonstrate an oncogenic role of DUSP5 in BRAF-mutant thyroid cancer cells, and combined analysis of its expression and BRAF V600E mutation can accurately diagnose thyroid cancer. In addition, inhibition of DUSP5 improves the response of BRAF-mutant thyroid cancer cells to sorafenib., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

BRAF V600E represents a constitutively active onco-kinase and stands as the most prevalent genetic alteration in thyroid cancer. However, the clinical efficacy of small-molecule inhibitors targeting BRAF V600E is often limited by acquired resistance. Here, we find that nerve/glial antigen 2 (NG2), also known as chondroitin sulfate proteoglycan 4 (CSPG4), is up-regulated in thyroid cancers, and its expression is increased with tumor progression in a BRAF V600E -driven thyroid cancer mouse model. Functional studies show that NG2 knockout almost does not affect tumor growth, but significantly improves the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4720. Mechanistically, the blockade of ERK-dependent feedback by BRAF inhibitor can activate receptor tyrosine kinase (RTK) signaling, causing the resistance to this inhibitor. NG2 knockout attenuates the PLX4720-mediated feedback activation of several RTKs, improving the sensitivity of BRAF-mutant thyroid cancer cells to this inhibitor. Based on this finding, we propose and demonstrate an alternative strategy for targeting NG2 to effectively treat BRAF-mutant thyroid cancers by combining multiple kinase inhibitor (MKI) Sorafenib or Lenvatinib with PLX4720. Thus, this study uncovers a new mechanism in which NG2 contributes to the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitor, and provides a promising therapeutic option for BRAF-mutant thyroid cancers., (© 2024. The Author(s).)

BRAF V600E mutation is one of the most therapeutic targets in thyroid cancers. However, its specific inhibitors have shown little clinical benefit because they can reactivate the MAPK/ERK and PI3K/AKT pathways by feedback upregulating the transcription of HER3. Peptidyl-prolyl cis/trans isomerase Pin1 has been proven to be closely associated with tumor progression. Here, we aimed to determine antitumor activity of Pin1 inhibitor API-1 in thyroid cancer and its effect on cellular response to BRAF inhibitors. The results showed that API-1 exhibited strong antitumor activity against thyroid cancer. Meanwhile, it improved the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4032 and there was a synergistic effect between them. Specially, a combination therapy of API-1 and PLX4032 significantly inhibited cell proliferation, colony formation, and the growth of xenograft tumors as well as induced cell apoptosis in BRAF-mutant thyroid cancer cells compared with API-1 or PLX4032 monotherapy. Similar results were also observed in transgenic mice with Braf V600E -driven thyroid cancer. Mechanistically, API-1 enhanced XPO5 ability to export pre-microRNA 20a (pre-miR-20a) from the nucleus to cytoplasm, thereby promoting the maturation of miR-20a-5p. Further studies showed that miR-20a-5p specifically targeted and down-regulated HER3, thereby blocking the reactivation of MAPK/ERK and PI3K/AKT signaling pathways caused by PLX4032. These results, taken together, demonstrate that Pin1 inhibitor API-1 significantly improves the sensitivity of BRAF-mutant thyroid cancer cells to PLX4032. Thus, this study not only determines the potential antitumor activity of Pin1 inhibitor API-1 in thyroid cancer but also offers an alternative therapeutic strategy for BRAF-mutant thyroid cancers by a combination of Pin1 inhibitor and BRAF kinase inhibitor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

STAG2, an important subunit in cohesion complex, is involved in the segregation of chromosomes during the late mitosis and the formation of sister chromatids. Mutational inactivation of STAG2 is a major cause of the resistance of BRAF-mutant melanomas to BRAF/MEK inhibitors. In the present study, we found that STAG2 was frequently down-regulated in thyroid cancers compared with control subjects. By a series of in vitro and in vivo studies, we demonstrated that STAG2 knockdown virtually had no effect on malignant phenotypes of BRAF-mutant thyroid cancer cells such as cell proliferation, colony formation and tumorigenic ability in nude mice compared with the control. In addition, unlike melanoma, STAG2 knockdown also did not affect the sensitivity of these cells to MEK inhibitor. However, we surprisingly found that STAG2-knockdown cells exhibited more sensitive to glutamine deprivation or glutaminase inhibitor BPTES compared with control cells. Mechanistically, knocking down STAG2 in BRAF-mutant thyroid cancer cells decreases the protein stability of c-Myc via the ERK/AKT/GSK3β feedback pathway, thereby impairing glutamine metabolism of thyroid cancer cells by down-regulating its downstream targets such as SCL1A5, GLS and GLS2. Our data, taken together, demonstrate that STAG2 inactivation reprograms glutamine metabolism of BRAF-mutant thyroid cancer cells, thereby improving their cellular response to glutaminase inhibitor. This study will provide a potential therapeutic strategy for BRAF-mutant thyroid cancers., (© 2023. The Author(s).)

BRAF V600E , the most common genetic alteration, has become a major therapeutic target in thyroid cancer. Vemurafenib (PLX4032), a specific inhibitor of BRAF V600E kinase, exhibits antitumor activity in patients with BRAF V600E -mutated thyroid cancer. However, the clinical benefit of PLX4032 is often limited by short-term response and acquired resistance via heterogeneous feedback mechanisms. Disulfiram (DSF), an alcohol-aversion drug, shows potent antitumor efficacy in a copper (Cu)-dependent way. However, its antitumor activity in thyroid cancer and its effect on cellular response to BRAF kinase inhibitors remain unclear. Antitumor effects of DSF/Cu on BRAF V600E -mutated thyroid cancer cells and its effect on the response of these cells to BRAF kinase inhibitor PLX4032 were systematically assessed by a series of in vitro and in vivo functional experiments. The molecular mechanism underlying the sensitizing effect of DSF/Cu on PLX4032 was explored by Western blot and flow cytometry assays. DSF/Cu exhibited stronger inhibitory effects on the proliferation and colony formation of BRAF V600E -mutated thyroid cancer cells than DSF treatment alone. Further studies revealed that DSF/Cu killed thyroid cancer cells by ROS-dependent suppression of MAPK/ERK and PI3K/AKT signaling pathways. Our data also showed that DSF/Cu strikingly increased the response of BRAF V600E -mutated thyroid cancer cells to PLX4032. Mechanistically, DSF/Cu sensitizes BRAF -mutant thyroid cancer cells to PLX4032 by inhibiting HER3 and AKT in an ROS-dependent way and subsequently relieving feedback activation of MAPK/ERK and PI3K/AKT pathways. This study not only implies potential clinical use of DSF/Cu in cancer therapy but also provides a new therapeutic strategy for BRAF V600E -mutated thyroid cancers.

Background: Metastatic disease is a major cause of thyroid cancer-related death. However, the mechanisms responsible for thyroid cancer metastasis are unclear. Dipeptidyl peptidase-4 (DPP4) is a multifunctional cell surface glycoprotein that has been reported to be a negative prognostic factor in thyroid cancer. We explored the molecular mechanism of the role of DPP4 in thyroid cancer cell metastasis. Methods: The effects of DPP4 on thyroid cancer cell migration/invasion in vitro were assessed by transwell assays. A lung metastatic mouse model was also established to determine the effect of DPP4 on tumor metastasis in vivo . DPP4 inhibitor sitagliptin was used to test its effect on thyroid cancer cell metastasis. The mechanism of which DPP4 promotes thyroid cancer cell metastasis was explored by a series of molecular and biochemical experiments. Results: We observed that DPP4 was significantly upregulated in papillary thyroid cancers compared with control subjects, and its expression was positively associated with lymph node metastasis and BRAF V600E mutation. Functional studies showed that DPP4 knockdown significantly inhibited metastatic potential of thyroid cancer cells, and vice versa . However, DPP4 inhibitor sitagliptin did not affect the metastatic ability of thyroid cancer cells, indicating that the promoting effect of DPP4 on tumor metastasis was independent of its enzymatic activity. Mechanistically, DPP4 interacted with the α4 and β1 integrin subunits, and stabilized the formation of integrin α4β1 complex. DPP4-mediated integrin signal activation promoted the nuclear localization of c-Jun through the FAK/AKT pathway, thereby inducing the transcription of transforming growth factor-beta 1 ( TGFB1 coding for protein TGF-β1). TGF-β1 then facilitated tumor metastasis by inducing the epithelial-mesenchymal transition. Conclusions: DPP4 promotes thyroid cancer cell metastasis through the integrins/FAK/AKT/c-Jun/TGF-β1 signaling axis. These findings may have implications for an alternative therapeutic strategy for thyroid cancer.

