The study was aimed at evaluating the correlation between central cervical lymph node metastasis (CLNM) in papillary thyroid carcinoma (PTC) patients and ultrasound (US) features, immunohistochemical factors and BRAF

Papillary thyroid carcinoma (PTC) is the most common endocrine cancer with a significantly increase of the incidence recently. Several cytokines, such as thyroid peroxidase (TPO), cluster of differentiation 56 (CD56), Galectin-3, mesothelial cell (MC), cytokeratin 19 (CK19) and BRAF (B-raf) were recommended to be tested by immunohistochemistry (IHC) for a definitive diagnosis, but were still limited in clinical use because of their relative lower sensitivity and specificity. MicroRNA (miRNA), as a new molecular biomarkers, however, has not been reported yet so far. To address this, hsa-miR-200a-5p, a miRNA, was selected and detected in PTC patients by in situ hybrization with benign thyroid tumor with papillary hyperplasia as a control, and the differential expression of hsa-miR-200a-5p between fresh PTC tissues and control was detected by qRT-PCR. Expressive levels of cytokines of TPO, CD56, Galectin-3, MC, CK19 and B-raf were also detected by immunohistochemistry. The correlation was analyzed by SPSS software using Spearman methods. As expected, the hsa-miR-200a-5p expressive level was significantly increased in PTC patients, compared to that of control, and was consistent with that of TPO, CD56, Galectin-3, MC, CK19 and B-raf. In addition, expression of hsa-miR-200a-5p showed negative correlation to that of TPO (rs = - 0.734; **: P < 0.01) and CD56 (rs = - 0.570; **: P < 0.01), but positive correlation to that of Galectin-3 (rs = 0.601; **: P < 0.01), MC (rs = 0.508; **: P < 0.01), CK19 (rs = 0.712; **: P < 0.01) and B-raf (rs = 0.378; **: P < 0.01). PTC and papillary benign thyroid papillary hyperplasia are difficult to distinguish in morphology, so requiring immunohistochemistry to further differentiate the diagnosis, however, for the existing clinical common diagnostic marker for immunohistochemistry, the sensitivity and accuracy are low, it is easy to miss diagnosis. Therefore, there is an urgent need for a rapid and sensitive molecular marker. So miR-200a-5p can be used to assist in the diagnosis of PTC at the molecular level, and as a biomarker, can be effectively used to distinguish between PTC and benign thyroid tumor with papillary hyperplasia.

Previous studies have suggested that thyroid function is associated with breast cancer risk, which could have an important clinical impact, as one in eight women will develop a thyroid disorder during her lifetime. However, the underlying pathomechanism behind the association is still unknown. We used the Malmö Diet and Cancer Study (a population-based prospective study consisting of 17,035 women) to examine 17 single nucleotide polymorphisms (SNPs) previously related to levels of free thyroxine (free T4) and thyroid peroxidase antibodies (TPO-Ab) as potential genetic risk factors for breast cancer. A baseline examination including free T4 and TPO-Ab levels was conducted at the time of inclusion. Genotyping was performed on 901 breast cancer patients and 3335 controls. Odds ratios (95% confidence intervals) for high free T4, TPO-Ab positivity, and breast cancer were calculated by logistic regression and adjusted for confounders. We identified one free T4-related SNP (rs2235544, D101 gene) that was significantly associated with both free T4 level and breast cancer risk. There was a suggested association between rs11675434 (TPO gene) and TPO-Ab level, and TPO-Ab-related rs11675434 (TPO), rs3094228 (HCP5), rs1033662 (no registered gene), and rs301806 (RERE) were associated with breast cancer risk. There was an indicated interaction between rs6485050 (no registered gene) and free T4 level in regards to breast cancer risk. This is the first study to suggest an association between thyroid-related SNPs and breast cancer risk. All SNPs have a biological plausibility of being associated with breast cancer risk, and may contribute to the genetic predisposition to breast cancer.

BACKGROUND: Anaplastic thyroid cancer (ATC) has a very poor prognosis due to its aggressive nature and resistance to conventional treatment. Radiotherapy and chemotherapy are not fully effective because of the undifferentiated phenotype and enhanced drug resistance of ATC. The objective of this study was to evaluate the involvement of Krüppel-like factor 4 (KLF4), a stemness-associated transcription factor, in the undifferentiated phenotype and drug resistance of ATC.
METHODS: ATC cells were compared to papillary thyroid cancer cells in drug resistance and gene expression. The effects of KLF4 knockdown in ATC cells on in vitro and in vivo drug resistance were measured. The effects of KLF4 overexpression and knockdown on ABC transporter activity were determined.
RESULTS: ATC cells, such as HTH83, 8505C, and SW1736, exhibited higher resistance to the anticancer drug paclitaxel and higher expression of KLF4 than TPC-1 papillary thyroid cancer cells. Knockdown of KLF4 expression in ATC cells increased the expression of the thyroid-specific differentiation genes, such as thyrotropin receptor, thyroid peroxidase, thyroglobulin, and sodium-iodide symporter. Knockdown of KLF4 expression in ATC cells decreased the resistance to doxorubicin and paclitaxel, and reduced ABC transporter expression. Luciferase reporter assay results showed that KLF4 overexpression increased ABCG2 promoter activity, which was abolished by KLF4 knockdown. A tumorigenicity assay showed that the combination of paclitaxel treatment and KLF4 knockdown significantly decreased tumor mass originated from HTH83 cells in mice.
CONCLUSIONS: ATC cells show high expression of KLF4, and KLF4 expression is necessary for maintaining the undifferentiated phenotype and drug resistance in vitro and in vivo. The present study identifies KLF4 as a potential therapeutic target for eliminating ATC cells.

