Thyroid Tumor Cells Research Articles

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.

ENHANCED BETA-CATENIN EXPRESSION IS ASSOCIATED WITH RECURRENCE OF PAPILLARY THYROID CARCINOMA

A direct role of β-catenin 1 (β-cat) in the proliferation of human thyroid tumor cells has been identified. This study aimed to determine if there is an association between β-cat gene expression and the staging, recurrence, metastasis, and disease-free survival of papillary thyroid cancer. A retrospective cohort study was conducted using data from available information in the medical records and paraffin blocks of 81 of 400 patients referred to the endocrine clinic over a 10-year period. Real-time polymerase chain reaction was used to evaluate β-cat gene expression. Disease-free survival was assessed using the Kaplan-Meier method. The 10-year survival rate in these patients was 98.25%, and disease-free survival was 48.1%. Cumulative dose of radioactive iodine that patients received was significantly and positively correlated with β-cat gene expression ( r = -0.2; P = .03). Also, in patients with recurrence, β-cat gene expression was higher and statistically significant (5-fold increase; P = .002). Patients in more advanced stage and those with recurrence/distant metastasis had higher β-cat gene expression. We found that the patients had a better survival (lower recurrence) if they had a lower β-cat gene expression (SD, 0.142 to 0.052) (Mantel-Cox test, P = .002). We conclude that β-cat gene expression is positively correlated with recurrence, distant metastasis, and tumor-node-metastasis stage. β-cat = β-catenin 1; CI = confidence interval; PTC = papillary thyroid carcinoma; ROC = receiver operating characteristic.

Progress in immune evasion mechanism of papillary thyroid cancer

Thyroid cancer is the most common endocrine malignant tumor of human body. Thyroid cancer has close relationship with tumor cell surrounding immuno microenvironment in the process of formation, development, invade and metastasis. Their combined effect promotes body immune tolerance thus helps avoid from immune surveillance. Papillary thyroid cancer is the most common thyroid tumor on clinical. This article focuses on the interaction of papillary thyroid cancer and tumor cell surrounding immuno microenvironment, and the mechanism of their combined effect promotes immune evasion of tumor cells, helps to find a new approach for the diagnose and therapy of papillary thyroid cancer. Key words: Papillary thyroid cancer; Tumor cell surrounding immunomicroenvironment; Immune evasion

Upregulation of Tyrosine Kinase FYN in Human Thyroid Carcinoma: Role in Modulating Tumor Cell Proliferation, Invasion, and Migration.

Tyrosine kinase FYN-a member of the SRC family of kinases-is associated with mediating mitogenic signals and regulating cell cycle entry, growth, and proliferation. It was hypothesized that FYN is upregulated in thyroid carcinoma, which plays a critical role in tumorigenesis. FYN expression level in thyroid carcinoma tissue and tumor cell lines was determined. Also, the effects of FYN on thyroid cancer (TC) growth, signaling, cell cycle, and apoptosis were evaluated in vitro. FYN was knocked down in thyroid cancer cells (TPC-1) by siRNA to investigate the effects on cell proliferation, invasion, and migration. First, FYN was upregulated both in thyroid carcinoma tissue and in tumor cell lines. Loss of FYN by siRNA weakened proliferation, invasion, and migration of PTC-1 cells. Furthermore, it was demonstrated that knockdown of FYN can inhibit the G0/G1 phase of cell cycle. It was also observed that reduced expression of FYN could increase the level of NF-κB/P65 and IκBα. This study was the first to demonstrate critical positive regulation of thyroid tumorigenesis by FYN, which could be a potential target gene for thyroid carcinoma treatment. In addition, findings from this study highlighted the potential role of FYN inhibitors in TC therapy.

Vemurafenib-resistance via de novo RBM genes mutations and chromosome 5 aberrations is overcome by combined therapy with palbociclib in thyroid carcinoma with BRAFV600E.

PurposePapillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAFV600E represents the PTC hallmark and is targeted with selective inhibitors (e.g. vemurafenib). Although there have been promising results in clinical trials using these inhibitors, most patients develop resistance and progress. Tumor clonal diversity is proposed as one mechanism underlying drug resistance. Here we have investigated mechanisms of primary and secondary resistance to vemurafenib in BRAFWT/V600E–positive PTC patient-derived cells with P16-/- (CDKN2A-/-).Experimental DesignFollowing treatment with vemurafenib, we expanded a sub-population of cells with primary resistance and characterized them genetically and cytogenetically. We have used exome sequencing, metaphase chromosome analysis, FISH and oligonucleotide SNP-microarray assays to assess clonal evolution of vemurafenib-resistant cells. Furthermore, we have validated our findings by networks and pathways analyses using PTC clinical samples.ResultsVemurafenib-resistant cells grow similarly to naïve cells but are refractory to apoptosis upon treatment with vemurafenib, and accumulate in G2-M phase. We find that vemurafenib-resistant cells show amplification of chromosome 5 and de novo mutations in the RBM (RNA-binding motifs) genes family (i.e. RBMX, RBM10). RBMX knockdown in naïve-cells contributes to tetraploidization, including expansion of clones with chromosome 5 aberrations (e.g. isochromosome 5p). RBMX elicits gene regulatory networks with chromosome 5q cancer-associated genes and pathways for G2-M and DNA damage-response checkpoint regulation in BRAFWT/V600E-PTC. Importantly, combined therapy with vemurafenib plus palbociclib (inhibitor of CDK4/6, mimicking P16 functions) synergistically induces stronger apoptosis than single agents in resistant-cells and in anaplastic thyroid tumor cells harboring the heterozygous BRAFWT/V600E mutation.ConclusionsCritically, our findings suggest for the first time that targeting BRAFWT/V600E and CDK4/6 represents a novel therapeutic strategy to treat vemurafenib-resistant or vemurafenib-naïve radioiodine-refractory BRAFWT/V600E-PTC. This combined therapy could prevent selection and expansion of aggressive PTC cell sub-clones with intrinsic resistance, targeting tumor cells either with primary or secondary resistance to BRAFV600E inhibitor.

Targeting COPZ1 non-oncogene addiction counteracts the viability of thyroid tumor cells

Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer.

Bioeffect of different parameters on four tumor cell lines by ultrasound-mediated microbubble destruction

Objective To explore the bioeffect of different parameters on 4 cell lines by ultrasound-mediated microbubble destruction. Methods The orthogonal experimental design was used to investigate the effect of three factors on the bioeffects of four cell lines under three levels. Three factors included microbubble concentration, sound intensity, irradiation time. Human breast tumor (MCF-7) cells, ovarian tumor (A2780) cells, liver tumor (Bel7402) cells and thyroid tumor (ARO) cells were exposed to ultrasound in the presence of SonoVue. The cell survival rate was determined by MTT methods and the cell luminosity factor was detected by flow cytometry. Results The optimum parameters for Bel7402 and ARO cell were the same(A2B3C2), and they were different from those from MCF-7(A3B1C1) and A2780(A1B3C3) cell. The cell survival rates for 4 cell lines were above 75%, and the cell luminosity factors were different among 4 cell lines. Conclusions The optimum parameters by ultrasound-mediated microbubble destruction for different cell lines are different, and under the optimum parameters the bioeffects of different cell lines are different. Key words: Ultrasonic therapy; Microbubbles; Bioeffect; Cell membrane permeability

Overexpression miR-211-5p hinders the proliferation, migration, and invasion of thyroid tumor cells by downregulating SOX11.

This study was aimed to investigate the relationship between miR-211-5p and SOX11, and the effects of their interaction on the proliferation, viability, and invasion of human thyroid cancer (TC) cells. We used quantitative real-time PCR (qRT-PCR) to determine the expression of miR-211-5p and SOX11mRNA in the thyroid tumorous and the adjacent tissues. The target relationship between miR-211-5p and SOX11 was confirmed using dual luciferase reporter gene assay. Flow cytometry, colony formation assay, Transwell assay, and MTT assay were performed to determine the cell-cycle progression, cell apoptosis, proliferation and invasion, respectively. In addition, the tumor formation assay in nude mice was done to assess the effect of miR-211-5p on TC development in vivo. MiR-211-5p was underexpressed, whereas SOX11 was overexpressed in TC. The overexpression of miR-211-5p inhibited the expression of SOX11. The cell cycle was arrested and the proliferation as well as invasiveness was suppressed by exogenous miR-211-5p in TC cell line. The antitumor role of miR-211-5p was proved by the animal experiment. MiR-211-5p affected the viability, proliferation and invasion of TC by negatively regulating SOX11 expression.