Purpose: Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancers and it is rapidly fatal without any effective therapeutic regimens. There are some clinical trials showing that paclitaxel-based chemotherapy for ATC can achieve a relatively high response rate and low incidence of adverse reaction. The aim of this study was to evaluate potential therapeutic activity of novel taxoids in ATC cells., Methods: We evaluated antitumor activity of five novel 3'-difluorovinyltaxoids (DFV-taxoids) in anaplastic thyroid cancer cells by a series of in vitro and in vivo experiments. Besides, we also explored the potential mechanism underlying the difference among the taxoids and paclitaxel by molecular docking and tubulin polymerization assays., Results: Our data showed that these novel DFV-taxoids were more effective than paclitaxel in ATC cell lines and xenografts, as reflected by the inhibition of cell proliferation, colony formation and tumorigenic potential in nude mice, and the induction of G 2 /M phase arrest and cell apoptosis. Using tubulin polymerization assays and molecular docking analysis, we found that these DFV-taxoids promoted more rapid polymerization of β-tubulin than paclitaxel., Conclusions: Our data demonstrate that these novel taxoids exhibit stronger antitumor activity in ATC cells than paclitaxel, thereby providing a promising therapeutic strategy for the patients with ATC., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Mannose, a natural hexose existing in daily food, has been demonstrated to preferentially inhibit the progression of tumors with low expression of phosphate mannose isomerase (PMI). However, its function in thyroid cancer still remains elusive., Methods: MTT, colony formation and flow cytometry assays were performed to determine the response of thyroid cancer cells to mannose. Meanwhile, mouse models of subcutaneous xenograft and primary papillary thyroid cancer were established to determine in vivo anti-tumor activity of mannose. The underlying mechanism of mannose selectively killing thyroid cancer cells was clarified by a series of molecular and biochemical experiments., Results: Our data demonstrated that mannose selectively suppressed the growth of thyroid cancer cells, and found that enzyme activity of PMI rather than its protein expression was negatively associated with the response of thyroid cancer cells to mannose. Besides, our data showed that zinc ion (Zn 2+ ) chelator TPEN clearly increased the response of mannose-insensitive cells to mannose by inhibiting enzyme activity of PMI, while Zn 2+ supplement could effectively reverse this effect. Further studies found that the expression of zinc transport protein ZIP10, which transport Zn 2+ from extracellular area into cells, was negatively related to the response of thyroid cancer cells to mannose. Knocking down ZIP10 in mannose-insensitive cells significantly inhibited in vitro and in vivo growth of these cells by decreasing intracellular Zn 2+ concentration and enzyme activity of PMI. Moreover, ectopic expression of ZIP10 in mannose-sensitive cells decrease their cellular response to mannose. Mechanistically, mannose exerted its anti-tumor effect by inhibiting cellular glycolysis; however, this effect was highly dependent on expression status of ZIP10., Conclusion: The present study demonstrate that mannose selectively kills thyroid cancer cells dependent on enzyme activity of PMI rather than its expression, and provide a mechanistic rationale for exploring clinical use of mannose in thyroid cancer therapy., (© 2021. The Author(s).)

BRAF V600E is the most common genetic alteration and has become a major therapeutic target in thyroid cancers; however, intrinsic feedback mechanism limited clinical use of BRAF V600E specific inhibitors. Synthetic lethal is a kind of interaction between two genes, where only simultaneously perturbing both of the genes can lead to lethality. Here, we identified CYP2S1 as a synthetic lethal partner of BRAF V600E in thyroid cancers. First, we found that CYP2S1 was highly expressed in papillary thyroid cancers (PTCs) compared to normal thyroid tissues, particularly in conventional PTCs (CPTCs) and tall-cell PTCs (TCPTCs), and its expression was positively associated with BRAF V600E mutation. CYP2S1 knockdown selectively inhibited cell proliferation, migration, invasion and tumorigenic potential in nude mice, and promoted cell apoptosis in BRAF V600E mutated thyroid cancer cells, but not in BRAF wild-type ones. Mechanistically, BRAF V600E -mediated MAPK/ERK cascade upregulated CYP2S1 expression by an AHR-dependent pathway, while CYP2S1 in turn enhanced transcriptional activity of AHR through its metabolites. This AHR/CYP2S1 feedback loop strongly amplified oncogenic role of BRAF V600E in thyroid cancer cells, thereby causing synthetic lethal interaction between CYP2S1 and BRAF V600E . Finally, we demonstrated CYP2S1 as a potential therapeutic target in both BRAF V600E -drived xenograft and transgenic mouse models by targetedly delivering CYP2S1-specific siRNA. Altogether, our data demonstrate CYP2S1 as a synthetic lethal partner of BRAF V600E in thyroid cancers, and indicate that targeting CYP2S1 will provide a new therapeutic strategy for BRAF V600E mutated thyroid cancers.

NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor, currently in phase 1/2 clinical trials, exhibiting clinical efficiency in treatment of numerous malignancies including thyroid cancer. Cancer cells harboring mutant p53 was widely reported to be blunt to pharmaceutical therapies. However, whether this genotype dependent effect also presents in thyroid cancer when treated with NVP-BEZ235 remains unknown. Therefore, in this study, the tumor suppressing effects of NVP-BEZ235 in thyroid cancer cell lines and in-vivo xenograft mouse model harboring different p53 status were examined. The antitumor effects were confirmed in p53 mutant thyroid cancer cells, though less prominent than p53 wild type cells. And for the p53 mutant cells, p53-independent upregulation of p21 plays a critical role in their response to NVP-BEZ235. Moreover, GSK3β/β-catenin signaling inhibition was implicated in the p21-mediated G 0 /G 1 cell cycle arrest in both p53 wild type and mutant thyroid cancer cells treated with NVP-BEZ235., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background : Vitamin C has been demonstrated to kill BRAF mutant colorectal cancer cells selectively. BRAF mutation is the most common genetic alteration in thyroid tumor development and progression; however, the antitumor efficacy of vitamin C in thyroid cancer remains to be explored. Methods : The effect of vitamin C on thyroid cancer cell proliferation and apoptosis was assessed by the MTT assay and flow cytometry. Xenograft and transgenic mouse models were used to determine its in vivo antitumor activity of vitamin C. Molecular and biochemical methods were used to elucidate the underlying mechanisms of anticancer activity of vitamin C in thyroid cancer. Results : Pharmaceutical concentration of vitamin C significantly inhibited thyroid cancer cell proliferation and induced cell apoptosis regardless of BRAF mutation status. We demonstrated that the elevated level of Vitamin C in the plasma following a high dose of intraperitoneal injection dramatically inhibited the growth of xenograft tumors. Similar results were obtained in the transgenic mouse model. Mechanistically, vitamin C eradicated BRAF wild-type thyroid cancer cells through ROS-mediated decrease in the activity of EGF/EGFR-MAPK/ERK signaling and an increase in AKT ubiquitination and degradation. On the other hand, vitamin C exerted its antitumor activity in BRAF mutant thyroid cancer cells by inhibiting the activity of ATP-dependent MAPK/ERK signaling and inducing proteasome degradation of AKT via the ROS-dependent pathway. Conclusions : Our data demonstrate that vitamin C kills thyroid cancer cells by inhibiting MAPK/ERK and PI3K/AKT pathways via a ROS-dependent mechanism and suggest that pharmaceutical concentration of vitamin C has potential clinical use in thyroid cancer therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: TBX1 is a member of the T-box family of transcription factors characterized by a conserved DNA binding domain termed T-box. TBX1 has been reported to be downregulated in mouse skin tumors and is considered a negative regulator of tumor cell growth in mice. However, its role and exact mechanism in human cancers, including thyroid cancer, remain totally unknown., Methods: Quantitative reverse transcription polymerase chain reaction and Western blot assays were performed to evaluate the expression of investigated genes. Methylation-specific polymerase chain reaction and pyrosequencing were used to analyze TBX1 promoter methylation. The biological functions of TBX1 in thyroid cancer cells were determined by a series of in vitro and in vivo experiments. Chromatin immunoprecipitation sequencing and dual-luciferase reporter assays were used to identify its downstream targets., Results: This study demonstrates that TBX1 is frequently downregulated by promoter methylation in both papillary thyroid cancers and thyroid cancer cell lines. Ectopic expression of TBX1 in thyroid cancer cells dramatically inhibits cell viability, colony formation, and tumorigenic potential in nude mice, and induces cell-cycle arrest and apoptosis through modulating a panel of cell-cycle and apoptosis-related genes. In addition, ectopic expression of TBX1 significantly decreases the migration and invasion potential of thyroid cancer cells through inhibiting the process of epithelial-mesenchymal transition and the expression of matrix metalloproteinases. On the other hand, TBX1 knockdown markedly promotes thyroid cancer cell viability and invasiveness. Mechanistically, TBX1 exerts its tumor suppressor function in thyroid cancer cells through inhibiting phosphorylation of AKT at Ser473 and ERK via regulating its downstream targets such as RNF41, PARK2, and PHLPP2., Conclusions: The data show that TBX1 is frequently inactivated by promoter methylation and functions as a potential tumor suppressor in thyroid cancer through inhibiting the activities of the PI3K/AKT and MAPK/ERK signaling pathways.

