Thyroid Tumor Progression Research Articles

SUN-338 Inflammation Promotes Aggressive Thyroid Cancer Progression in a Mouse Model

Thyroid cancer is the most common endocrine malignancy. The association between inflammation and thyroid cancer has long been recognized. However, a cause-effect relationship at the molecular level has yet to be elucidated. We have created a mutant mouse, which expresses a dominant negative thyroid hormone receptor β (denoted as TRβPV) and deficiency in one allele of the Pten gene (ThrbPV/PVPten+/- mice). This mutant mouse exhibits aggressive follicular thyroid cancer similarly as in patients. We explored how inflammation could contribute to thyroid carcinogenesis in ThrbPV/PVPten+/- mice. Using FACS analysis, we found significantly increased inflammatory monocytes in thyroid tumors of ThrbPV/PVPten+/- mice. We next identified alterations in inflammation-related genes during thyroid carcinogenesis by cDNA microarrays to compare global gene expression profiles between thyroids of wild-type and ThrbPV/PVPten+/- mice. Gene array data revealed that a total 2,410 genes were differentially altered in thyroid tumors of ThrbPV/PVPten+/- mice (1,097 genes were up-regulated, and 1,313 genes were down-regulated). Analysis of the expression data showed an enrichment of genes that were associated with immune cell markers, cytokines, and chemokines, including Ptgs1, Sphk1, OPN, Tnfrsf18, CCL12, and IL6. Using Q/PCR, we validated the array data to show the significant increase in the expression of pro-inflammatory target genes [such as Ptgs1 (3 folds); Sphk1 (7.5 folds); OPN (5.4 folds), MCP1 (2.5 folds), IL17 (10.3 folds) and Tnfrsf18 (9.4 folds)] and significant decrease in the expression of anti-inflammatory genes [Ephx2 (2.3 folds) and CD163 (5.9 folds)] in thyroid tumors of ThrbPV/PVPten+/- mice. We further delineated the inflammatory role of osteopontin (OPN) in the thyroid carcinogenesis of ThrbPV/PVPten+/- mice. OPN is thought to facilitate the recruitment of monocytes/macrophages and to mediate cytokine secretion in leukocytes. Extracellular OPN functions through its interaction with multiple cell surface receptors (e.g., integrins and CD44), leading to the activation of PI3K-AKT and NF-kB signaling. We found that the protein abundance of OPN and integrin β1 was highly increased and concordantly, the downstream effectors, AKT and NF-kB were significantly elevated to drive thyroid tumor progression of ThrbPV/PVPten+/- mice. These results demonstrated that increased inflammation driven by elevated expression of immune-related genes and cytokines promoted thyroid cancer progression and that OPN played important roles in thyroid carcinogenesis. These preclinical findings suggest that OPN can be a potential target for thyroid cancer therapy.

Vitamin C kills thyroid cancer cells through ROS-dependent inhibition of MAPK/ERK and PI3K/AKT pathways via distinct mechanisms.

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.

Molecular evaluation of BRAF gene mutation in thyroid tumors: Significant association with papillary tumors and extra thyroidal extension indicating its role as a biomarker of aggressive disease

BackgroundSomatic mutation of the BRAF gene is one to be the most commonly known genetic change in thyroid tumors especially papillary thyroid cancers. The T1799A activating point mutation is detected in >98% of the thyroid tumors, and result in substitution of amino acid valine at position 600 to glutamic acid. MethodIn this study, we evaluated BRAF mutation in 95 Indian thyroid tumors by pyrosequencing assay. ResultsOverall, 36 cases (38%) showed presence of BRAF V600E mutation, while none of the cases showed V600 K mutation. BRAF mutation was found predominantly in female patients in comparison to males (38.4% vs. 36.4%, p = .86). Likewise, smaller sized tumors (≤2.0 cm) showed increased frequency of BRAF mutation as compared to larger sized tumors which were greater than equal to 2 cm (46% vs. 34.4%, p = .64). Furthermore, the frequency of BRAF mutations was significantly higher in conventional PTC tumor type in comparison to non-conventional and other than PTC tumor type (56% vs. 35% vs. 4%, p = .0007). Notably, a significant correlation between presence of BRAF mutation and extra-thyroidal extension was noted. Nevertheless, presence of BRAF mutation was neither associated with capsular/vascular invasion, nor with tumor necrosis. ConclusionsPyrosequencing assay was found to be highly sensitive and accurate method for detecting BRAF point mutations. The frequency and distribution pattern of BRAF mutations is similar to global reports. Furthermore, association of BRAF mutation with extra thyroidal extension indicates its aggressive nature and thus can provide insights into the progression of thyroid tumors from less aggressive to poorly differentiated subtype.