BACKGROUND: Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed.
METHODS: In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs.
RESULTS: We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs.
CONCLUSIONS: Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.

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BACKGROUND: The thyroperoxidase (TPO) genetic variants in thyroid carcinoma is scarcely reported.
OBJECTIVE: We report on a pedigree of thyroid papillary carcinoma and hypoechoic thyroid nodules with the TPO gene mutations.
METHODS: The compound heterozygotic mutations of the TPO gene (c.2268-2269 insT and c.2090 G>A) in two patients with congenital goiters hypothyroidism were demonstrated. Fifteen family members of the proband and 105 control individuals were enrolled. The participants underwent clinical examination and molecular screening for TPO mutation. The hypoechoic thyroid nodules underwent fine needle aspiration biopsy.
RESULTS: The mutation c.2268-2269 insT was detected in the four family members with normal thyroid hormone levels. The other two members harbored the c.2090 G>A mutation. The heterozygotes had degeneratively hypoechoic thyroid nodules. The control individuals showed no mutation. The maternal grandfather developed a multifocal papillary thyroid carcinoma with lymph gland and nerve invasion in the left lobe of the thyroid gland. The maternal grandfather harbored the TPO c.2268-2269 insT mutation but without BRAFV600E mutation. Malignant cells were not observed in other members by fine needle aspiration biopsy.
CONCLUSION: TPO genetic variants may be associated with thyroid carcinoma and hypoechoic thyroid nodules in a few cases. Long-term follow-up in the pedigree with congenital goiter is reasonable.

BACKGROUND: Thyroid cancer incidence has dramatically increased worldwide over the last two decades. The rise is mostly due to an increased detection of small papillary thyroid carcinomas (PTCs) (≤20  mm), predominantly microPTC (≤10  mm). Although small tumors generally have an excellent outcome, a considerable percentage may have a more aggressive disease and worse prognosis. The clinical challenge is to preoperatively identify those tumors that are more likely to recur.
AIM: To improve risk stratification and patient management, we sought to determine the prognostic value of BRAF V600E, NRAS or RET/PTC mutations in patients with PTC measuring <20  mm, mainly microPTC.
METHODS: The prevalence of RET/PTC fusion genes was examined by quantitative RT-PCR. BRAF V600E and NRAS Q61 mutations were determined by PCR sequencing. To further elucidate why some small PTC are less responsive to radioactive iodine treatment therapy, we explored if these genetic alterations may modulate the expression of iodine metabolism genes (NIS, TPO, TG, TSHR and PDS) and correlated with clinico-pathological findings that are predictors of recurrence.
RESULTS: This study shows that tumors measuring ≤20  mm exhibited higher prevalence of BRAF V600E mutation, which correlated with aggressive histopathological parameters, higher risk of recurrence, and lower expression of NIS and TPO. Although this correlation was not found when microPTC were evaluated, we show that tumors measuring 7-10  mm, which were positive for BRAF mutation, presented more aggressive features and lower expression of NIS and TPO.
CONCLUSION: We believe that our findings will help to decide the realistic usefulness of BRAF V600E mutation as a preoperative marker of poor prognosis in small PTC, primarily in microPTC.

BACKGROUND: Whereas 40 % to 70 % of papillary thyroid carcinomas (PTCs) are characterized by a BRAF mutation (BRAFmut), unified biomarkers for the genetically heterogeneous group of BRAF wild type (BRAFwt) PTCs are not established yet. Using state-of-the-art technology we compared RNA expression profiles between conventional BRAFwt and BRAFmut PTCs.
METHODS: Microarrays covering 36,079 reference sequences were used to generate whole transcript expression profiles in 11 BRAFwt PTCs including five micro PTCs, 14 BRAFmut PTCs, and 7 normal thyroid specimens. A p-value with a false discovery rate (FDR) < 0.05 and a fold change > 2 were used as a threshold of significance for differential expression. Network and pathway utilities were employed to interpret significance of expression data. BRAF mutational status was established by direct sequencing the hotspot region of exon 15.
RESULTS: We identified 237 annotated genes that were significantly differentially expressed between BRAFwt and BRAFmut PTCs. Of these, 110 genes were down- and 127 were upregulated in BRAFwt compared to BRAFmut PTCs. A number of molecules involved in thyroid hormone metabolism including thyroid peroxidase (TPO) were differentially expressed between both groups. Among cancer-associated molecules were ERBB3 that was downregulated and ERBB4 that was upregulated in BRAFwt PTCs. Two microRNAs were significantly differentially expressed of which miR492 bears predicted functions relevant to thyroid-specific molecules. The protein kinase A (PKA) and the G protein-coupled receptor pathways were identified as significantly related signaling cascades to the gene set of 237 genes. Furthermore, a network of interacting molecules was predicted on basis of the differentially expressed gene set.
CONCLUSIONS: The expression study focusing on affected genes that are differentially expressed between BRAFwt and BRAFmut conventional PTCs identified a number of molecules which are connected in a network and affect important canonical pathways. The identified gene set adds to our understanding of the tumor biology of BRAFwt and BRAFmut PTCs and contains genes/biomarkers of interest.