SIRT7 is an NAD + -dependent histone/non-histone deacetylase, which is highly expressed in different types of cancer including thyroid cancer; however, its biological function in thyroid cancer is still undiscovered. In this study, we found that SIRT7 expression was elevated in papillary thyroid cancers (PTCs), and demonstrated that SIRT7 knockdown dramatically inhibited the proliferation, colony formation, migration and invasion of thyroid cancer cells, and induced thyroid cancer cell cycle arrest and apoptosis. Conversely, SIRT7 re-expression markedly enhanced thyroid cancer cell growth, invasiveness and tumorigenic potential in nude mice. Further studies revealed that SIRT7 exerted an oncogenic function in thyroid tumorigenesis by phosphorylation of Akt and p70S6K1. Mechanistically, SIRT7 binds to the promoter of deleted in breast cancer-1 (DBC1), an endogenous inhibitor of SIRT1, and represses its transcription via deacetylation of H3K18Ac. This results in enhanced interactions between SIRT1 and Akt or p70S6K1, thereby promoting deacetylation and subsequent phosphorylation of Akt and p70S6K1 through a SIRT1-dependent manner. Altogether, our results show that DBC1 is a downstream target of SIRT7, and first uncover that SIRT7 promotes thyroid tumorigenesis through phosphorylation and activation of Akt and p70S6K1 via the modulation of DBC1/SIRT1 axis.

Background: c-Myc is overexpressed in different types of cancer, including thyroid cancer, and has been considered undruggable. There is evidence showing that MLN8237, a type of aurora A kinase (AURKA) inhibitor, destabilizes c-Myc proteins in liver cancer cells through disruption of the c-Myc/AURKA complex. However, the role of MLN8237 in thyroid cancer remains largely unclear. The aims of this study were to test the therapeutic potential of MLN8237 in thyroid cancer, and to analyze determinant factors affecting the response of thyroid cancer cells to MLN8237 and clarify the corresponding mechanism. Methods: The phenotypic effects of MLN8237 in thyroid cancer cells were evaluated through a series of in vitro and in vivo experiments, and the mechanism of c-Myc affecting MLN8237 response were explored using Western blot, ubiquitination, and cycloheximide chase assays. Results: The data show that the levels of c-Myc protein were strongly associated with MLN8237 cellular response in thyroid cancer cells. Only the cells with high c-Myc expression exhibited growth inhibition upon MLN8237 treatment. However, MLN8237 barely affected the growth of those with low c-Myc expression. Mechanistically, MLN8237 dramatically promoted proteasomal degradation of c-Myc proteins through disruption of the c-Myc/AURKA complex in the cells with high c-Myc expression. A similar antitumor activity of MLN8237 was also found in xenograft tumor models. Conclusions: The data demonstrate that c-Myc is a major determinant for MLN8237 responsiveness in thyroid cancer cells. Thus, indirectly targeting c-Myc by MLN8237 may be an effective strategy for thyroid cancer overexpressing c-Myc.

The zinc finger protein 677 (ZNF677) belongs to the zinc finger protein family, which possesses transcription factor activity by binding sequence-specific DNA. Previous studies have reported its downregulated by promoter methylation in non-small cell lung cancer. However, its biological role and exact mechanism in human cancers, including thyroid cancer, remain unknown. In this study, we demonstrate that ZNF677 is frequently downregulated by promoter methylation in primary papillary thyroid cancers (PTC) and show that decreased expression of ZNF677 is significantly associated with poor patient survival. Ectopic expression of ZNF677 in thyroid cancer cells dramatically inhibited cell proliferation, colony formation, migration, invasion, and tumorigenic potential in nude mice and induced cell-cycle arrest and apoptosis. Conversely, knockdown of ZNF677 promoted thyroid cancer cell proliferation and colony formation. ZNF677 exerted its tumor suppressor functions in thyroid cancer cells through transcriptional repression of two targets CDKN3 and HSPB1 (or HSP27), thereby inhibiting phosphorylation and activation of Akt via distinct mechanisms. Taken together, our data show that ZNF677 functions as a tumor suppressor and is frequently silenced via promoter methylation in thyroid cancer. Significance: These findings report a tumor suppressive role of the zinc-finger protein ZNF677 in primary papillary thyroid cancer through inhibition of Akt phosphorylation. Cancer Res; 78(18); 5216-28. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Papillary thyroid cancer (PTC) is the most common epithelial thyroid tumor, accounting for more than 80% of all thyroid cancers. Though the fine needle aspiration biopsy (FNAB) represents as the golden standard for the diagnostics of thyroid nodules, there is a ∼25% risk of indeterminate cytological features. TMPRSS4 is a newly found transmembrane serine protease which was overexpressed in papillary thyroid cancer (PTC)., Aims: The aim of this study was to determine its potential as a diagnostic marker to improve the diagnostic accuracy of thyroid cancer., Methods: We used pyrosequencing and quantitative real-time PCR (qRT-PCT) approaches to examine BRAF V600E mutation and TMPRSS4 mRNA level in FNAB specimens of thyroid nodules. The detection and analysis were respectively applied to training group with 91, and test group with 88 samples., Results: We demonstrated that PTC patients had an increased TMPRSS4 mRNA level as compared with benign subjects. The diagnostic sensitivity, specificity, and accuracy of TMPRSS4 were 93.33, 100, and 96.70%, respectively. Notably, compared with BRAF V600E mutation testing alone, combining with TMPRSS4 mRNA level significantly increased the diagnostic sensitivity and accuracy., Conclusions: Our findings indicated BRAF V600E mutation combination with TMPRSS4 mRNA analysis can dramatically improve the sensitivity and accuracy of preoperative diagnosis of thyroid nodules., (Copyright © 2018 IMSS. Published by Elsevier Inc. All rights reserved.)

