Tatiana Rogounovitch

Oncogenic role of miR-17-92 cluster in anaplastic thyroid cancer cells

Micro RNAs (miRNAs) are non‐coding small RNAs and constitute a novel class of negative gene regulators that are found in both plants and animals. Several miRNAs play crucial roles in cancer cell growth. To identify miRNAs specifically deregulated in anaplastic thyroid cancer (ATC) cells, we performed a comprehensive analysis of miRNA expressions in ARO cells and primary thyrocytes using miRNA microarrays. MiRNAs in a miR‐17‐92 cluster were overexpressed in ARO cells. We confirmed the overexpression of those miRNAs by Northern blot analysis in ARO and FRO cells. In 3 of 6 clinical ATC samples, miR‐17‐3p and miR‐17‐5p were robustly overexpressed in cancer lesions compared to adjacent normal tissue. To investigate the functional role of these miRNAs in ATC cells, ARO and FRO cells were transfected with miRNA inhibitors, antisense oligonucleotides containing locked nucleic acids. Suppression of miR‐17‐3p caused complete growth arrest, presumably due to caspase activation resulting in apoptosis. MiR‐17‐5p or miR‐19a inhibitor also induced strong growth reduction, but only miR‐17‐5p inhibitor led to cellular senescence. On the other hand, miR‐18a inhibitor only moderately attenuated the cell growth. Thus, we have clarified functional differences among the members of the cluster in ATC cells. In conclusion, these findings suggest that the miR‐17‐92 cluster plays an important role in certain types of ATCs and could be a novel target for ATC treatment. (Cancer Sci 2008; 99: 1147–1154)

The FOXE1 locus is a major genetic determinant for radiation-related thyroid carcinoma in Chernobyl

Papillary thyroid cancer (PTC) among individuals exposed to radioactive iodine in their childhood or adolescence is a major internationally recognized health consequence of the Chernobyl accident. To identify genetic determinants affecting individual susceptibility to radiation-related PTC, we conducted a genome-wide association study employing Belarusian patients with PTC aged 0-18 years at the time of accident and age-matched Belarusian control subjects. Two series of genome scans were performed using independent sample sets, and association with radiation-related PTC was evaluated. Meta-analysis by the Mantel-Haenszel method combining the two studies identified four SNPs at chromosome 9q22.33 showing significant associations with the disease (Mantel-Haenszel P: mhp = 1.7 x 10(-9) to 4.9 x 10(-9)). The association was further reinforced by a validation analysis using one of these SNP markers, rs965513, with a new set of samples (overall mhp = 4.8 x 10(-12), OR = 1.65, 95% CI: 1.43-1.91). Rs965513 is located 57-kb upstream to FOXE1, a thyroid-specific transcription factor with pivotal roles in thyroid morphogenesis and was recently reported as the strongest genetic risk marker of sporadic PTC in European populations. Of interest, no association was obtained between radiation-related PTC and rs944289 (mhp = 0.17) at 14p13.3 which showed the second strongest association with sporadic PTC in Europeans. These results show that the complex pathway underlying the pathogenesis may be partly shared by the two etiological forms of PTC, but their genetic components do not completely overlap each other, suggesting the presence of other unknown etiology-specific genetic determinants in radiation-related PTC.

Polymorphisms of DNA damage response genes in radiation-related and sporadic papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) etiologically occurs as a radiation-induced or sporadic malignancy. Genetic factors contributing to the susceptibility to either form remain unknown. In this retrospective case-control study, we evaluated possible associations between single-nucleotide polymorphisms (SNPs) in the candidate DNA damage response genes (ATM, XRCC1, TP53, XRCC3, MTF1) and risk of radiation-induced and sporadic PTC. A total of 255 PTC cases (123 Chernobyl radiation-induced and 132 sporadic, all in Caucasians) and 596 healthy controls (198 residents of Chernobyl areas and 398 subjects without history of radiation exposure, all Caucasians) were genotyped. The risk of PTC and SNPs interactions with radiation exposure were assessed by logistic regressions. The ATM G5557A and XRCC1 Arg399Gln polymorphisms, regardless of radiation exposure, associated with a decreased risk of PTC according to the multiplicative and dominant models of inheritance (odds ratio (OR) = 0.69, 95% confidence interval (CI) 0.45-0.86 and OR = 0.70, 95% CI 0.59-0.93 respectively). The ATM IVS22-77 T > C and TP53 Arg72Pro SNPs interacted with radiation (P = 0.04 and P = 0.01 respectively). ATM IVS22-77 associated with the increased risk of sporadic PTC (OR = 1.84, 95% CI 1.10-3.24) whereas TP53 Arg72Pro correlated with the higher risk of radiogenic PTC (OR = 1.80, 95% CI 1.06-2.36). In the analyses of ATM/TP53 (rs1801516/rs664677/rs609429/rs1042522) combinations, the GG/TC/CG/GC genotype strongly associated with radiation-induced PTC (OR = 2.10, 95% CI 1.17-3.78). The GG/CC/GG/GG genotype displayed a significantly increased risk for sporadic PTC (OR = 3.32, 95% CI 1.57-6.99). The results indicate that polymorphisms of DNA damage response genes may be potential risk modifiers of ionizing radiation-induced or sporadic PTCs.