CONTEXT: The aberrant silencing of iodide-handling genes accompanied by up-regulation of glucose metabolism presents a major challenge for radioiodine treatment of papillary thyroid cancer (PTC).
OBJECTIVE: This study aimed to evaluate the effect of tyrosine kinase inhibitors on iodide-handling and glucose-handling gene expression in BHP 2-7 cells harboring RET/PTC1 rearrangement.
MAIN OUTCOME MEASURES: In this in vitro study, the effects of sorafenib or cabozantinib on cell growth, cycles, and apoptosis were investigated by cell proliferation assay, cell cycle analysis, and Annexin V-FITC apoptosis assay, respectively. The effect of both agents on signal transduction pathways was evaluated using the Western blot. Quantitative real-time PCR, Western blot, immunofluorescence, and radioisotope uptake assays were used to assess iodide-handling and glucose-handling gene expression.
RESULTS: Both compounds inhibited cell proliferation in a time-dependent and dose-dependent manner and caused cell cycle arrest in the G0/G1 phase. Sorafenib blocked RET, AKT, and ERK1/2 phosphorylation, whereas cabozantinib blocked RET and AKT phosphorylation. The restoration of iodide-handling gene expression and inhibition of glucose transporter 1 and 3 expression could be induced by either drug. The robust expression of sodium/iodide symporter induced by either agent was confirmed, and (125)I uptake was correspondingly enhanced. (18)F-fluorodeoxyglucose accumulation was significantly decreased after treatment by either sorafenib or cabozantinib.
CONCLUSIONS: Sorafenib and cabozantinib had marked effects on cell proliferation, cell cycle arrest, and signal transduction pathways in PTC cells harboring RET/PTC1 rearrangement. Both agents could be potentially used to enhance the expression of iodide-handling genes and inhibit the expression of glucose transporter genes.

The aim of this study is to determine whether circulating epithelial cells (CEC) detected in patients with differentiated thyroid cancer (DTC) stem from the thyroid gland. CEC have been described to increase in patients with progressive cancer disease and thus have been used as a marker of tumour cell dissemination. CEC were selected from venous blood samples of five DTC patients and analysis of thyroid-specific mRNA (i.e. Tg, TSH-R, TPO, NIS) was performed on a single cell level. 16/48 cells were positive for at least three different thyroid-mRNA transcripts, predominantly found in patients with detectable serum thyroglobulin. In conclusion, evidence was found that in patients with detectable serum thyroglobulin, most of the CECs originate from the thyroid gland. However, further investigations including a larger sample size are needed to validate the clinical impact of this method.

CONTEXT: It was shown in the rat thyroid that thyroglobulin (Tg) stored in the follicular lumen is a potent regulator of thyroid-specific gene expression to maintain the function of individual follicles. However, the actions of Tg as a regulatory molecule in human thyroid have not been studied.
OBJECTIVE: Our objective was to determine the effect of Tg on gene expression in normal and diseased human thyroid and to examine whether the proposed model of negative-feedback autocrine regulation of thyroid function by Tg is applicable in the human as well as the rat.
DESIGN: Primary cultures of human thyrocytes were established from normal thyroid, Graves' disease thyroid, adenomatous goiter, follicular adenoma, and papillary carcinoma tissues obtained during surgery. Cells were stimulated with physiologic (ie, follicular) concentrations of Tg, and mRNA and protein expression of genes involved in thyroid hormonogenesis were evaluated. The effects of Tg on thyroid-specific gene expression were also assessed in 2 human papillary carcinoma cell lines.
RESULTS: Transcript levels of genes participating in thyroid hormone biosynthesis were significantly reduced by Tg in thyrocyte cultures derived from normal and Graves' thyroid, but not in cultures derived from thyroid neoplasms and adenomatous goiter.
CONCLUSION: It was confirmed that Tg acts as a negative-feedback regulator of gene expression in human thyrocytes, suggesting that Tg signaling may constitute a common mechanism for maintaining thyroid homeostasis in species with follicular thyroid morphology. However, certain diseases of intrinsic thyroid overgrowth appear to be associated with an escape from the regulatory mechanism of Tg.