Thyroid cancer is the most common endocrine malignant tumor with rapidly increasing incidence in recent decades. Although the majority of thyroid cancers are relatively indolent, some cases still have a risk of developing into more aggressive and lethal forms of thyroid cancers. Similar to other malignancies, thyroid tumorigenesis is a multistep process involving the accumulation of a large number of genetic and epigenetic alterations. Thus, determination of the mechanisms of tumorigenesis is an urgent need for thyroid cancer treatment. Long noncoding RNAs (LncRNAs) have recently been demonstrated to participate in cancer progression. However, their role and molecular mechanism in thyroid cancer remain largely unclear. In this review, we focus on the dysregulation of lncRNAs in thyroid cancer, summarize the latest findings regarding the functions and mechanism of lncRNAs in thyroid cancer, and discuss their potential clinical significance in diagnosis and prognosis of thyroid cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Proteasome inhibitors are promising antitumor drugs with preferable cytotoxicity in malignant cells and have exhibited clinical efficiency in several hematologic malignancies. P53-dependent apoptosis has been reported to be a major mechanism underlying. However, apoptosis can also be found in cancer cells with mutant-type p53, suggesting the involvement of p53-independent mechanism. Tumor suppressor forkhead Box O3 is another substrate of proteasomal degradation, which also functions partially through inducing apoptosis. The aim of this study was to explore the effect of proteasome inhibition on the expression and activity of forkhead Box O3 in thyroid cancer cells. Using flow cytometry, western blot, immunofluorescence staining and quantitative RT-PCR assays, we assessed proteasome inhibitor MG132-induced apoptosis in thyroid cancer cells and its effect on the expression and activity of forkhead Box O3. The resulted showed that MG132 induced significant apoptosis, and caused the accumulation of p53 protein in both p53 wild-type and mutant-type thyroid cancer cell lines, whereas the proapoptotic targets of p53 were transcriptionally upregulated only in the p53 wild-type cells. Strikingly, upon MG132 administration, the accumulation and nuclear translocation of transcription factor forkhead Box O3 as well as transcriptional upregulation of its proapoptotic target genes were found in thyroid cancer cells regardless of p53 status. Cell apoptosis was enhanced by ectopic overexpression while attenuated by silencing of forkhead Box O3. Altogether, we demonstrated that proteasome inhibitor MG132 induces thyroid cancer cell apoptosis at least partially through modulating forkhead Box O3 activity.

Context: Although neuronal cell adhesion molecule (NrCAM) has been reported to be overexpressed in papillary thyroid cancer (PTCs), its role in this disease remains largely unclear., Objectives: The aim of this study was to explore the biological functions of NrCAM and its potential as a diagnostic marker and therapeutic target in thyroid cancer., Experimental Design: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate messenger RNA expression of investigated genes. The functions of knockdown and ectopic expression of NrCAM in thyroid cancer cells were determine by a series of in vitro and in vivo experiments., Results: We found that NrCAM was highly expressed in PTCs and demonstrated that NrCAM might be a potential marker for preoperative diagnosis of PTC. Moreover, NrCAM depletion dramatically inhibited thyroid cancer cell growth, invasiveness and tumorigenic potential in nude mice. On the other hand, ectopic expression of NrCAM significantly enhanced its tumor-promoting effects. Mechanically, NrCAM exerted its oncogenic function by activating MAPK/Erk and PI3K/Akt pathways via its ectodomain shedding and binding to EGFR and α4β1 integrins. In turn, these 2 pathways were also responsible for NrCAM overexpression through GSK3β/β-catenin signaling axis in thyroid cancer cells. Similar results were also found in transgenic mice with thyroid-specific knock-in of oncogenic BrafV600E (TPO-Cre/LSL-BrafV600E)., Conclusions: Our data first reveal positive feedback loops between NrCAM and major signaling pathways contributing to thyroid tumorigenesis by modulating tumor microenvironment, and suggest that NrCAM may represent a potential diagnostic marker and therapeutic target for thyroid cancer., (Copyright © 2017 by the Endocrine Society)

Epithelial-mesenchymal transition (EMT), a crucial step in disease progression, plays a key role in tumor metastasis. N-cadherin, a well-known EMT marker, acts as a major oncogene in diverse cancers, whereas its functions in thyroid cancer remains largely unclear. This study was designed to explore the biological roles and related molecular mechanism of N-cadherin in thyroid tumorigenesis. Quantitative RT-PCR (qRT-PCR) and immunohistochemistry assays were used to evaluate N-cadherin expression. A series of in vitro studies such as cell proliferation, colony formation, cell cycle, apoptosis, migration and invasion assays were performed to determine the effect of N-cadherin on malignant behavior of thyroid cancer cells. Our results showed that N-cadherin was significantly upregulated in papillary thyroid cancers (PTCs) as compared with non-cancerous thyroid tissues. N-cadherin knockdown markedly inhibited cell proliferation, colony formation, cell migration and invasion, and induced cell cycle arrest and apoptosis. On the other hand, ectopic expression of N-cadherin promoted thyroid cancer cell growth and invasiveness. Mechanically, our data demonstrated that tumor-promoting role of N-cadherin in thyroid cancer was closely related to the activities of the MAPK/Erk, the phosphatidylinositol-3-kinase (PI3K)/Akt and p16/Rb signaling pathways in addition to affecting the EMT process. Altogether, our findings suggest that N-cadherin promotes thyroid tumorigenesis by modulating the activities of major signaling pathways and EMT process, and may represent a potential therapeutic target for this cancer.

E26 transformation-specific (ETS) transcription factor EHF plays a tumor suppressor role in prostate cancer and esophageal squamous cell carcinoma (ESCC), whereas it is overexpressed and may act as an oncogene in ovarian and mammary cancers. However, its biological role in thyroid cancer remains totally unknown. The aim of this study was to explore the biological functions of EHF and its potential as a therapeutic target in thyroid cancer. Using quantitative RT-PCR (qRT-PCR) assay, we evaluated mRNA expression of EHF in a cohort of primary papillary thyroid cancers (PTCs) and matched non-cancerous thyroid tissues. The functions of knockdown and ectopic expression of EHF in thyroid cancer cells were determine by a series of in vitro and in vivo experiments. Moreover, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays were performed to identify its downstream targets. Our data showed that EHF expression was significantly increased in PTCs compared with matched non-cancerous thyroid tissues. EHF knockdown significantly inhibited thyroid cancer cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice and induced cell cycle arrested and apoptosis by modulating the PI3K/Akt and MAPK/Erk signaling pathways. On the other hand, ectopic expression of EHF in thyroid cancer cells notably promoted cell growth and invasiveness. Importantly, EHF was identified as a new transcription factor for HER2 and HER3, contributing to thyroid tumorigenesis. Altogether, our findings suggest that EHF is a novel functional oncogene in thyroid cancer by transcriptionally regulating HER2 and HER3, and may represent a potential therapeutic target for this cancer., Competing Interests: The authors declare no conflicts of interest.

Paired box 3 (PAX3) is expressed early during embryonic development in spatially restricted domains in the nervous system and in some mesodermally-derived structure. In recent years, it is found to be overexpressed in different types of cancer tissues and cell lines including glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas and gastric cancers, suggesting that it may function as an oncogene in these cancers. However, its role in thyroid cancer remains totally unclear. The aim of this study was to explore the functions and related molecular mechanism of PAX3 in thyroid tumorigenesis. Using quantitative RT-PCR (qRT-PCR) and Methylation-specific PCR (MSP) assays, we demonstrated that PAX3 was frequently down-regulated by promoter methylation in both primary thyroid cancer tissues and thyroid cancer cell lines. In addition, our data showed that ectopic expression of PAX3 dramatically inhibited thyroid cancer cell proliferation, colony formation, migration and invasion, induced cell cycle arrest and apoptosis and retarded tumorigenic potential in nude mice. Mechanically, PAX3 exerted its tumor suppressor function by inhibiting the activity of major signaling pathways including the phosphatidylinositol-3-kinase (PI3K)/Akt and MAPK/Erk pathways, and enhancing expression and activity of transcription factor FOXO3a. Altogether, our findings provided insight into the role of PAX3 as a novel functional tumor suppressor in thyroid cancer through modulating the activities of PI3K/Akt and MAPK signaling pathways and transcription factor FOXO3a, and demonstrated that epigenetic alterations such as promoter methylation should be a major mechanism of PAX3 inactivation in this cancer.