Aminophenols increase proliferation of thyroid tumor cells by inducing the transcription factor activity of estrogen receptor α

Aminophenols, which are widely used as components of hair dye and medicine, may function as environmental endocrine disruptors by regulating the proliferation of endocrine-related cancers. Estrogen receptor α (ERα) is a key regulator of breast cancer. Recently, it was found that ERα may also participate in the transformation and progression of thyroid tumors, but its interaction with aminophenols and its function in thyroid tumors is not clear. In this study, the transcription factor activity of ERα in BHP10-3 cells (a thyroid tumor cell line) was examined using luciferase assays. The promoter recruitment of ERα was examined using chromatin co-precipitation (ChIP). Additionally, in an in vivo study, BHP10-3 cells were transplanted into nude mice. Upon administration of aminophenols, the transcription factor activity of ERα was significantly increased in BHP10-3 cells, and the recruitment of ERα to the promoter of its target gene was increased. Aminophenols enhanced the in vitro and in vivo proliferation of BHP10-3 cells. By discovering that aminophenols induce the onco-promoting activity of ERα, our study extends the understanding of the function of aminophenols and suggests that ERα is a potential therapeutic target for the treatment of thyroid tumors.

The mitochondrial DNA control region might have useful diagnostic and prognostic biomarkers for thyroid tumors

Background: It is currently present in the literature that mitochondrial DNA (mtDNA) defects are associated with a great number of diseases including cancers. The role of mitochondrial DNA (mtDNA) variations in the development of thyroid cancer is a highly controversial topic. In this study, we investigated the role of mt-DNA control region (CR) variations in thyroid tumor progression and the influence of mtDNA haplogroups on susceptibility to thyroid tumors. Material & method: For this purpose, totally 108 hot thyroid nodules (HTNs), 95 cold thyroid nodules (CTNs), 48 papillary thyroid carcinoma (PTC) samples with their surrounding tissues and 104 healthy control subject9s blood samples were screened for entire mtDNA CR variations by using Sanger sequencing. The obtained DNA sequences were anaysed with the mistomaster, a web-based bioinformatics tool. Results: MtDNA haplogroup U was significantly associated with susceptibility to benign and malign thyroid entities on the other hand J haplogroup was associated with a protective role for benign thyroid nodules. Besides, 8 SNPs (T146C, G185A, C194T, C295T, G16129A, T16304C, A16343G and T16362C) in mtDNA CR region were associated with the occurrence of benign and malign thyroid nodules in Turkish population. By contrast with the healthy Turkish population and HTNs, frequency of C7 repeats in D310 polycytosine sequence was found higher in cold thyroid nodules and PTC samples. Beside this, the frequency of somatic mutations in mtMSI regions including T16189C and D514 CA dinucleotide repeats were found higher in PTC samples than the benign thyroid nodules. Conversely, the frequency of somatic mutations in D310 was detected higher in HTNs than CTNs and PTCs. Conclusion: mtDNA D310 instability do not play a role in tumorogenesis of the PTC but the results indicates that it might be used as a diagnostic clonal expansion biomarker for premalignant thyroid tumor cells. Beside this, D514 CA instability might be used as prognostic biomarker in PTCs. Also, we showed that somatic mutation rate is less frequent in more aggressive tumors when we examined micro- and macro carcinomas as well as BRAFV600E mutation.

Long Non-coding RNA Linc-ROR Is Upregulated in Papillary Thyroid Carcinoma.