Erk dimers inhibition is a therapeutic target in thyroid cancer

Thyroid carcinoma is the most common endocrine malignancy, and its incidence is rapidly rising in the world. Its initiation and progression involves multiple genetic and epigenetic alterations whereby BRAF and RAS mutations lead to the activation of the ERK signaling pathway. Recently, significant advances have been accomplished by developing pharmacological agents directed against the kinases of the RAS-ERK pathway. However, most of the molecules tested have undesired side effects and promote drug resistance. Consequently, it is imperative to find alternative RAS-ERK pathway inhibitors. It has been shown that by inhibiting ERKs dimerization it is possible to suppress tumor progression. DEL22379, a small molecule inhibitor for ERK dimerization, has been identified to impede the growth of melanoma tumor cells driven by RAS-ERK pathway oncogenes, without affecting ERK phosphorylation (Cancer Cell 28:17082 2015). The aim of this work is to study the role played by ERK dimerization and its inhibition using DEL22379 in thyroid cancer progression. We have used an in vitro model of thyroid tumor cells harboring oncogenic drivers (RAS or BRAF) to complete viability, migration and invasion assays as well as an orthotopic mouse model with anaplastic cells as an in vivo model. We observed that in BRAF mutated cells, ERK dimer formation is sustained for longer compared to the RAS mutated or control cells, resulting in the altered activation of the effected signaling pathway. RAS mutated cells are resistant to DEL22379 in vitro, while BRAF mutated cells are not able to form ERK dimers upon inhibitor addition. Consequently, these cells lose their invasive and migratory potential as well as displaying low viability. Preliminary results suggest DEL22379 treatment inhibits tumor growth in orthotopic mice. These results describe a new molecule that could be effectively used as a therapy in thyroid cancers harboring BRAF or RAS mutated genes. We have observed it is able to partially revert the tumorigenic phenotype, which may result in an improved prognosis in thyroid cancer patients.

Stage-Specific Embryonic Antigen-1 (SSEA-1) Expression in Thyroid Tissues.

Stage-specific embryonic antigen-1 (SSEA-1), also known as CD15, is a member of a cluster of differentiation antigens that have been identified in various normal tissues and in different types of cancers including papillary and medullary thyroid carcinoma. SSEA-1 may be expressed in normal stem cells and cancer stem-like cells. To evaluate the potential diagnostic and prognostic utility of SSEA-1 in thyroid tumors, we analyzed the expression of SSEA-1 in normal and neoplastic thyroid tissues by immunohistochemistry (IHC) using a tissue microarray with 158 different tissue cores. To evaluate the potential utility of SSEA-1 as a surface marker, we also assessed the expression of SSEA-1 in thyroid cell lines by flow cytometric analysis. SSEA-1 immunoreactivity was identified in malignant thyroid follicular epithelial cancers but not in the benign thyroid tissues. Anaplastic thyroid (ATC) (80%) and conventional papillary thyroid carcinoma (PTC) (60.7%) showed significantly higher percentage of cases that were SSEA-1 immunoreactive than follicular variant of papillary thyroid carcinoma (FVPTC) (20.6%) and follicular carcinoma (FCA) (32.1%). Flow cytometric analysis of cultured thyroid cell lines showed that a small subpopulation of ATC and PTC thyroid tumor cells had SSEA-1 immunoreactivity which may represent thyroid cancer stem-like cells. The ATC cells expressed more SSEA-1 immunoreactive cells than the PTC cell lines. Our findings suggest that expression of SSEA-1 immunoreactivity in thyroid neoplasms was associated with more aggressive thyroid carcinomas. SSEA-1 is a marker that detects malignant thyroid neoplasms in formalin-fixed paraffin-embedded thyroid tissue sections and may be a useful marker for thyroid cancer stem-like cells.

439 - Targeting COPZ1 in thyroid tumour cells

439 - Targeting COPZ1 in thyroid tumour cells

Resistance of papillary thyroid cancer stem cells to chemotherapy.

Thyroid carcinoma is the most common endocrine neoplasm, with the highest mortality rate of all the endocrine cancers. Among the endocrine malignancies, ~80% are papillary thyroid carcinomas (PTCs). In the initiation and progression of this tumor, genetic alterations in the mitogen-associated protein kinase pathway, including RAS point mutations, RET/PTC oncogene rearrangements and BRAF point mutations, play an important role, particularly in deciding targeted therapy. In the present study, a small population of thyroid tumor cells, known as tumor spheres, were isolated and characterized from PTC surgical samples. These spheres can be expanded indefinitely in vitro and give rise to differentiated adherent cells when cultivated in differentiative conditions. The present study showed by reverse transcription-polymerase chain reaction and flow cytometric analysis that the undifferentiated PTC cells exhibited a characteristic antigen expression profile of adult progenitor/stem cells. The cells were more resistant to chemotherapeutics, including bortezomib, taxol, cisplatin, etoposide, doxorubicin and vincristine, than differentiated PTC cells and the majority possessed a quiescent status, as revealed by the various cell cycle characteristics and anti-apoptotic protein expression. Such advances in cancer thyroid stem cell biology may provide relevant information for future targeted therapies.