Cyclin D1 overexpression in thyroid tumours from a radio-contaminated area and its correlation with Pin1 and aberrant β-catenin expression

Cyclin D1 is a target molecule transcriptionally activated by aberrant β‐catenin in Wnt signalling, while prolyl isomerase Pin1 promotes cyclin D1 overexpression directly or through accumulation of β‐catenin in cancer cells. This study aimed to elucidate whether Pin1 was involved in cyclin D1 overexpression and aberrant β‐catenin in thyroid tumourigenesis by examining 14 follicular adenomas (FAa) and 14 papillary thyroid carcinomas (PTCs). All PTCs displayed cyclin D1 overexpression and strong cytoplasmic β‐catenin and/or decreased membrane β‐catenin expression by immunohistochemistry. Overexpression of cyclin D1 mRNA was observed in 45.5% of FAs and 54.5% of PTCs by TaqMan real‐time PCR. Pin1 expression was observed in PTC by immunostaining and was confirmed by reverse transcriptase‐PCR. There was a strong correlation between cyclin D1 and Pin1/cytoplasmic/membrane β‐catenin expression (p < 0.001), and between Pin1 and cytoplasmic (p < 0.001)/membrane (p = 0.002) β‐catenin expression in thyroid tumours. Mutation of the β‐catenin gene could not be detected in PTC. Western blot analysis demonstrated high levels of cyclin D1 and β‐catenin as well as Pin1 expression in a human PTC cell line possessing wild‐type β‐catenin and APC genes. This study suggests that both cyclin D1 overexpression and aberrant β‐catenin expression are of significance in thyroid tumours. Pin1 expression appears to correlate closely with the level of cyclin D1 and aberrant β‐catenin expression in thyroid tumours such as FA and PTC. Pin1 may be an important factor in regulating cyclin D1 and β‐catenin expression during thyroid carcinogenesis. Copyright

The FOXE1 and NKX2-1 loci are associated with susceptibility to papillary thyroid carcinoma in the Japanese population

Background FOXE1 and NKX2-1 are two known genetic risk factors for the predisposition to sporadic papillary thyroid carcinoma (PTC) in Europeans, but their association in other ethnicities is still unknown. Objective We aim to examine the association of the two genes with Japanese sporadic PTC, which exhibits high BRAFV600E mutation rate. Methods 507 Japanese sporadic PTC cases and 2766 controls were genotyped for rs965513 (FOXE1) and rs944289 (NKX2-1). PTC cases were also examined for their BRAFV600E mutational status. Results The association of both rs965513 (p=1.27×10−4, OR=1.69, 95% CI 1.29 to 2.21) and rs944289 (p=0.0121, OR=1.21, 95% CI 1.04 to 1.39) with the risk of sporadic PTC was confirmed. Subgroup analysis based on the BRAF mutational status showed strong association of rs965513 with BRAFV600E-positive cases (p=2.26×10−4, OR=1.72, 95% CI 1.29 to 2.29), but not with BRAFV600E-negative cases (p=0.143, OR=1.52, 95% CI 0.87 to 2.65). However, there was no difference in the observed effect size between both subgroups. For rs944289, both subgroups showed marginal association (p=0.0585, OR=1.17, 95% CI 0.99 to 1.37 for BRAFV600E-positive cases; p=0.0492, OR=1.35, 95% CI 1.00 to 1.81 for BRAFV600E-negative cases). Conclusions Both FOXE1 and NKX2-1 were associated with the increased risk of sporadic Japanese PTC. No clear associations were observed for either SNP with BRAFV600E status.

Different structural components of conventional papillary thyroid carcinoma display mostly identical BRAF status

Activating BRAFT1799A mutation is closely associated with a papillary thyroid carcinoma (PTC) histotype. The transversion is frequently detected in the conventional type, Warthin‐like and tall cell variants, but is rare in the follicular variant of PTC. Conventional PTC is often presented with tumors of mixed architecture, which besides the papillary structures also contain areas with follicular and solid morphology in which the details of BRAF mutational status are unknown. We set out to differentially investigate the presence of mutated BRAF in the individual structural components microdissected from 44 formalin‐fixed, paraffin‐embedded PTC tissues from 40 patients. The mutation was detected in at least 1 structural component in 23 tumors (52%). Different structural components of the same tumor had identical BRAF status in 41/44 tumors (93%). In 3 tumors the BRAFT1799A mutation was found only in the papillary, but not in the follicular component. Mutational patterns identical to those in the primary tumors were found in 11/12 lymph node metastases (92%, including both BRAFT1799A‐positive and ‐negative cases). The high concordance of the BRAF mutational status in structurally distinct areas suggests a rather homogeneous distribution of neoplastic epithelial cells in a conventional PTC tumor in most cases. These results imply the reliability of preoperative molecular diagnosis of PTC regardless of the type of tumor component at the site of biopsy sampling and suggest that the majority of patients with BRAF mutation‐positive PTC may benefit from the targeted pharmacotherapy.