Long non-coding RNAs (lncRNAs) may contribute to carcinogenesis and tumor progression by regulating transcription and gene expression. The role of lncRNAs in the regulation of thyroid cancer progression is being extensively examined. Here, we analyzed three lncRNAs that were overexpressed in papillary thyroid carcinomas, long intergenic non-protein coding RNA, regulator of reprogramming (Linc-ROR, ROR) PVT1 oncogene (PVT1), and HOX transcript antisense intergenic RNA (HOTAIR) to determine their roles in thyroid tumor development and progression. ROR expression has not been previously examined in thyroid carcinomas. Tissue microarrays (TMAs) of formalin-fixed paraffin-embedded tissue sections from 129 thyroid cases of benign and malignant tissues were analyzed by in situ hybridization (ISH), automated image analysis, and real-time PCR. All three lncRNAs were most highly expressed in the nuclei of PTCs. SiRNA experiments with a PTC cell line, TPC1, showed inhibition of proliferation with siRNAs for all three lncRNAs while invasion was inhibited with siRNAs for ROR and HOTAIR. SiRNA experiments with ROR also led to increased expression of miR-145, supporting the role of ROR as an endogenous miR-145 sponge. After treatment with TGF-β, there was increased expression of ROR, PVT1, and HOTAIR in the PTC1 cell line compared to control groups, indicating an induction of their expression during epithelial to mesenchymal transition (EMT). These results indicate that ROR, PVT1, and HOTAIR have important regulatory roles during the development of PTCs.

Aggressive differentiated thyroid cancer

Differentiated thyroid cancer is characteristically associated with an innocuous clinical course, but a minority of cases may manifest surprisingly aggressive behaviour. Such aggressive DTC are directly responsible for the majority of thyroid cancer related deaths. Moreover, they contribute indirectly to increased DTC-related morbidity, because our inability to differentiate these tumours from innocuous DTC at an early stage fuels a significant degree of DTC overtreatment around the globe. In the present paper we describe how improved understanding of the clinicopathological thyroid tumour progression model and optimization of clinical staging systems continues to improve our ability to diagnose and treat aggressive DTC. Early recognition of aggressive DTC allows instillation of an aggressive management strategy which is based upon surgical-oncologic completeness, and minimization of treatment-related sequelae through continued development of reconstructive options and focussed delivery of adjuvant treatments.

Role of YAP-1 in Thyroid Tumor Progression and Outcome.

Yes-associated protein-1 (YAP-1) is a player of the Hippo pathway and is involved in regulating cell proliferation. YAP-1 is overexpressed in papillary and anaplastic thyroid cancers. However, a correlation between YAP-1 expression and outcome in thyroid carcinoma has not been conclusively demonstrated. This study was designed to clarify whether YAP-1 may be considered a marker of worse prognosis and outcome in thyroid cancer. A large series of cases of thyroid cancer with a long follow-up were investigated for YAP-1 expression. The study was carried out in the Pathology section of a referral Italian center for Endocrine Surgery and Endocrinology. The study included a consecutive series of 105 patients who underwent thyroidectomy from 1985 to 1992. The mean follow-up was 15 years. For all patients, clinicopathologic features were considered. All patients completed the study.The study also included a consecutive series of 52 patients who underwent thyroidectomy from 2012 to 2013 in order to analyze more deeply the correlation of YAP-1expression with BRAF mutation. The 105 thyroid tumors were immunohistochemically investigated for YAP-1 expression. We expected a correlation between YAP-1 expression and worse prognosis. Among 105 tumors, 77 scored positive for YAP-1 expression, of which 68 papillary thyroid carcinomas and 9 anaplastic thyroid carcinomas were YAP-1 positive. The correlation of YAP-1 expression with clinicopathologic characteristics was significant for the absence of a tumoral capsule, gender, and extrathyroid invasion.Interestingly, significant correlations were found between YAP-1 and both persistence of disease and death from carcinoma. The data show an association of YAP-1 expression with worse clinicopathologic features of thyroid tumors that seem to have a specific impact on outcome.