The sonic hedgehog signaling pathway stimulates anaplastic thyroid cancer cell motility and invasiveness by activating Akt and c-Met.

The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.

TNF-α increases the membrane expression of the chemokine receptor CCR6 in thyroid tumor cells, but not in normal thyrocytes: potential role in the metastatic spread of thyroid cancer.

The chemokine receptor CCR6, selectively bound by CCL20, is involved in the metastatic spread of cancer cells. Tumor necrosis factor-α (TNF-α) displays a complex pro-tumorigenic actions, but it is unknown whether this cytokine could modulate the expression of chemokine receptors in thyroid tumors. The membrane expression of CCR6 was assessed by flow cytometry and immunofluorescence, in primary cultures of normal human thyroid (NHT) cells and in thyroid cancer cell lines (TPC-1 and BCPAP), both in basal conditions and after stimulation with TNF-α. In basal conditions, CCR6+ cells were virtually absent in NHT cells (0.4 ± 0.4%), while they were detected in TPC-1 (23.6 ± 6.6%) and in BCPAP (12.9 ± 9.4%) tumor cells (ANOVA F: 10.534; p < 0.005). The incubation with TNF-α significantly increased the percentage of CCR6+ cells in TPC-1 (23.6 ± 6.6% vs. 33.1 ± 8.7; p < 0.033) and in BCPAP (12.9 ± 9.4% vs. 18.1 ± 11.5; p < 0.030), but not in NHT (0.4 ± 0.4% vs. 0.2 ± 0.3; NS) cells. The magnitude of the TNF-α effect was similar for TPC-1 and BCPAP (∼40% vs. baseline) cells. TPC-1 cells were characterized by a greater amount of CCR6 per cell as compared with BCPAP cells, both in basal conditions (148.3 ± 33.7 fluorescence intensity vs. 102.5 ± 22.1 p < 0.016) and after TNF-α stimulation (147.8 ± 46.3 fluorescence intensity vs. 95.3 ± 18.5; p < 0.025). Cell migration assays showed that TNF-α treatment significantly increased the rate of migrated cells in those cells in which it also increased the membrane expression of CCR6 (TPC-1 and BCPAP) as compared to basal condition (p < 0.05 for both TPC-1 and BCPAP cells). No effect was observed in NHT cells in which TNF-α stimulation had no effect in terms of CCR6 expression. We first report that TNF-α enhances the expression of CCR6 in thyroid tumor cells, thus providing evidence that TNF-α increases the metastatic potential of thyroid tumors.

Normal human thyroid cells, BCPAP, and TPC-1 thyroid tumor cell lines display different profile in both basal and TNF-α-induced CXCL8 secretion.

CXCL8 is secreted by both normal human thyrocytes (NHT) and thyroid cancer cell lines. CXCL8 displays several tumor-promoting effects and recent evidences indicate that its concentrations within the tumor microenvironment can impact the clinical course of the malignancy. Aim of this study was to compare the basal secretion of CXCL8 among NHT and thyroid cancer cell lines (TPC-1 and BCPAP), and to assess the specific cell response to TNF-α in terms of CXCL8 secretion. NHT primary cultures, TPC-1 and BCPAP cell lines were cultured with or without TNF-α (0, 0.1, 1, 10, and 100ng/ml). CXCL8 levels were measured in the cell supernatants after 24 h. In basal condition, significant differences in the mean levels of CXCL8 were observed among the three cell types: NHT (110.5±56.2pg/ml), TPC1 (467.4±43.2pg/ml), and BCPAP (1731.8±493.3pg/ml), (F=35.06; p<0.0001). TNF-α significantly and in a dose-response manner induced CXCL8 secretion in NHT (F=25.53; p<0.00001), TPC-1 (F=13.38; p<0.0001), and BCPAP (F=9.88; p<0.001) cells. The magnitude of the TNF-α effect (fold-increase vs. basal level of CXCL8) differed significantly among the three cell types (F=10.47; p<0.0001). BCPAP were identified as the cells showing the highest basal secretion of CXCL8 and the less responsive to TNF-α. NHT, TPC-1, and BCPAP display significant differences in the secretion of both basal and TNF-α-induced CXCL8 secretion. These results indicate that the mechanisms regulating the secretion of CXCL8 differ in tumor cells harboring different genetic alterations suggesting that specific strategies aimed at inhibiting CXCL8 secretion will be required.

Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening.

The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3-1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies.

Cyclin D1 and CHEK2 polymorphic variants as low risk susceptibility alleles in DTC patients

Differentiated thyroid cancer (DTC) originates from thyroid follicular epithelial cells and belongs to a group of slowly progressing tumors with relatively good prognosis. However, optimal results in the treatment of these tumors via coordinated multimodal therapy are achieved mostly by early diagnosis. A serious problem in course of DTC are recurrences and metastasis. Conversion from well differentiated thyroid cancer to aggressive anaplastic carcinoma is possible, which underlines the importance of effective diagnosis and characterisation at the molecular level as well. A genetic background of DTC is not clear. There are known several mutations detected in the thyroid tumor cells, but genetic factors predisposing to promotion of carcinogenesis of the thyroid gland are still unexplained. The CHEK2 gene (checkpoint kinase 2) is located in position 22q12.1 and spans 54.6 kb. It encodes the human ortholog of the yeast checkpoint kinases Cds1 and Rad53. In response to the damage of genetic material and inhibition of the replication process, the protein is activated by phosphorylation. Mutations in the CHEK2 gene are evidenced to be a multicancer predisposition factor. The protein product of this gene is a cell cycle checkpoint regulator, tumor suppressor and is a member of subfamily of serine/threonine protein kinases. The CCND1 gene (11q13) encodes the cyclin D1, a protein which belongs to the highly conserved cyclin family, that serve as regulators of CDK kinases. Cyclin D1 is a key regulatory protein which plays an important role in the transition from the G1 to the S phase of cell cycle during cell division. The cancer relevance of the cyclin D1 gene was apparent upon its identification in 1991. The cyclin D1 locus was initially identified based on its involvement in a chromosomal rearrangement of benign parathyroid tumors. The CCND1 gene alterations which influence cell cycle progression are frequently observed in a variety of tumors and may contribute to tumorigenesis. The aim of the study was to examine two sequence variants in CHEK2 p.R145W (c.433C>T, rs137853007 and p. I157T c.470T>C, rs17879961)and CCND1 (c.723G>A (rs9344; p.Pro241= and c.669C>T, rs3862792; p.Phe223=) genes in a group of Polish patients with differentiated thyroid cancer and control individuals from Polish population. We examined a cohort of 647 Polish patients diagnosed with differentiated thyroid carcinoma and Polish population included 799 subjects (615 females and 184 males). As a genotyping technique we used pyrosequencing. In order to determine statistical significance of differences in genotypic and allelic frequency, observed among compared groups, we used GraphPad Prism 4 (chi-square distribution and Fisher exact test). We did not observe noteworthy variability in p.R145W (c.433C>T, rs137853007), but in analysis of p.I157T sequence variant (c.470T>C, rs17879961) we demonstrated statistically significant differences in allele frequencies (p=0.005, OR=2.014, C.I.=[1.348-3.010]). In a case of CCND1 gene we observed statistically higher frequency of allele A in rs9344 locus in DTC patients (p=0.02498, OR=1.191, C.I.=[1.022-1.387]). We observed also a statistically significant difference between genotypes in subjected group. Genotype AA occurs significantly to be more frequent in patients with DTC (p=0.02023, OR=1.452, C.I.=[1.059-1.989]) than in control population group. We did not notice any statistically important differences in frequencies of genotypes or alleles in rs3862792 locus in subjected groups. * Correspondence: szymon.h@amu.edu.pl The NanoBioMedical Center, Adam Mickiewicz University, Poznan, Poland Full list of author information is available at the end of the article Hryhorowicz et al. Hereditary Cancer in Clinical Practice 2015, 13(Suppl 1):A15 http://www.hccpjournal.com/content/13/S1/A15

EBP1 suppresses growth, migration, and invasion of thyroid cancer cells through upregulating RASAL expression