Sulforaphane (SFN), a natural compound derived from broccoli/broccoli sprouts, has been demonstrated to be used as an antitumor agent in different types of cancers. However, its antitumor effect in thyroid cancer remains largely unknown. The aim of the study was to investigate the therapeutic potential of SFN for thyroid cancer and explore the mechanisms underlying antitumor effects of SFN by in vitro and in vivo studies. Our data demonstrated that SFN significantly inhibited thyroid cancer cell proliferation in a dose- and time-dependent manner, induced G2/M phase cell cycle arrest and apoptosis, and inhibited thyroid cancer cell migration and invasion by suppressing epithelial-mesenchymal transition (EMT) process and expression of Slug, Twist, MMP-2 and -9. Mechanically, SFN inhibited thyroid cancer cell growth and invasiveness through repressing phosphorylation of Akt, enhancing p21 expression by the activation of Erk and p38 signaling cascades, and promoting mitochondrial-mediated apoptosis via reactive oxygen species (ROS)-dependent pathway. Growth of xenograft tumors derived from thyroid cancer cell line FTC133 in nude mice was also significantly inhibited by SFN. Importantly, we did not find significant effect of SFN on body weight and liver function of mice. Collectively, we for the first time demonstrate that SFN is a potentially effective antitumor agent for thyroid cancer.

Fine-needle aspiration biopsy remains the mainstay for preoperative examination of thyroid nodules; however, it does not provide a definite diagnosis in up to 25% of nodules. Considerable studies have been performed to identify molecular markers to resolve this diagnostic dilemma. The aim of this study was to establish the distribution and frequency of common genetic alterations in a comprehensive set of benign and malignant thyroid nodules, and to determine the feasibility and role of testing for a panel of genetic alterations in improving the accuracy of cytology diagnosis in a Chinese population. This study was conducted in 314 thyroid nodules comprising 104 papillary thyroid carcinomas, 13 suspicious nodules, 52 indeterminate nodules, and 145 benign nodules. Point mutations and RET/PTC rearrangements, were evaluated by pyrosequencing and TaqMan real-time PCR, respectively. After surgery, 115 nodules were confirmed as conventional papillary thyroid carcinoma and 102 (88.70%) of these nodules harbored either the BRAF(V600E) mutation (76.52%) or RET/PTC rearrangements (12.17%). RAS mutation was found in 1 (33.33%) follicular thyroid carcinoma, 1 (14.29%) follicular thyroid adenoma and 4 (10%) goiter nodules. With cytology and molecular testing, the diagnostic accuracy was further increased to 98.82% in papillary thyroid carcinoma diagnosis, and was preoperatively increased to 76.92% and 84.00%, respectively, in nodules with suspicious and indeterminate cytology. In conclusion, molecular testing of a panel of genetic alterations in fine-needle aspiration biopsy can be effectively performed in clinical practice. It enhances the accuracy of cytology and is of particular value for indeterminate nodules in the Chinese population., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The new finding of the heterogeneous distribution of BRAF(V600E) mutation in primary papillary thyroid carcinoma suggested the percentage of BRAF(V600E) alleles should be taken into consideration when evaluating its association with clinicopathological features of papillary thyroid carcinoma. The aim of this study was to detect both the presence and the percentage of BRAF(V600E) alleles in fine-needle aspiration biopsy samples and to assess its association with clinicopathological characteristics of papillary thyroid carcinoma in a Chinese population., Materials and Methods: Fine needle aspiration samples were collected in a total of 182 patients (132 conventional papillary thyroid carcinomas and 50 goiters). The associations of the presence and percentage of BRAF(V600E) alleles genotyped by pyrosequencing with clinicopathological characteristics were evaluated in papillary thyroid carcinomas., Results: 80 (60.61%) of papillary thyroid carcinomas exhibited BRAF(V600E) mutation in a range of 7.7% to 46.3% of the total BRAF alleles. The presence of BRAF(V600E) mutation was significantly associated with extrathyroidal invasion. There was no significant difference between the presence of BRAF(V600E) mutation and other clinicopathological features. It was not found that the significant relationship between percentage of BRAF(V600E) alleles and clinicopathological characteristics., Conclusion: We concluded that the presence of BRAF(V600E) could be preoperatively predictive of extrathyroidal invasion in a Chinese population.

Context: ZIC1 has been reported to be overexpressed and plays an oncogenic role in some brain tumors, whereas it is inactivated by promoter hypermethylation and acts as a tumor suppressor in gastric and colorectal cancers. However, until now, its biological role in thyroid cancer remains totally unknown., Objectives: The aim of this study is to explore the biological functions and related molecular mechanism of ZIC1 in thyroid carcinogenesis., Setting and Design: Quantitative RT-PCR (qRT-PCR) was performed to evaluate mRNA expression of investigated genes. Methylation-specific PCR was used to analyze promoter methylation of the ZIC1 gene. The functions of ectopic ZIC1 expression in thyroid cancer cells were determined by cell proliferation and colony formation, cell cycle and apoptosis, as well as cell migration and invasion assays., Results: ZIC1 was frequently down-regulated by promoter hypermethylation in both primary thyroid cancer tissues and thyroid cancer cell lines. Moreover, our data showed that ZIC1 hypermethylation was significantly associated with lymph node metastasis in patients with papillary thyroid cancer. Notably, restoration of ZIC1 expression in thyroid cancer cells dramatically inhibited cell proliferation, colony formation, migration and invasion, and induced cell cycle arrest and apoptosis by blocking the activities of the phosphatidylinositol-3-kinase (PI3K)/Akt and RAS/RAF/MEK/ERK (MAPK) pathways, and enhancing FOXO3a transcriptional activity., Conclusions: Our data demonstrate that ZIC1 is frequently inactivated by promoter hypermethyaltion and functions as a tumor suppressor in thyroid cancer through modulating PI3K/Akt and MAPK signaling pathways and transcription factor FOXO3a.

The p53 tumor suppressor pathway blocks carcinogenesis by triggering apoptosis and cellular senescence in response to oncogenic stress. Over 50% of human cancers including thyroid cancer carry loss-of-function mutations in the p53 gene. Recently, the identification of mutant p53-reactivating small molecules such as PRIMA-1 (p53 reactivation and induction of massive apoptosis) renders possibilities for the development of more efficient anticancer drugs. Although PRIMA-1 has been widely used for cancer therapy and exhibits a promising anticancer activity, its biological effect, particularly the epigenetic aspect, remains to be well elucidated. The present study attempts to explore the effect of PRIMA-1 on DNA methylation in a panel of thyroid cancer cell lines using luminometric methylation assay (LUMA). Our results showed that only p53 mutant-type cells were inhibited upon PRIMA-1 treatment. Conversely, p53 wild-type cells were non-sensitive to PRIMA-1. Moreover, our data demonstrated that PRIMA-1 selectively induced significant global DNA demethylation in p53 mutant-type cells. Mechanically, PRIMA-1 induced global DNA demethylation in these cells mainly through inhibiting the expression of DNA methyltransferase (DNMT) 1, 3a and 3b, and upregulating the expression of GADD45a. Notably, PRIMA-1 dramatically increased the expression of the ten-eleven translocation (TET) family of 5mC-hydroxylases, particularly TET1, in p53 mutant-type cells, further contributing to DNA demethylation. Thus, this study uncovered a previously unrecognized and prominent biological effect of PRIMA-1 through which it can cause global DNA demethylation in p53 mutant-type cancer cells mainly by rescuing the function of mutant p53 protein.