Abstract 3973: Role of thyroid tumor microenvironment secretome in cancer initiation and progression

Abstract The incidence of thyroid cancer in the United States is on a rise with an appreciably high disease recurrence rate of 20-30%. Tumor associated macrophages (TAMs) release cytokines, chemokines and other secretory components like exosomes which aid in thyroid tumor progression and metastasis. In our previous study on thyroid tumor microenvironment, we established that M1 polarized pro-inflammatory macrophages modulate thyroid cancer phenotype. TAMs secretome, consisting of pro-inflammatory cytokines such as TGF-β, IL6, TNF-α, IL-1β, amongst others, induce epithelial to mesechymal transition (EMT) in thyroid cancer cells promoting tumor metastasis and propagation. In our present study, we used an in vitro model system to assess the effects of secretory molecules called exosomes, in the thyroid tumor microenvironment. We used thyroid cancer cell lines: BCPAP (papillary), 8505C (anaplastic) and CGTHW-1 (follicular) to represent the spectrum of clinically observed thyroid cancers. THP-1 monocyte/macrophage cell line was used to denote the inflammatory component. Thyroid cancer cells treated with activated M1 polarized THP-1 macrophage exosomes showed halt in proliferation, transformation to mesenchymal phenotype as well as modulation in expression of EMT markers, such as vimentin and NFk-B, indicative of induction of EMT in thyroid cancer cells. Interestingly, secretory exosomes from anaplastic thyroid cancer cells led to activation of THP-1 monocytes. Since the exosomal cargo is the reflection and fingerprint of its originating parental cells, we profiled exosomal miRNA derived from thyroid cancer cells and THP1 cells. Modulation of various miRNA in follicular and anaplastic thyroid cancer exosomes was observed, when compared to papillary thyroid cancer exosomes. We detected a down regulation of miR-138-5p, miR-146a-5p, miR-26a-5p, miR-26b-5p, miR-34a-5p and miR-31-5p in anaplastic and follicular cancer exosomes, which may play a role in promoting cancer invasion and metastasis. In comparison to papillary thyroid cancer exosomes, an upregulation of miR-214-3p, miR-200a-3p, miR-298, miR-299-3p, and miR-302a-3p was ascertained in follicular thyroid cancer exosomes. Conversely, we noted a down regulation of these miRNAs in anaplastic thyroid cancer exosomes. These distinct miRNA expressions in cancer secretome will provide new insights into the tumor development and dissemination. Our findings suggest an important crosstalk between the secretome of thyroid cancer cells and inflammatory cells in tumor microenvironment, defining thyroid cancer phenotype. These exosomal miRNA serve as early diagnostic markers of thyroid cancer differentiation as well as targets for novel therapies specifically for anaplastic thyroid cancer. Citation Format: Neha Yashpal Tuli, Ameet Kamat, Rachana Maniyar, Ghada Ben Rahoma, Sanjukta Chakraborty, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Role of thyroid tumor microenvironment secretome in cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3973. doi:10.1158/1538-7445.AM2017-3973

Identification of somatic TERT promoter mutations in familial nonmedullary thyroid carcinomas.

The genes causing familial nonmedullary thyroid carcinoma (FNMTC) identified to date are only involved in a small fraction of the families. Recently, somatic mutations in TERT promoter region and in EIF1AX gene were reported in thyroid tumours of undefined familial status. The aim of this study was to investigate the role of TERT and EIF1AX mutations in familial thyroid tumours. The promoter region of TERT was sequenced in leucocyte DNA of the probands from 75 FNMTC families. In thyroid tumours from 54 familial cases, we assessed somatic TERT promoter, RAS and BRAF hotspot mutations, and the whole EIF1AX gene. No potentially pathogenic germline variants were identified in TERT in the 75 FNMTC families' probands. In the 54 carcinomas, we identified five cases (9%) with hotspot somatic TERT promoter mutations. BRAF mutations were found in 41% of the tumours. All TERT-positive samples were also positive for BRAF p.Val600Glu, and this co-occurrence was found to be statistically significant (P=.008). RAS mutations were detected in four tumours wild-type for TERT (7%). Evaluation of tumour mutation data together with the patients' clinicopathological features revealed a significant correlation between TERT plus BRAF mutations and advanced tumour stage (T4) (P=.020). No mutations were identified in EIF1AX. The results of this study suggest that TERT promoter and EIF1AX mutations are not frequently involved in FNMTC aetiology. However, we show for the first time that TERT alterations are associated with familial thyroid tumour progression. Our data also suggest that TERT mutations are more often found in concomitance with BRAF mutations in advanced stages of FNMTC.