Ebp1, a protein identified by its interactions with the ErbB3 receptor, has been characterized as a negative regulator of cancers. RAS GTPase-activating protein (RasGAP), RASAL1, was recently identified as a major tumor suppressor in thyroid cancer. In this study, we examined EBP1 expression in papillary and follicular thyroid cancer cells. We found that compared with normal thyroid cells, TPC1, WRO, and FTC133 thyroid tumor cells exhibited lower EBP1 expression at messenger RNA (mRNA) and protein levels. We then investigated the effects of forced EBP1 expression on growth, migration, and invasiveness of thyroid tumor cells. By using MTT and Boyden chamber assays, we showed that EBP1 overexpression dramatically reduced growth rate, migration, and invasiveness of K1 and FTC133 thyroid tumor cells. Furthermore, we explored the molecular mechanism underlying the effects of EBP1 on the cells by disclosing the correlation of EBP1 and RASAL1 expression. RASAL expression was elevated in thyroid tumor cells overexpressing EBP1. Knockdown RASAL by transduction of RASAL1 shRNA lentiviral particles markedly reduced RASAL levels with restoration of EBP1, and RASAL1 knockdown abrogated the effects of forced EBP1 expression on cell growth, migration, and invasiveness of thyroid tumor cells. These findings suggest that Ebp1 suppressed thyroid cancer cell lines by upregulating RASRAL expression.

Mitochondrial dynamics protein Drp1 is overexpressed in oncocytic thyroid tumors and regulates cancer cell migration.

Oncocytic cell tumors are characterized by the accumulation of morphologically abnormal mitochondria in their cells, suggesting a role for abnormal mitochondrial biogenesis in oncocytic cell transformation. Little is known about the reason for the dysmorphology of accumulated mitochondria. The proteins regulating the morphology of mitochondria, the "mitochondria-shaping" proteins, can modulate their size and number; however, nothing is known hitherto about a possible involvement of mitochondrial dynamics in oncocytic cell transformation in tumors. Our aim was to assess the status of the mitochondria morphology and its role in oncocytic cell transformation. We therefore evaluated the expression pattern of the main mitochondrial fusion and fission proteins in a series of thyroid cell tumor samples, as well as in thyroid tumor cell lines, with and without oncocytic cell features. The expression of mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1 and Fis1) proteins were evaluated by immunohistochemistry (IHC) in a series of 88 human thyroid tumors. In vitro studies, for comparative purposes and to deepen the study, were performed using TPC1 - a papillary thyroid carcinoma derived cell line—and XTC.UC1, an oncocytic follicular thyroid carcinoma-derived cell line. Both IHC and in vitro protein analyses showed an overall increase in the levels of "mitochondrial-shaping" proteins in oncocytic thyroid tumors. Furthermore, overexpression of the pro-fission protein Drp1 was found to be associated with malignant oncocytic thyroid tumors. Interestingly, genetic and pharmacological blockage of Drp1 activity was able to influence thyroid cancer cells’ migration/invasion ability, a feature of tumor malignancy. In this study we show that unbalanced mitochondrial dynamics characterize the malignant features of thyroid oncocytic cell tumors, and participate in the acquisition of the migrating phenotype.

Expression of stanniocalcin 1 in thyroid side population cells and thyroid cancer cells.

Mouse thyroid side population (SP) cells consist of a minor population of mouse thyroid cells that may have multipotent thyroid stem cell characteristics. However the nature of thyroid SP cells remains elusive, particularly in relation to thyroid cancer. Stanniocalcin (STC) 1 and 2 are secreted glycoproteins known to regulate serum calcium and phosphate homeostasis. In recent years, the relationship of STC1/2 expression to cancer has been described in various tissues. Microarray analysis was carried out to determine genes up- and down-regulated in thyroid SP cells as compared with non-SP cells. Among genes up-regulated, stanniocalcin 1 (STC1) was chosen for study because of its expression in various thyroid cells by Western blotting and immunohistochemistry. Gene expression analysis revealed that genes known to be highly expressed in cancer cells and/or involved in cancer invasion/metastasis were markedly up-regulated in SP cells from both intact as well as partial thyroidectomized thyroids. Among these genes, expression of STC1 was found in five human thyroid carcinoma-derived cell lines as revealed by analysis of mRNA and protein, and its expression was inversely correlated with the differentiation status of the cells. Immunohistochemical analysis demonstrated higher expression of STC1 in the thyroid tumor cell line and thyroid tumor tissues from humans and mice. These results suggest that SP cells contain a population of cells that express genes also highly expressed in cancer cells including Stc1, which warrants further study on the role of SP cells and/or STC1 expression in thyroid cancer.