Thyroid cancer risk involves the interaction of genetic and environmental factors. The thyroperoxidase (TPO) has a key role in the iodine metabolism, being essential for the thyroid function. Mutations in the TPO gene are common in congenital hypothyroidism, and there are also signs of the implication of TPO in thyroid cancer. We performed a case-control association study of genetic variants in TPO and differentiated thyroid carcinoma (DTC) in 1,586 DTC patients and 1,769 controls including two European populations (Italy: 1,190 DTC and 1,290 controls; Spain: 396 DTC and 479 controls). Multivariate logistic regression analyses were performed separately for each population and each single-nucleotide polymorphism (SNP). From the three studied polymorphisms, significant associations were detected between DTC and rs2048722 and rs732609 in both populations (p < 0.05). In the Italian population, both SNPs showed a negative association (rs2048722, odds ratio [OR] = 0.79, 95% confidence interval [CI] = 0.63-1.00, p = 0.045; rs732609, OR = 0.72, 95% CI = 0.55-0.94, p = 0.016), whereas in the Spanish population, these SNPs showed a positive association (rs2048722, OR = 1.39, 95% CI = 1.03-1.89, p = 0.033; rs732609, OR = 1.41, 95% CI = 1.06-1.87, p = 0.018). The corresponding associations for papillary or follicular thyroid cancer were similar to those for all DTC, within population. No association was detected for the third TPO polymorphism in the Italian and the Spanish populations. Our results, for the first time, point to TPO as a gene involved in the risk of DTC, and suggest the importance of interactions between TPO variants and other unidentified population-specific factors in determining thyroid cancer risk.

INTRODUCTION: Dedifferentiated thyroid cancer is often incurable because it does not respond to radioiodine. This study aimed to investigate iodide uptake and the expressions of thyroid-specific molecules after the transfection of human thyrotropin receptor (hTSHR) gene in poorly differentiated follicular thyroid cancer cell line (FTC-133).
METHODS: pGC-FU-hTSHR-GFP-lentivirus and pGC-FU-GFP-lentivirus were added into FTC-133 cells respectively. The parental cells were defined as the blank group. Cells transduced with pGC-FU-GFP and pGC-FU-hTSHR-GFP were defined as the control group and experimental group respectively. The efficiency of transfection was observed under a fluorescence microscope. (125)I uptake by FTC-133 was analyzed by measuring the radioactivity. Real time-PCR, western blotting and radioimmunoassay were applied to detect the expressions of mRNAs and proteins of Na(+)/I(-) symporter (NIS), thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO) and thyroglobulin (Tg) in FTC-133.
RESULTS: The green fluorescence was present in 80% of the transduced cells under fluorescence microscope. The iodine uptake of cells transduced with pGC-FU-TSHR-GFP was 3.3 times higher than that in the other two groups (P<0.01). NIS, TSHR, TPO and Tg had been significantly up-regulated in the experimental group as compared to the control group (P<0.01) and the blank group (P<0.01).
CONCLUSION: The hTSHR transfection in FTC-133 improved the expression of thyroid-specific molecules including TSHR, NIS, TPO and Tg and radioiodide uptake.

Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role for the development of megakaryocyte and considered to regulate megakaryocytopoiesis. Previously we reported that TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter and the expression of the c-mpl gene is modulated by transcription through a protein kinase C (PKC)-dependent pathway in the megakaryoblastic cells. In this research, to elucidate the required elements in c-mpl promoter, the promoter activity of the deletion constructs and site-directed mutagenesis were measured by a transient transfection assay system. Destruction of -77GATA in c-mpl promoter decreased the activity by 22.8%. Our study elucidated that -77GATA involved in TPO-induced c-mpl gene expression in a human megakaryoblastic cell line, CMK.

Anaplastic thyroid cancer (ATC) is an aggressive, fatal disease unresponsive to traditional therapies, generating a need to develop effective therapies. The PAX8/PPARγ fusion protein (PPFP) has been shown to favorably modulate tumor growth in follicular thyroid cancer, prompting our evaluation of its efficacy to inhibit ATC cell and tumor growth in vitro and in vivo. PPFP was constitutively expressed in five ATC cell lines: BHT-101, FRO, C-643, KTC-2 and KTC-3, and inhibited cell growth in four of five cell lines and xenograft tumor growth in four of four cell lines. PPFP-mediated growth inhibition involved multiple mechanisms, including upregulation of miR-122 and miR-375, associated with decreased angiogenesis and AKT pathway inactivation, respectively. Also, PPFP expression resulted in marked increase of thyroid-specific marker transcripts, including PAX8, thyroid peroxidase (TPO), sodium iodide symporter (NIS) and thyroglobulin, to varying degrees by activating their respective promoters, suggesting that PPFP induced cellular redifferentiation. Functional studies demonstrate that increased NIS messenger RNA is not associated with increased 125I uptake. However, ectopic expression of wild-type NIS-induced perchlorate-sensitive iodine uptake, suggesting that endogenous NIS in ATC cell lines is defective. As current treatment for ATC is only palliative, overexpression of PPFP may offer a novel therapeutic strategy for the treatment of ATC.

BACKGROUND: Numerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R) agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues.
METHODS: Breast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and for TPO-R protein expression by immunohistochemistry (IHC). Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot.
RESULTS: MPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43%) and malignant (3-17%) breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines.
CONCLUSIONS: Multiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and megakaryocyte precursors.