Bromodomain and Extraterminal Protein Inhibitor JQ1 Suppresses Thyroid Tumor Growth in a Mouse Model.

New therapeutic approaches are needed for patients with thyroid cancer refractory to radioiodine treatment. An inhibitor of bromodomain and extraterminal domain (BET) proteins, JQ1, shows potent antitumor effects in hematological cancers and solid tumors. To evaluate whether JQ1 is effective against thyroid cancer, we examined antitumor efficacy of JQ1 using the ThrbPV/PVKrasG12D mouse, a model of anaplastic thyroid cancer. We treated ThrbPV/PVKrasG12D mice with vehicle or JQ1 at a dose of 50 mg/kg body weight/day starting at the age of 8 weeks for a 10-week period and monitored thyroid tumor progression. JQ1 markedly inhibited thyroid tumor growth and prolonged survival of these mice. Global differential gene expression analysis showed that JQ1 suppressed the cMyc (hereafter referred to as Myc) transcription program by inhibiting mRNA expression of Myc, ccnd1, and other related genes. JQ1-suppressed Myc expression was accompanied by chromatin remodeling as evidenced by increased expression of histones and hexamethylene bis-acetamide inducible 1, a suppressor of RNA polymerase II transcription elongation. Analyses showed that JQ1 decreased MYC abundance in thyroid tumors and attenuated the cyclin D1-CDK4-Rb-E2F3 signaling to decrease tumor growth. Further analysis indicated that JQ1 inhibited the recruitment of BDR4 to the promoter complex of the Myc and Ccnd1 genes in rat thyroid follicular PCCL3 cells, resulting in decreased MYC expression at the mRNA and protein levels to inhibit tumor cell proliferation. These preclinical findings suggest that BET inhibitors may be an effective agent to reduce thyroid tumor burden for the treatment of refractory thyroid cancer. Clin Cancer Res; 23(2); 430-40. ©2016 AACR.

HAND1 gene expression is negatively regulated by the High Mobility Group A1 proteins and is drastically reduced in human thyroid carcinomas.

HMGA1 proteins exert their major physiological function during embryonic development and play a critical role in neoplastic transformation. Here, we show that Hand1 gene, which codes for a transcription factor crucial for differentiation of trophoblast giant cells and heart development, is upregulated in hmga1 minus embryonic stem cells. We demonstrate that HMGA1 proteins bind directly to Hand1 promoter both in vitro and in vivo and inhibit Hand1 promoter activity. We have also investigated HAND1 expression in human thyroid carcinoma cell lines and tissues, in which HMGA proteins are overexpressed, with respect to normal thyroid; an inverse correlation between HMGA1 and HAND1 expression was found in all thyroid tumor histotypes. A correlation between HAND1 gene repression and promoter hypermethylation was found in anaplastic carcinomas but not in other thyroid tumor histotypes. Therefore, we can hypothesize that HMGA1 overexpression plays a key role on HAND1 silencing in differentiated thyroid carcinomas and that promoter hypermethylation occurs in later stages of thyroid tumor progression. Finally, the restoration of the HAND1 gene expression reduces the clonogenic ability of two human thyroid carcinoma-derived cell lines, suggesting that HAND1 downregulation may have a role in the process of thyroid carcinogenesis.