Context: 3-Deazaneplanocin A (DZNep) depletes enhancer of zeste homolog 2 (EZH2), a core component of polycomb repressive complex 2 (PRC2), which is frequently overexpressed in human cancers. DZNep exhibits promising antitumor activity, and its responsiveness in cancer cells is determined by certain genetic factors., Objectives: Our aims were (1) to test the therapeutic potential of DZNep and explore the genetic determinants affecting the DZNep response in thyroid cancer cells and (2) to test the combined therapeutic effect of DZNep and PRIMA-1, a mutant p53 reactivator, in thyroid cancer., Experimental Design: We evaluated the phenotypic effects of DZNep in thyroid cancer cells and examined the effects of DZNep alone or in combination with PRIMA-1 on cell proliferation, the cell cycle, apoptosis, and xenograft tumor growth., Results: DZNep induced enhancer of zeste homolog 2 depletion and trimethylated lysine 27 in H3 histone (H3K27me3) mark reduction in all thyroid cancer cells; however, only TP53 wild-type cells exhibited growth inhibition with DZNep treatment. In these cells, DZNep caused p53 protein accumulation through up-regulation of USP10 expression, resulting in activation of the p53 pathway, contributing to inhibition of cell growth. Conversely, TP53 mutant-type cells were resistant to DZNep. Strikingly, the combination of DZNep with PRIMA-1 restored the sensitivity of TP53 mutant-type cells to DZNep. A similar antitumor effect of DZNep and PRIMA-1 alone or in combination was also seen in xenograft tumor models., Conclusion: Our data demonstrated that DZNep responsiveness was strongly associated with TP53 genomic status in thyroid cancer cells. Reactivation of p53 restored the sensitivity of TP53 mutant-type cells to DZNep. Thus, a combined therapeutic strategy may be effective in treating thyroid cancer cells (or patients) harboring mutant p53.

Context: Shikonin, which is an active naphthoquinone isolated from traditional Chinese herbal medicine Zi Cao, has been recently developed to use as an antitumor agent in colorectal cancer, melanoma, leukemia, breast cancer, and hepatocellular cancer. However, its antitumor effect in thyroid cancer remains largely unknown., Objectives: The aim of the study was to test the therapeutic potential of shikonin for thyroid cancer and explore the mechanisms underlying antitumor effects of shikonin., Experimental Design: We examined the effects of shikonin on proliferation, cell cycle, apoptosis, migration, invasion, and xenograft tumor growth in thyroid cancer cells and the effect of shikonin on proliferation of primary thyroid cancer cells., Results: Shikonin inhibited thyroid cancer cell proliferation in a dose- and time-dependent manner and induced cell cycle arrest. Moreover, shikonin induced cell apoptosis through reactive oxygen species-mediated DNA damage and activation of the p53 signaling pathway. Our data also showed that shikonin dramatically inhibited thyroid cancer cell migration and invasion by suppressing epithelial-mesenchymal transition and downregulating expression of Slug and MMP-2, -9, and -14. Further elucidation of the mechanisms involved revealed that shikonin markedly repressed the phosphorylation of Erk and Akt and activated the p16/Retinoblastoma protein (Rb) pathway in thyroid cancer cells. Growth of xenograft tumors derived from the thyroid cancer cell line FTC133 in nude mice was significantly inhibited by shikonin. Importantly, we did not find the effect of shikonin on liver function in mice., Conclusion: We for the first time demonstrated that shikonin is a potentially effective antitumor agent for thyroid cancers.

Background: MT1G inactivation mediated by promoter methylation has been reported in thyroid cancer. However, the role of MT1G in thyroid carcinogenesis remains unclear. The aim of this study is to examine the biological functions and related molecular mechanisms of MT1G in thyroid cancer., Methods: Methylation-specific PCR (MSP) was performed to analyze promoter methylation of MT1G and its relationship with clinicopathological characteristics of papillary thyroid cancer (PTC) patients. Conventional and real-time quantitative RT-PCR assays were used to evaluate mRNA expression. The functions of ectopic MT1G expression were determined by cell proliferation and colony formation, cell cycle and apoptosis, as well as cell migration and invasion assays., Results: MT1G expression was frequently silenced or down-regulated in thyroid cancer cell lines, and was also significantly decreased in primary thyroid cancer tissues compared with non-malignant thyroid tissues. Promoter methylation, along with histone modification, contributes to MT1G inactivation in thyroid tumorigenesis. Moreover, our data showed that MT1G hypermethylation was significantly positively associated with lymph node metastasis in PTC patients. Importantly, restoring MT1G expression in thyroid cancer cells dramatically suppressed cell growth and invasiveness, and induced cell cycle arrest and apoptosis through inhibiting phosphorylation of Akt and Rb., Conclusions: We have for the first time revealed that MT1G appears to be functional tumor suppressor involved in thyroid carcinogenesis mainly through modulating the phosphatidylinositol-3-kinase (PI3K)/Akt pathway and partially through regulating the activity of Rb/E2F pathway in this study.

The BRAF V600E mutation plays an important role in the tumorigenesis of papillary thyroid cancer (PTC). To explore an epigenetic mechanism involved in this process, we performed a genome-wide DNA methylation analysis using a methylated CpG island amplification (MCA)/CpG island microarray system to examine gene methylation alterations after shRNA knockdown of BRAF V600E in thyroid cancer cells. Our results revealed numerous methylation targets of BRAF V600E mutation with a large cohort of hyper- or hypo-methylated genes in thyroid cancer cells, which are known to have important metabolic and cellular functions. As hypomethylation of numerous genes by BRAF V600E was particularly a striking finding, we took a further step to examine the selected 59 genes that became hypermethylated in both cell lines upon BRAF V600E knockdown and found them to be mostly correspondingly under-expressed (i.e. they were normally maintained hypomethylated and over-expressed by BRAF V600E in thyroid cancer cells). We confirmed the methylation status of selected genes revealed on MCA/CpG microarray analysis by performing methylation-specific PCR. To provide proof of concept that some of the genes uncovered here may play a direct oncogenic role, we selected six of them to perform shRNA knockdown and examined its effect on cellular functions. Our results demonstrated that the HMGB2 gene played a role in PTC cell proliferation and the FDG1 gene in cell invasion. Thus, this study uncovered a prominent epigenetic mechanism through which BRAF V600E can promote PTC tumorigenesis by altering the methylation and hence the expression of numerous important genes.

Objective: To detect the BRAF mutation on fine needle aspiration biopsy (FNAB) specimens in papillary thyroid cancer (PTC)., Study Design: Eight fresh FNAB specimens and 10 patients' FNAB slides with cytologic examination were examined for the presence of BRAF V600E mutation by direct DNA sequencing., Results: Genomic DNA was all successfully extracted from both fresh FNAB specimens and patients' FNAB slides. Moreover, BRAF V600E mutation was detected in 2 of 8 fresh FNAB specimens and 6 of 10 patients' FNAB slides., Conclusion: We demonstrated for the first time that it is feasible to detect a BRAF mutation from a small number of fresh FNAB specimens or slides in China, suggesting that detection of BRAF mutation on FNAB samples is useful not only for preoperative diagnosis of PTC but also for accurate pathologic diagnosis and prognosis.

Context: Radioiodine ablation is commonly used to treat thyroid cancer, but a major challenge is often the loss of radioiodine avidity of the cancer caused by aberrant silencing of iodide-handling genes., Objectives: This study was conducted to test the therapeutic potential of targeting the aberrantly activated MAPK and PI3K/Akt/mTOR pathways and histone deacetylase to restore radioiodine avidity in thyroid cancer cells., Experimental Design: We tested the effects of specific inhibitors targeting these pathways/molecules that had established clinical applicability, including the MAPK kinase inhibitor RDEA119, mTOR inhibitor temsirolimus, Akt inhibitor perifosine, and histone deacetylase inhibitor SAHA, individually or in combinations, on the expression of iodide-handling genes and radioiodide uptake in a large panel of thyroid cancer cell lines., Results: The expression of a large number of iodide-handling genes could be restored, particularly the sodium/iodide symporter, TSH receptor, and thyroperoxidase, by treating cells with these inhibitors. The effect was particularly robust and synergistic when combinations of inhibitors containing SAHA were used. Robust expression of sodium/iodide symporter in the cell membrane, which plays the most important role in iodide uptake in thyroid cells, was confirmed by immunofluorescent microscopy. Radioiodide uptake by cells was correspondingly induced under these conditions. Thyroid gene expression and radioiodide uptake could both be further enhanced by TSH., Conclusions: Targeting major signaling pathways could restore thyroid gene expression and radioiodide uptake in thyroid cancer cells. Further studies are warranted to test this therapeutic potential in restoring radioiodine avidity of thyroid cancer cells for effective ablation treatment.