BACKGROUND: The occurrence of thyroid carcinoma in patients with congenital hypothyroidism (CH) caused by dyshormonogenesis is very rare, and has only been reported in one patient harboring mutations in the thyroid peroxidase (TPO) gene.
PATIENT FINDINGS: We report on a 29-year follow-up of two consanguineous siblings with CH due to total iodide organification defect who also had sensorineural hearing loss. Molecular analysis revealed a novel biallelic mutation of the TPO gene in which phenylalanine substitutes serine at codon 292 (c.875C>T, p.S292F) in exon 8. Despite early initiation, adequate doses of levothyroxine treatment and consequently normal thyrotropin (TSH) levels, the proposita developed a huge multinodular goiter (MNG) and underwent total thyroidectomy due to tracheal compression. Pathological examination revealed a unifocal follicular thyroid carcinoma without vascular invasion in the left lobe of the thyroid gland.
SUMMARY: Our finding of follicular thyroid carcinoma arising from dyshormonogenetic MNG in a patient without elevated serum TSH levels indicates that genetic and environmental factors other than TSH level might be involved in the development of thyroid carcinoma in dyshormonogenetic MNG.
CONCLUSIONS: Despite the rare occurrence of thyroid carcinoma in dyshormonogenetic MNG, we recommend long-term follow-up and regular neck ultrasound imaging to prevent delayed diagnosis of thyroid carcinoma.

INTRODUCTION: Differentiated thyroid carcinoma (DTC) usually has a high iodine uptake. However, dedifferentiation of DTC with decreased or no radioiodine ((131)I) uptake is observed in clinical practice, with poor prognosis. The aim of this study was to investigate the effects of (131)I radiation on radioiodine uptake (RAIU) and the expression of thyroid-specific molecules.
METHODS: FTC-133 cells were treated with (131)I, the dosage dictated by methylthiazol tetrazolium test results and preliminary experiments. The experimental cell group was incubated with (131)I for 48 h and then cultured for 3 months in (131)I-free medium. The control group was set without (131)I. Primary cells were defined as the blank group. Following treatment, RAIU was measured with a gamma counter as the counts/cell number. Na(+)/I(-) symporter (NIS), thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO) and thyroglobulin (Tg) levels were detected by Western blotting and radioimmunoassay, and their mRNAs were detected by real-time polymerase chain reaction.
RESULTS: RAIU of FTC-133 cells decreased gradually after coincubation with (131)I and did not recover even if (131)I was removed. The relative RAIU of the control and experimental groups was 0.567 and 0.182, respectively, a statistically significant difference (P<.01). Expression of NIS, TSHR, TPO and Tg decreased in the experimental group to a statistically significant degree compared to that of controls (P<.05).
CONCLUSION: Changes in the mRNA levels were in accordance with the expression of thyroid-specific proteins. Thus, FTC-133 cells undergo dedifferentiation during long-term culture in vitro, and (131)I may promote this progress.

Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter, an excellent tumor-specific promoter, has potential value for targeted gene therapy of glioma. We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma. The U87 and U251 cells were co-transfected with two adenoviral vectors, in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter, respectively. Then, we performed Western blot, 125I intake and efflux assays, and clonogenic assay with cancer cells. We also did 99mTc tumor imaging of nude mice models. After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO, glioma cells showed the 125I intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone. Western blots revealed bands of approximately 70 kDa and 110 kDa, consistent with the hNIS and hTPO proteins. In clonogenic assay, approximately 90% of co-transfected cells were killed, compared to 50% of control cells after incubated with 37 MBq of 131I. These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro. Co-transfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine. Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.

INTRODUCTION: Radioiodine is a routine therapy for differentiated thyroid cancers. In principle, undifferentiated thyroid cancers as well as nonthyroid cancers can concentrate and, thus, be treated with radioiodine after transfection with the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter is an effective tumor-specific promoter of gene expression and, thus, may be useful in targeted gene therapy of cancer.
METHODS: We used hTERT promoter-modulated expression of the hNIS and human thyroperoxidase (hTPO) genes in an experimental model of radioiodine-based treatment of malignant glioma. Cells were cotransfected by adenovirus in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the human cytomegalovirus (CMV) promoter (Ad-hTERT-hNIS and Ad-CMV-hTPO, respectively), and they were evaluated in cells thus transfecting transgene expression by western blots, (125)I uptake and influx, and clonogenecity after (131)I treatment.
RESULTS: After cotransfection with two adenovirus, cells showed about 31-34 times higher (125)I uptake than the control cells transfected with Ad-CMV-EGFP (enhanced green fluorescent protein) and almost 1.3-1.4 times higher (125)I uptake than cells only transfected with Ad-hTERT-hNIS. Western blots revealed two bands of ∼70 and 110 kDa, respectively. The in vitro clonogenic assay indicated that, after exposure to 100-1000 μCi of (131)I-iodide for 12 hours, 91%-94% of cells cotransfected with the hNIS and hTPO genes, 88%-93% of cells transfected with the hNIS gene, and only 62%-68% of control (nontransfected) cells were killed.
CONCLUSIONS: The experiments demonstrated that an effective therapy of (131)I was achieved in malignant glioma cell lines after induction of tumor-specific iodide uptake activity by the hTERT promoter-directed NIS expression in vitro. Cotransfection of the hNIS and hTPO genes can lead to longer retention of radioiodide, but did not increase cell killing over that achieved with transfection with the hNIS gene alone.