Coexistence of chronic lymphocytic thyroiditis and papillary thyroid carcinoma. Impact on presentation, management, and outcome

BackgroundThe association between chronic lymphocytic thyroiditis (CLT) and papillary thyroid carcinoma (PTC) has been investigated for several years from different perspectives. In spite of that, there were only few attempts to design a common frame of references to understand the complex mutual interactions between the various pathways of inflammatory response and of thyroid tumor induction and progression. This study compares two independent groups of patients aiming to determine the frequency and the prognostic significance of CLT in patients with PTC. Material and methodsFrom January 2005 to September 2013, we conducted a retrospective study on 160 patients with PTC who underwent thyroidectomy. CLT was diagnosed histopathologically. Age, sex, tumor features (dimensions, angioinvasion, capsular infiltration, mono/multifocality and lymph node metastases) pathologic findings and outcome were considered.Mean follow-up (metastasis, completeness-of-resection, serum thyroglobulin levels, tumor recurrence) period was 61 months (ranged from 18 to 132 months). A p < 0.05 was considered statistically significant. ResultsPatients were divided in 2 groups. In group A there were 90 patients affected by PTC alone, and in group B there were 70 patients affected with PTC associated with CLT.Our data showed that the presence of CLT correlate with a lower grade of PTC (p < 0.05). Considering the sex of the patients there were a statistically significant correlation (p < 0.02) and the presence of CLT associated with PTC was most representative in female patients. ConclusionsThe presence of CLT in patients with PTC correlated with a lower grade of PTC, but it does not affect the overall survival of papillary thyroid cancers.

Risk factors and the progression of thyroid malignancies

Thyroid cancer is the most common endocrine malignancy and the fifth most common malignant neoplasm of the female sex. During the past several decades, an increasing incidence of thyroid cancer has been reported. The mortality from thyroid cancer is comparatively low and remains almost stable showing a slight increase. It is currently unclear whether the observed increase in thyroid cancer is real or is due to overdiagnosis, as clinical and pathology findings may be sometimes doubtful in diagnosing the incidence and mortality of thyroid cancer. The cancer has different distribution depending on gender, race, age and environmental conditions. Despite considerable progress in the understanding of the biology and molecular pathways of carcinogenesis in the thyroid gland, less progress has been made in terms of defining a risk profile for thyroid cancer. The only risk factor which is systematically documented as carcinogenic for thyroid is exposure to ionizing radiation during childhood. Recently several studies are examining as risk factors diet and exercise, benign thyroid diseases as well as a genetic factors that influence the incidence and mortality of the disease. To the best of our knowledge there are only few epidemiological studies examining the effects of exposure to chemical agents on thyroid cancer's "behavior". The effect of risk factors for the onset and progression of malignant thyroid tumors constitutes a field that requires further study in order to provide answers on the pathogenesis of the disease, so as to take preventing measures and finally manage to decrease thyroid cancer incidence and mortality.

Klotho inhibits human follicular thyroid cancer cell growth and promotes apoptosis through regulation of the expression of stanniocalcin-1.

The new anti-aging gene Klotho has been identified as a multi-functional humoral factor which influences multiple biological processes, including tumor progression. Although ample evidence indicates that Klotho plays important roles in cervical, lung and breast cancer, the role and mechanism of Klotho in thyroid cancer are still unclear. The present study aimed to investigate the effects and mechanisms of Klotho in human thyroid cancer cell lines FTC133 and FTC238. Klotho overexpression markedly reduced thyroid cancer FTC133 and FTC238 cell proliferation and enhanced apoptosis, whereas, Klotho silencing in the FTC133 and FTC238 cells increased cell growth. Moreover, soluble human KL1 (sKL) and Klotho overexpression had a similar effect on FTC133 and FTC238 cell growth. A high level of Klotho was also found to be associated with a low level of stanniocalcin 1 (STC1) in both the FTC133 and FTC238 cell lines. STC1 silencing significantly inhibited thyroid cancer cell proliferation, whereas recombinant human STC1 (hSTC1) markedly enhanced cell proliferation. In addition, our study demonstrated that hSTC1 treatment attenuated Klotho-induced inhibition of cell proliferation and promotion of apoptosis. Our data revealed the existence of a moderating effect between Klotho and STC1, where Klotho may inhibit thyroid tumor progression by inhibiting the tumor marker level of STC1.

Autotaxin is an inflammatory mediator and therapeutic target in thyroid cancer.