We investigated the genotype-dependent therapeutic potential of targeting the phosphoinositide 3-kinase (PI3K)/Akt pathway for thyroid cancer. Proliferation of TPC1, Hth7, FTC133, OCUT1, K1, and BCPAP cells that harbored PI3K/Akt-activating genetic alterations was potently inhibited by the Akt inhibitor perifosine, whereas SW1736, Hth74, WRO, KAT18, and TAD2 cells that harbored no genetic alterations had no or only modest responses. Inhibition of Akt phosphorylation by perifosine was seen in these cells. Genetic-dependent apoptosis was induced by perifosine in cells selectively tested. Similarly, potent inhibition of cell proliferation by the mammalian target of rapamycin (mTOR) inhibitor temsirolimus occurred in virtually all the cells harboring genetic alterations, whereas modest inhibition was seen in some of the cells not harboring genetic alterations. Temsirolimus inhibited the phosphorylation of p70S6K, a substrate of mTOR. Knockdown of Akt1/2 or mTOR by shRNA approach inhibited the proliferation and colony formation of FTC133 and OCUT1 cells that harbored genetic alterations in the PI3K/Akt pathway but had no effect on SW1736 and KAT18 cells that did not. Transfection with PIK3CA mutants greatly sensitized SW1736 cells to perifosine and temsirolimus. Growth of xenograft tumors derived from FTC133 cells but not SW1736 cells in nude mice was dramatically inhibited by perifosine. Thus, this work for the first time shows that genetic alterations in the PI3K/Akt pathway confer thyroid cancer cells addiction to this pathway and their sensitivity to inhibition by targeting Akt and mTOR. This genotype-based targeting of the PI3K/Akt pathway using Akt and mTOR inhibitors may offer an effective therapeutic strategy for thyroid cancer and warrants further studies.

Context: Epidemiological studies have indicated that high iodine intake might be a risk factor for papillary thyroid cancer (PTC), which commonly harbors the oncogenic T1799A BRAF mutation., Objective: The objective of the study was to investigate the relationship between BRAF mutation in PTC and iodine intake in patients., Subjects and Methods: We analyzed and compared the prevalences of the T1799A BRAF mutation in classical PTC of 1032 patients from five regions in China that uniquely harbor different iodine contents in natural drinking water, ranging from normal (10-21 microg/liter) to high (104-287 microg/liter). The BRAF mutation was identified by direct DNA sequencing., Results: The prevalence of BRAF mutation was significantly higher in any of the regions with high iodine content than any of the regions with normal iodine content. Overall, BRAF mutation was found in 387 of 559 PTC with high iodine content (69%) vs. 252 of 473 PTC with normal iodine content (53%), with an odds ratio of 1.97 (95% confidence interval 1.53-2.55) for the association of BRAF mutation with high iodine content (P < 0.0001). In addition, clinicopathological correlation analysis, the largest one of its type ever, showed that BRAF mutation was significantly associated with extrathyroidal invasion, lymph node metastasis, and advanced tumor stages of PTC., Conclusions: High iodine intake seems to be a significant risk factor for the occurrence of BRAF mutation in thyroid gland and may therefore be a risk factor for the development of PTC. This large study also confirmed the association of BRAF mutation with poorer clinicopathological outcomes of PTC.

Background: The phosphatidylinositol 3-kinase (PI3K)/AKT pathway plays an important role in thyroid tumorigenesis and progression. Genetic alterations, particularly PIK3CA amplification and mutations and ras mutations, are the major cause of aberrant activation of this pathway in thyroid tumors. Epigenetic silencing of the PTEN gene, a negative regulator of the PI3K/AKT pathway, also occurs in thyroid tumors, but its relationship with genetic alterations in this pathway is unclear., Methods: By using quantitative methylation-specific polymerase chain reaction, the authors examined PTEN methylation and its relationship with genetic alterations in the PI3K/AKT pathway in various types of thyroid tumors., Results: The authors found PTEN methylation to become progressively higher from benign thyroid adenoma to follicular thyroid cancer and to aggressive anaplastic thyroid cancer, which harbored activating genetic alterations in the PI3K/AKT pathway correspondingly with a progressively higher prevalence. The association of PTEN methylation was seen with both overall genetic alterations and individual genetic alterations, particularly PIK3CA alterations and ras mutations, in the PI3K/AKT pathway within each of the 3 types of thyroid tumors. In contrast, no such relationship was observed for the tumor suppressor gene RASSF1A., Conclusions: The authors found an interesting association of PTEN methylation with the activating genetic alterations in the PI3K/AKT pathway in thyroid tumors. This finding is consistent with a model in which aberrant methylation and hence silencing of the PTEN gene, which coexists with activating genetic alterations of the PI3K/AKT pathway, may enhance the signaling of this pathway aberrantly activated by genetic alterations and hence contribute to the progression of thyroid tumors. Cancer 2008.

Context: Genetic alterations in receptor tyrosine kinases (RTKs) and phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK pathways have not been fully defined in anaplastic and follicular thyroid cancers [anaplastic thyroid cancer (ATC), follicular thyroid cancer (FTC)]., Objective: The objective of the study was to explore a wide-range genetic basis for the involvement of these pathways in ATC., Design: We examined mutations and copy number gains of a large panel of genes in these pathways and corresponding phosphorylation of ERK (p-ERK) and Akt., Results: We found frequent copy gains of RTK genes, including EGFR, PDGFRalpha and -beta, VEGFR1 and 2, KIT, and MET and in PIK3Ca, PIK3Cb, and PDK1 genes in the PI3K/Akt pathway. Mutations of Ras, PIK3Ca, PTEN, and BRAF genes and RET/PTC rearrangements were common, whereas mutations in PDK1, Akt1, Akt2, and RTK genes were uncommon in ATC. Overall, 46 of 48 ATC (95.8%) harbored at least one genetic alteration, and coexistence of two or more was seen in 37 of 48 ATC (77.1%). These genetic alterations were somewhat less common in FTC. Genetic alterations that could activate both the PI3K/Akt and MAPK pathways were found in 39 of 48 ATC (81.3%). RTK gene copy gains were preferentially associated with p-Akt, suggesting their dominant role in activating the PI3K/Akt pathway. The phosphorylation of Akt was far more common than p-ERK in FTC, and both were relatively common and often coexisted in ATC., Conclusions: Genetic alterations in the RTKs and PI3K/Akt and MAPK pathways are extremely prevalent in ATC and FTC, providing a strong genetic basis for an extensive role of these signaling pathways and the development of therapies targeting these pathways for ATC and FTC, particularly the former.

Background: It remains to be investigated whether the aberrant methylation of DNA repair genes plays a pathogenic role in BRAF mutation-promoted tumorigenesis of papillary thyroid cancer (PTC)., Methods: In the current study, the promoter methylation status of 23 DNA repair genes in relation to clinicopathologic characteristics and BRAF mutation was examined in PTC tumors using methylation-specific polymerase chain reaction., Results: Among the 38 PTC tumors examined, 3 of 23 DNA repair genes were hypermethylated, including the hMLH1 gene in 8 of 38 samples (21%), the PCNA gene in 5 of 38 samples (13%), and the OGG1 gene in 2 of 38 samples (5%). Methylation of these genes was also found in some thyroid cancer cell lines. Methylation of the hMLH1 gene in particular was found to be associated with lymph node metastasis of PTC (5 of 8 samples [63%] in the methylation group vs 3 of 30 samples [10%] in the nonmethylation group; P = .0049). Methylation of the hMLH1 gene was also found to be associated with the T1799A BRAF mutation in PTC (6 of 19 samples (32%) in the BRAF mutation-positive group vs 2 of 19 samples (11%) in the BRAF mutation-negative group; P = .042)., Conclusions: The data from the current study suggest that, as shown previously in colon cancer, aberrant methylation of the hMLH1 gene may play a role in BRAF mutation-promoted thyroid tumorigenesis.