BACKGROUND: Lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator and autoantigen that is overexpressed in tumors, including prostate cancer (PCa). This oncoprotein promotes resistance to cell death induced by oxidative stress and chemotherapy by mechanisms that remain unclear. To get insights into these mechanisms we identified candidate target stress genes of LEDGF/p75 using pathway-specific gene expression profiling in PCa cells.
METHODS: A "Human oxidative stress and antioxidant defense" qPCR array was used to identify genes exhibiting significant expression changes in response to knockdown or overexpression of LEDGF/p75 in PC-3 cells. Validation of array results was performed by additional qPCR and immunoblotting.
RESULTS: Cytoglobin (CYGB), Phosphoinositide-binding protein PIP3-E/IPCEF-1, superoxidase dismutase 3 (SOD3), thyroid peroxidase (TPO), and albumin (ALB) exhibited significant transcript down- and up-regulation in response to LEDGF/p75 knockdown and overexpression, respectively. CYGB gene was selected for further validation based on its emerging role as a stress oncoprotein in human malignancies. In light of previous reports indicating that LEDGF/p75 regulates peroxiredoxin 6 (PRDX6), and that PRDXs exhibit differential expression in PCa, we also examined the relationship between these proteins in PCa cells. Our validation data revealed that changes in LEDGF/p75 transcript and protein expression in PCa cells closely paralleled those of CYGB, but not those of the PRDXs.
CONCLUSIONS: Our study identifies CYGB and other genes as stress genes potentially regulated by LEDGF/p75 in PCa cells, and provides a rationale for investigating their role in PCa and in promoting resistance to chemotherapy- and oxidative stress-induced cell death.

Anaplastic thyroid cancer represents one of the most aggressive cancers. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to have antiproliferative and/or redifferentiating properties in several malignancies, including thyroid cancer. The objective of this study was to investigate the effects of 1,25(OH)(2)D(3) and the superagonistic analog CD578 in anaplastic thyroid cancer, alone or in combination with paclitaxel, a taxane, and suberoylanilide hydroxamic acid (SAHA), a potent histone deacetylase inhibitor with promising effects in undifferentiated thyroid cancer. Four human thyroid cancer cell lines (FTC-133, C643, 8505C and HTh74) were treated with 1,25(OH)(2)D(3) or CD578, alone or in combination with paclitaxel or SAHA. Effects on cell growth and differentiation were evaluated. Clear effects on growth arrest were observed in a clonogenic assay, and absolute cell counts demonstrated a 24-36% reduction in all cell lines after 72h treatment with 1,25(OH)(2)D(3) (10(-6)M) and a 60% inhibition after 120h in the most sensitive cell line HTh74. A similar growth inhibition was shown after treatment with a 1000-fold lower concentration of analog CD578. This growth arrest was explained by antiproliferative effects, further supported by an increased % of cells in the G(0)-G(1) phase of the cell cycle and by a decreased transcription factor E2F1 mRNA expression. Combination treatments of 1,25(OH)(2)D(3) or CD578 with paclitaxel or SAHA resulted in an additive and in some conditions a synergistic effect on the inhibition of proliferation. Redifferentiation analysis revealed only a modest increase in sodium iodide symporter and thyroglobulin mRNA expression after treatment with 1,25(OH)(2)D(3), without additive effect after combination treatment. No effects were observed on TSH-receptor or thyroid peroxidase mRNA expression. Our in vitro findings demonstrate that the superagonistic vitamin D analog CD578 holds promise as adjuvant antiproliferative therapy of anaplastic thyroid cancer, especially in combination with other drugs such as paclitaxel or SAHA.

BACKGROUND: Subclinical hyperthyroidism is usually associated with Graves' disease or toxic nodular goiter. Here we report a family with hereditary subclinical hyperthyroidism caused by a constitutively activating germline mutation of the thyrotropin receptor (TSHR) gene.
METHODS: The proband was a 64-year-old Japanese woman who presented with a thyroid nodule and was found to be euthyroid with a suppressed serum TSH. The nodule was not hot. Although antibodies to thyroid peroxidase and thyroglobulin antibodies were present, TSHR antibodies were not detected by TSH-binding inhibition or by bioassay. Two of her middle-aged sons, but not her daughter, also had subclinical hyperthyroidism without TSHR antibodies. Without therapy, the clinical condition of the affected individuals remained unchanged over 3 years without development of overt hyperthyroidism.
RESULTS: A novel heterozygous TSHR point mutation causing a glutamic acid to lysine substitution at codon 575 (E575K) in the second extracellular loop was detected in the three family members with subclinical hyperthyroidism, but was absent in her one daughter with normal thyroid function. In vitro functional studies of the E575K TSHR mutation demonstrated a weak, but significant, increase in constitutive activation of the cAMP pathway.
CONCLUSION: Although hereditary nonautoimmune overt hyperthyroidism is very rare, TSHR activating mutations as a cause of subclinical hyperthyroidism may be more common and should be considered in the differential diagnosis, especially if familial.