Autotaxin is a secreted enzyme that converts extracellular lysophosphatidylcholine to lysophosphatidate (LPA). In cancers, LPA increases tumour growth, metastasis and chemoresistance by activating six G-protein coupled receptors. We examined >200 human thyroid biopsies. Autotaxin expression in metastatic deposits and primary carcinomas was four- to tenfold higher than in benign neoplasms or normal thyroid tissue. Autotaxin immunohistochemical staining was also increased in benign neoplasms with leukocytic infiltrations. Malignant tumours were distinguished from benign tumours by high tumour autotaxin, LPA levels and inflammatory mediators including IL1β, IL6, IL8, GMCSF, TNFα, CCL2, CXCL10 and platelet-derived growth factor (PDGF)-AA. We determined the mechanistic explanation for these results and revealed a vicious regulatory cycle in which LPA increased the secretion of 16 inflammatory modulators in papillary thyroid cancer cultures. Conversely, treating cancer cells with ten inflammatory cytokines and chemokines or PDGF-AA and PDGF-BB increased autotaxin secretion. We confirmed that this autotaxin/inflammatory cycle occurs in two SCID mouse models of papillary thyroid cancer by blocking LPA signalling using the autotaxin inhibitor ONO-8430506. This decreased the levels of 16 inflammatory mediators in the tumours and was accompanied by a 50-60% decrease in tumour volume. This resulted from a decreased mitotic index for the cancer cells and decreased levels of vascular endothelial growth factor and angiogenesis in the tumours. Our results demonstrate that the autotaxin/inflammatory cycle is a focal point for driving malignant thyroid tumour progression and possibly treatment resistance. Inhibiting autotaxin activity provides an effective and novel strategy for decreasing the inflammatory phenotype in thyroid carcinomas, which should complement other treatment modalities.

Autophagy in Thyroid Cancer: Present Knowledge and Future Perspectives

Thyroid cancer is the most common endocrine malignancy. Despite having a good prognosis in the majority of cases, when the tumor is dedifferentiated it does no longer respond to conventional treatment with radioactive iodine, the prognosis worsens significantly. Treatment options for advanced, dedifferentiated disease are limited and do not cure the disease. Autophagy, a process of self-digestion in which damaged molecules or organelles are degraded and recycled, has emerged as an important player in the pathogenesis of different diseases, including cancer. The role of autophagy in thyroid cancer pathogenesis is not yet elucidated. However, the available data indicate that autophagy is involved in several steps of thyroid tumor initiation and progression as well as in therapy resistance and therefore could be exploited for therapeutic applications. The present review summarizes the most recent data on the role of autophagy in the pathogenesis of thyroid cancer and we will provide a perspective on how this process can be targeted for potential therapeutic approaches and could be further explored in the context of multimodality treatment in cancer and personalized medicine.

Changes in the expression pattern of apoptotic molecules (galectin-3, Bcl-2, Bax, survivin) during progression of thyroid malignancy and their clinical significance.

Papillary carcinoma of the thyroid (PTC) is generally a slow growing tumor with favorable prognosis, while anaplastic thyroid carcinoma (ATC) is highly aggressive malignancy. Genetic defects in apoptotic pathways may contribute to differences in their biological behavior. In this study, we analyzed immunohistochemically the expression of apoptosis-related molecules: galectin-3, Bcl-2, survivin (antiapoptotic), and Bax (pro-apoptotic), in archival tissue sections of PTC (n = 69) and ATC (n = 30) and correlated the results with clinicopathological parameters of these tumors. Galectin-3 and Bcl-2 showed a similar trend of down-regulation from high levels of both in PTC to low levels in ATC (p < 0.05). Bax was expressed at high levels in both type of thyroid carcinoma. Expression of survivin increased from PTC to ATC (p < 0.05), which may, at least in part, further facilitate the ability of malignant thyroid cell of ATC to escape programmed cell death despite high Bax expression. Only survivin, but not galectin-3, Bcl-2, or Bax, correlated significantly with lymph node metastasis presence and advanced stages of malignancy. In conclusion, this study documented down-regulation of galectin-3 and Bcl-2 (antiapoptotic molecules) and stepwise increase of survivin (inhibitor of apoptosis), during thyroid tumor progression from PTC to ATC. Correlation of high survivin expression with aggressive behavior implies its role in progression of thyroid tumor malignancy and suggests that survivin could be a useful tool in the prediction of aggressiveness of a subset of papillary carcinomas and a possible target for molecular therapy for ATC patients.