The relationship among BRAF mutation, platelet counts, and platelet-derived growth factor (PDGF) with respect to clinicopathological outcomes of papillary thyroid cancer (PTC) may play a role in PTC pathogenesis but remains undefined. We examined the T1799A BRAF mutation by direct genomic DNA sequencing in 108 primary PTC samples from a Chinese cohort and analyzed its relationship with clinicopathological, hematological, and other laboratory results as well as the levels of expression of PDGF in tumors. We found that the BRAF mutation was significantly associated with extrathyroidal invasion and advanced tumor stages III and IV. Specifically, extrathyroidal invasion was seen in 30/54 (56%) PTC with BRAF mutation versus 18/54 (33%) PTC without the mutation (P=0.02). Tumor stages III and IV were seen in 16/54 (30%) PTC with BRAF mutation versus 7/54 (13%) PTC without the mutation (P=0.04). The BRAF mutation was also significantly associated with a higher platelet count, with 249.28+/-53.76 x 10(9)/l in the group of patients with BRAF mutation versus 207.79+/-58.98 x 10(9)/l in the group without the mutation (P=0.001). An association of higher platelet accounts with extrathyroidal invasion was also seen, with 242.66+/-51.85 x 10(9)/l in patients with extrathyroidal invasion versus 218.49+/-59.10 x 10(9)/l in patients without extrathyroidal invasion (P=0.03). The BRAF T1799A-positive PTC tissues harbored a significantly higher level of PDGF-B than BRAF T1799A-negative PTC tissues. The data suggest that the BRAF T1799A mutation is associated with aggressive pathological outcomes of PTC in which high platelet counts and increased PDGF production may play a role.

Context: It remains inconclusive whether mutations in thyroid hormone receptor (TR) genes naturally occur in thyroid cancer and whether these genes could be suppressors of this cancer., Objectives: Our objectives were to examine further mutations of TRalpha and TRbeta genes in thyroid cancer and also to examine their methylation as an epigenetic silencing mechanism in thyroid cancer., Experimental Design: Instead of using a cDNA sequencing approach used in previous studies, we used genomic DNA to sequence directly the coding regions of the TRalpha and TRbeta genes to search mutations in various differentiated thyroid tumors and used methylation-specific PCR to analyze promoter methylation of these genes. Allelic zygosity status at TRbeta was also analyzed., Results: We found no TRalpha gene mutation in 17 papillary thyroid cancers (PTCs) and 11 follicular thyroid cancers (FTCs), and no TRbeta gene mutation in 16 PTCs and 12 FTCs. We also found no methylation of the TRalpha gene in 33 PTCs, 31 FTCs, 20 follicular thyroid adenomas (FTAs), and 10 thyroid tumor cell lines. In contrast, we found hypermethylation of the TRbeta gene in 10 of 29 (34%) PTCs, 22 of 27 (81%) FTCs, five of 20 (25%) follicular thyroid adenomas, and three of 10 (30%) thyroid tumor cell lines, with the highest prevalence in FTC. We additionally examined loss of heterozygosity at TRbeta and found it in three of nine (33%) PTCs and three of nine (33%) FTCs., Conclusions: Mutation is not common in TR genes, whereas hypermethylation of the TRbeta gene as an alternative gene silencing mechanism is highly prevalent in thyroid cancer, particularly FTC, consistent with a possible tumor suppressor role of this gene for FTC.

Context: Genetic alterations in the phosphatidylinositol-3-kinase (PI3K)/Akt pathway and their role in thyroid tumor pathogenesis in Chinese people remain undefined., Objective: The objective of the study was to examine the major genetic alterations and their relationship in the PI3K/Akt pathway in differentiated thyroid tumors in a Chinese cohort., Design: We used real-time quantitative PCR for the analysis of PIK3CA copy gain and direct DNA sequencing for the detection of PIK3CA, RAS, and PTEN mutations on genomic DNA isolated from 234 thyroid tumors, including 31 follicular thyroid cancer (FTC), 141 papillary thyroid cancer (PTC), and 62 follicular thyroid adenoma (FTA)., Results: We found PIK3CA copy gain (defined as four or more copies) in nine of 31 FTC (29%), 20 of 141 PTC (14%), and five of 62 FTA (8%); PIK3CA gene mutations in four of 31 FTC (13%), one of 141 PTC (1%), and none of 62 FTA (0%); Ras mutations in three of 31 FTC (10%) and none of the 141 PTC and 62 FTA; and PTEN mutations in two of 31 FTC (6%) and none of 62 FTA (0%). Collectively, nine of 31 FTC (29%) vs. none of 62 FTA (0%) (P < 0.01) harbored one of the mutations, and when PIK3CA copy gain was included, 16 of 31 FTC (52%) vs. five of 62 FTA (8%) (P < 0.01) harbored any genetic alteration in the PI3K/Akt pathway. Mutual exclusivity was seen among all these PI3K/Akt pathway-related genetic alterations in all thyroid tumors except for two cases that harbored two genetic alterations., Conclusion: These data from a Chinese cohort provide further genetic evidence suggesting that dysregulated PI3K/Akt pathway plays a significant role in the pathogenesis of thyroid tumors, particularly FTC.

Purpose: The V600E BRAF mutant plays an important role in the pathogenesis of papillary thyroid cancer (PTC) and is associated with loss of expression of thyroid iodide-metabolizing genes. This study was done to investigate the restorability of expression of these genes by suppressing the BRAF/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway in V600E BRAF-harboring thyroid cells and to explore the mechanisms involved., Experimental Design: We used inducible expression of V600E BRAF, small interfering RNA transfection, and MEK-specific inhibitor to alter the MAP kinase pathway activities and subsequently examined the changes in expression, promoter activities, and methylation status of thyroid genes., Results: MEK inhibitor U0126 or cessation of V600E BRAF expression in PCCL3 cells restored expression of thyroid genes silenced by induced expression of V600E BRAF. U0126 also restored the expression of these genes in V600E BRAF-harboring PTC-derived NPA cells. Knockdown of BRAF by specific small interfering RNA restored expression of some of these genes in NPA cells. Luciferase reporter assay using thyroid-stimulating hormone receptor gene as a model showed that the promoter activity was modulated by the MAP kinase pathway. Promoter methylation in association with DNA methyltransferase expression played a role in gene silencing by MAP kinase pathway in NPA cells., Conclusions: We showed the restorability of expression of thyroid iodide-metabolizing genes silenced by V600E BRAF, and linked this process to gene methylation in PTC cells. The results provide clinical implications that therapeutic targeting at the BRAF/MEK/MAP kinase pathway may be a good approach in restoring thyroid gene expression for effective radioiodine therapy for BRAF mutation-harboring PTC.

Purpose: To investigate the overall occurrence and relationship of genetic alterations in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in thyroid tumors and explore the scope of this pathway as a therapeutic target for thyroid cancer., Experimental Design: We examined collectively the major genetic alterations and their relationship in this pathway, including PIK3CA copy number gain and mutation, Ras mutation, and PTEN mutation, in a large series of primary thyroid tumors., Results: Occurrence of any of these genetic alterations was found in 25 of 81 (31%) benign thyroid adenoma (BTA), 47 of 86 (55%) follicular thyroid cancer (FTC), 21 of 86 (24%) papillary thyroid cancer (PTC), and 29 of 50 (58%) anaplastic thyroid cancer (ATC), with FTC and ATC most frequently harboring these genetic alterations. PIK3CA copy gain was associated with increased PIK3CA protein expression. A mutual exclusivity among these genetic alterations was seen in BTA, FTC, and PTC, suggesting an independent role of each of them through the PI3K/Akt pathway in the tumorigenesis of the differentiated thyroid tumors. However, coexistence of these genetic alterations was increasingly seen with progression from differentiated tumor to undifferentiated ATC. Their coexistence with BRAF mutation was also frequent in PTC and ATC., Conclusions: The data provide strong genetic implication that aberrant activation of PI3K/Akt pathway plays an extensive role in thyroid tumorigenesis, particularly in FTC and ATC, and promotes progression of BTA to FTC and to ATC as the genetic alterations of this pathway accumulate. Progression of PTC to ATC may be facilitated by coexistence of PI3K/Akt pathway-related genetic alterations and BRAF mutation. The PI3K/Akt pathway may thus be a major therapeutic target in thyroid cancers.