CONTEXT: Children present a higher susceptibility to developing thyroid cancer after radioiodine exposure and also a higher frequency of functional metastases than adults.
OBJECTIVE: To assess the mRNA expression of the sodium/iodide (Na(+)/I(-)) symporter (NIS), the Pendred syndrome gene (PDS), thyroperoxidase (TPO), thyroglobulin (Tg) and TSH receptor (TSH-R) in normal thyroid tissues (NTTs) and papillary thyroid carcinomas (PTCs) among different age groups.
METHODS: Analysis included 59 samples: 21 NTTs and 38 PTCs, of which 21 were the classic type (CPTC) and 17 the follicular variant (FVPTC). Patients were divided into three age groups: I (n = 16) 5-21 years, II (n = 13) 22-59 years, and III (n = 10) 60-91 years. The relative mRNA expression of the five target genes was determinate by quantitative reverse transcription polymerase chain reaction (QRT-PCR).
RESULTS: Expression of all genes was significantly higher in NTTs than in PTCs, and it was not age dependent in the NTT group. Among PTCs, the mean expression of PDS, TPO and TSH-R was significantly lower in group II than in group I. PDS, TPO and Tg expression was significantly lower in classic PTCs than in FVPTCs. The difference was related to a higher frequency of the BRAF(V600E) mutation in the former group.
CONCLUSIONS: The finding of higher PDS, TPO and TSH-R mRNA expression in paediatric vs. adult primary tumour tissues supports the hypothesis that this might contribute to the increased functional activity of metastases in the paediatric group. The finding that mRNA expression of the target genes in NTT was not age dependent does not provide an explanation for the higher susceptibility in the paediatric group.

A low sodium iodide symporter (NIS) expression has been shown in papillary thyroid carcinomas (PTCs) harboring the BRAFV600E mutation. In the present study, we analyzed the mRNA expression of thyroid differentiation genes, glucose transporter (GLUT)-1 and GLUT-3, in 78 PTCs according to the presence of BRAFV600E or RET/PTC rearrangements. We found BRAFV600E and RET/PTC rearrangements in 35.8 and 19.4% of PTCs respectively. The mRNA expression of NIS and thyroperoxidase (TPO) genes were significantly lower (P<0.0001 and P=0.004 respectively) in BRAFV600E-positive PTC with respect to non-mutated samples. In support of this result, immunohistochemistry showed that the percentage of NIS-positive cells was significantly lower (P=0.005) in BRAFV600E-mutated PTC (mean 53.5%) than in negative cases (mean 72.6%). In contrast, no difference either in NIS or in any other thyroid differentiation genes' mRNA expression was found in PTC with or without RET/PTC rearrangements. When GLUT-1 and GLUT-3 mRNA expression was considered, no correlation was found either in BRAFV600E- nor in RET/PTC-mutated cases. In conclusion, this study confirmed the presence of a genetic alteration of BRAF and/or RET oncogenes in 64% of PTC cases and revealed a significant correlation of BRAFV600E mutation with a lower expression of both NIS and TPO. This latter finding could indicate that an early dedifferentiation process is present at the molecular level in BRAFV600E-mutated PTC, thus suggesting that the previously demonstrated poor prognostic significance of BRAFV600E mutation could be related to the dedifferentiation process more than to a more advanced stage at diagnosis.

The aim of this study was to comparatively investigate the effects of 5-azacytidine-C (5-Aza), trichostatin-A (TSA), and all-trans retinoic acid (ATRA) on the mRNA expressions of the sodium and iodine (Na/I) symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), and the thyroid-stimulating hormone receptor (TSH-R), as well as radioiodine (RAI) uptake in cancer (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines. Cell lines were treated with 10 ng/mL of TSA, 5 microM of 5-AZA, and 1 microM of ATRA, according to the MTT (methyl-thiazol-tetrazolium) test results. Additionally, recombinant thyroid-stimulating hormone (rTSH) was also applied, with a selected dose of 100 ng/mL. Following the treatment, NIS, Tg, TPO, and TSH-R mRNA levels were detected by real-time-polymerase chain reaction (RT-PCR) and RAI uptakes were measured by using a well counter as counts/cell number. 5-Aza increased TSH-R mRNA expression in both of the cell lines and decreased TPO, NIS, and Tg mRNA levels in the cancer cell line. In the normal thyroid cell line, 5-AZA increased TPO mRNA levels by 2-fold and made no differences in NIS and Tg mRNA levels. TSA treatment repressed NIS and Tg mRNA levels and made no change on other thyroid-specific genes that were investigated in the cancer cell line. In the normal thyroid cell line, TSA increased TSH-R mRNA levels in 72 hours and created no important difference in the other genes. ATRA repressed the TSH-R mRNA levels in the normal thyroid cell line and increased the TPO and Tg mRNA levels slightly in both the cell lines. Furthermore, in short-term treatment, ATRA repressed the NIS gene expression slightly, but in the long term, this repression turned to basal levels. 5-Aza, TSA, and ATRA did not make any changes in RAI uptake in the cancer cell line, but rTSH increased RAI uptake significantly. In the normal thyroid cell line, TSA and ATRA decreased RAI uptake (to 1/10 and 1/2, respectively), but 5-Aza and rTSH increased RAI uptake significantly (2- and 4-fold, respectively). In our study, we showed an increase in TSH-R gene expression and radioiodine uptake with 5-Aza. Further in vitro and in vivo studies are needed to support our findings and the potential clinical use of this agent.