MiRNA expression in anaplastic thyroid carcinomas.

Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents an end stage of thyroid tumor progression. No effective treatment exists so far. In this study, we analyzed the miRNA expression profiles of 11 ATC by microarrays and their relationship with the mRNA expression profiles of the same 11 ATC samples. ATC show distinct miRNA expression profiles compared to other less aggressive thyroid tumor types. ATC show 18 commonly deregulated miRNA compared to normal thyroid tissue (17 downregulated and 1 upregulated miRNA). First, the analysis of a combined approach of the mRNA gene expression and of the bioinformatically predicted mRNA targets of the deregulated miRNA suggested a role for these regulations in the epithelial to mesenchymal transition (EMT) process in ATC. Second, the direct interaction between one of the upregulated mRNA target, the LOX gene which is an EMT key player, and a downregulated miRNA, the miR-29a, was experimentally validated by a luciferase assay in HEK cell. Third, we confirmed that the ATC tissue is composed of about 50% of tumor associated macrophages (TAM) and suggested, by taking into account our data and published data, their most likely direct or paracrine intercommunication between them and the thyroid tumor cells, amplifying the tumor aggressiveness. Finally, we demonstrated by in situ hybridization a specific thyrocyte localization of 3 of the deregulated miRNA: let-7g, miR-29a and miR-30e and we pointed out the importance of identifying the cell type localization before drawing any conclusion on the physiopathological role of a given gene.

Helicase-like transcription factor: a new marker of well-differentiated thyroid cancers

BackgroundThe preoperative characterization of thyroid nodules is a challenge for the clinicians. Fine-needle aspiration (FNA) is the commonly used pre-operative technique for diagnosis of malignant thyroid tumor. However, many benign lesions, with indeterminate diagnosis following FNA, are referred to surgery. There is an urgent need to identify biomarkers that could be used with the FNA to distinguish benign thyroid nodules from malignant tumors. The purpose of the study is to examine the level of expression of the helicase-like transcription factor (HLTF) in relation to neoplastic progression of thyroid carcinomas.MethodsThe presence of HLTF was investigated using quantitative and semi-quantitative immunohistochemistry in a series of 149 thyroid lesion specimens. Our first clinical series was composed of 80 patients, including 20 patients presenting thyroid adenoma, 40 patients presenting thyroid papillary carcinoma, 12 patients presenting thyroid follicular carcinoma and 8 patients presenting anaplastic carcinoma. These specimens were assessed quantitatively using computer assisted microscopy. Our initial results were validated on a second clinical series composed of 40 benign thyroid lesions and 29 malignant thyroid lesions using a semi-quantitative approach. Finally, the HLTF protein expression was investigated by Western blotting in four thyroid cancer cell lines.ResultsThe decrease of HLTF staining was statistically significant during thyroid tumor progression in terms of both the percentage of mean optical density (MOD), which corresponds to the mean staining intensity (Kruskall-Wallis: p < 0.0005), and the labelling index (LI), which corresponds to the percentage of immunopositive cells (Kruskall-Wallis: p < 10−6). Adenomas presented very pronounced nuclear HLTF immunostaining, whereas papillary carcinomas exhibited HLTF only in the cytoplasm. The number of HLTF positive nuclei was clearly higher in the adenomas group (30%) than in the papillary carcinomas group (5%).The 115-kDa full size HLTF protein was immunodetected in four studied thyroid cancer cell lines. Moreover, three truncated HLTF forms (95-kDa, 80-kDa and 70-kDa) were also found in these tumor cells.ConclusionsThis study reveals an association between HLTF expression level and thyroid neoplastic progression. Nuclear HLTF immunostaining could be used with FNA in an attempt to better distinguish benign thyroid nodules from malignant tumors.