The aim of this study was to comparatively investigate the effects of 5-azacytidine-C (5-Aza), trichostatin-A (TSA), and all-trans retinoic acid (ATRA) on mRNA expressions of Na/I symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), and thyroid stimulating hormone receptor (TSH-R), and radioiodine (RAI) uptake in cancer (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines. Cell lines were treated with 10 ng/mL of TSA, 5 microM of 5-Aza, and 1 microM of ATRA, according to the MTT (methyl-thiazol-tetrazolium) test results. Additionally, recombinant thyroid stimulating hormone (rTSH) was also applied, with a selected dose of 100 ng/mL. Following the treatment, NIS, Tg, TPO, and TSH-R mRNA levels were detected by real-time-polymerase chain reaction (RT-PCR) and RAI uptakes were measured by using a well counter as the counts/cell number. 5-Aza increased TSH-R mRNA expression in both of the cell lines and decreased TPO, NIS, and Tg mRNA levels in the cancer cell line. In the normal thyroid cell line, 5-Aza increased TPO mRNA levels 2-fold and made no differences in NIS and Tg mRNA levels. TSA treatment repressed NIS and Tg mRNA levels, and made no differences on other thyroid specific genes investigated in the cancer cell line. In the normal thyroid cell line, TSA increased TSH-R mRNA levels in 72 hours and created no important differences in other genes. ATRA repressed the TSH-R mRNA levels in the normal thyroid cell line and increased the TPO and Tg mRNA levels slightly in both cell lines. Furthermore, in short-term treatment, ATRA repressed NIS gene expression slightly, but in the long term, this repression turned to basal levels. 5-Aza, TSA, and ATRA did not make any differences in RAI uptake in the cancer cell line, but rTSH increased RAI uptake significantly. In the normal thyroid cell line, TSA and ATRA decreased RAI uptake (to 1/10 and 1/2, respectively), but 5-Aza and rTSH increased RAI uptake significantly (2- and 4-fold, respectively). We have shown an increase in TSH-R gene expression and radioiodine uptake with 5-Aza. Further in vitro and in vivo studies are needed to support our findings and the potential clinical use of this agent.

Immunocytochemistry (ICC) of thyroid peroxidase (TPO) using the monoclonal antibody MoAb47 has been used as malignancy marker on thyroid fine needle aspiration. However, little is known about the fate of TPO in thyroid carcinoma. We performed a qualitative PCR (Q-PCR) analysis to measure the expression of variants of tpo mRNA in 13 normal tissue samples, 30 benign tumors (BT), 21 follicular carcinomas (FC), 20 classical papillary carcinomas (PCc), 12 follicular variants of papillary carcinomas (PCfv) and nine oncocytic carcinomas (OC). We also studied mutations involving the ras, Braf, ret or pax8 genes. Results of Q-PCR were closely correlated with those of ICC (P < 0.0001; R = 0.59) and showed that overall tpo expression was lower in all carcinomas than in normal and BT (P < 0.05). The ratio tpo2 or tpo3 to tpo1 was inversed in follicular tumors. Genetic mutations were observed in 90% of PCc, 61.9% of FC, 41.7% of PCfv, 0% of OC and 10% in BT. pax8-ppar gamma1 rearrangement was correlated with qualitative changes in tpo mRNA (P < 0.01). These results confirmed the decrease of TPO expression in 97% of thyroid carcinomas regardless of histological type and the overexpression of shorter splice variants in follicular tumors. Both reduction in quantity of TPO and impairment of its maturation process could account for the atypical immunohistochemical reaction of MoAb47 with TPO.

OBJECTIVE: Congenital primary hypothyroidism occurs in 1 of 4000 births. Whereas the majority of the cases are due to developmental defects of the thyroid gland, 20% carry a defect in thyroid hormonogenesis. We report a Turkish boy who had goitrous hypothyroidism due to a mutation in the thyroid peroxidase (TPO) gene.
DESIGN: The TPO gene was sequenced directly from genomic DNA and cDNA which was transcribed from three RNA samples harvested from different parts of the patient's excised thyroid gland. Patient The boy was thyroidectomized because of continuing growth of his thyroid gland and development of multiple nodes suspected of malignancy by ultrasound examination. Histopathological examination verified a dyshormonogenetic goiter with multiple follicular adenomas.
RESULTS: The patient had a novel homozygous 10-bp deletion of the TPO gene at position 2812 in exon 16. This frame shift mutation results in a severely altered intracellular part of the protein. The deletion identified in leucocyte DNA was also found in thyroid tissue cDNA - so that instability of the transcript or a splicing defect was excluded. Both unaffected parents were heterozygous carriers of the mutation whereas 50 healthy individuals of the same ethnic background did not harbour the mutation.
CONCLUSIONS: The identified TPO gene deletion is the first mutation coding for an inactive TPO molecule, which has a severely altered intracellular segment. Because the most likely reason for the enlarging goiter was poor compliance of the patient, this report underlines the importance of a careful and regular follow-up of patients with dyshormonogenesis.