BRAFV600E mutation is highly prevalent in thyroid carcinomas in the young population in Fukushima: a different oncogenic profile from Chernobyl

After the accident at the Fukushima Daiichi Nuclear Power Plant, the thyroid ultrasound screening program for children aged 0–18 at the time of the accident was started from October 2011. The prevalence of thyroid carcinomas in that population has appeared to be very high (84 cases per 296,253). To clarify the pathogenesis, we investigated the presence of driver mutations in these tumours. 61 classic papillary thyroid carcinomas (PTCs), two follicular variant PTCs, four cribriform-morular variant PTCs and one poorly-differentiated thyroid carcinoma were analysed. We detected BRAFV600E in 43 cases (63.2%), RET/PTC1 in six (8.8%), RET/PTC3 in one (1.5%) and ETV6/NTRK3 in four (5.9%). Among classic and follicular variant PTCs, BRAFV600E was significantly associated with the smaller size. The genetic pattern was completely different from post-Chernobyl PTCs, suggesting non-radiogenic etiology of these cancers. This is the first study demonstrating the oncogene profile in the thyroid cancers discovered by large mass screening, which probably reflects genetic status of all sporadic and latent tumours in the young Japanese population. It is assumed that BRAFV600E may not confer growth advantage on paediatric PTCs, and many of these cases grow slowly, suggesting that additional factors may be important for tumour progression in paediatric PTCs.

Radiation Exposure Does Not Significantly Contribute to the Risk of Recurrence of Chernobyl Thyroid Cancer

CONTEXT Papillary thyroid carcinoma (PTC) in patients exposed to environmental radioiodine after the Chernobyl accident is thought to have a relatively aggressive clinical course. Long-term results of treatment are not well known, especially in comparison with sporadic PTC. OBJECTIVE The determination of risk factors for PTC recurrence in a controlled for baseline factors group of patients with radiation-related and sporadic PTC. DESIGN Retrospective cohort study involving patients treated for PTC and followed-up in 1991-2008. Risk factors were assessed by stratified analysis using the proportional hazard model. SETTING Referral center-based. PATIENTS A total of 497 patients were enrolled. Patients exposed to radioiodine were 172 individuals with reconstructed individual radiation thyroid doses ranging 51-3170 mGy. Patients with sporadic PTC included 325 individuals matched to exposed patients for sex, age ± 5 yr and time to treatment ± 2 yr. MAIN OUTCOME MEASURE Cancer recurrence. RESULTS Nodal disease increased the recurrence rate (HR = 5.21; 95% CI = 1.63-16.7) while the presence of tumor capsule (HR = 0.17; 95% CI = 0.06-0.45) and, particularly, treatment according to the Revised American Thyroid Association Management Guidelines for Patients with Thyroid Nodules and Differentiated Thyroid Cancer significantly reduced it (HR = 0.16; 95% CI = 0.06-0.42). None of the tested variables interacted with radiation factor. CONCLUSIONS PTC developing after internal exposure to radioiodine does not display specific risk factors for recurrence different from those in sporadic PTC. Common treatment approaches for patients with PTC should be recommended regardless of a history of radiation exposure.

Inhibition of ABL Tyrosine Kinase Potentiates Radiation-Induced Terminal Growth Arrest in Anaplastic Thyroid Cancer Cells

Abstract Podtcheko, A., Ohtsuru, A., Namba, H., Saenko, V., Starenki, D., Palona, I., Sedliarou, I., Rogounovitch, T. and Yamashita, S. Inhibition of ABL Tyrosine Kinase Potentiates Radiation-Induced Terminal Growth Arrest in Anaplastic Thyroid Cancer Cells. Radiat. Res. 165, 35–42 (2006). Gleevec®, a selective tyrosine kinase inhibitor, retarded the growth of anaplastic thyroid cancer cell lines in vitro and in vivo through selective inhibition of ABL tyrosine kinase activity. In the present study, we investigated the ability of Gleevec® to modulate the in vitro and in vivo radiation response of anaplastic thyroid cancer cells. Cell growth assays, colony formation assays and xenograft models were used to quantify the radiosensitizing effect of Gleevec® in cells of the anaplastic thyroid cancer cell lines ARO and FRO. FACS, Western blotting and histochemical techniques were employed to study the mechanisms of radiation response after exposure to Gleevec®. Gleevec® (7.0 μM) increased the anti-proliferative effect of radiation on the growth ARO and FRO cells in vitro. Clonogenic analysis demonstrated that Gleevec® reduced cell survival after irradiation. Gleevec® combined with radiation produced an increase in tumor growth inhibition compared to treatment with either modality alone in mice bearing anaplastic thyroid cancer xenografts. The drug suppressed radiation-induced ABL activation and promoted CDKN1A (p21cip1) accumulation in irradiated anaplastic thyroid cancer cells. Gleevec® had an additional effect on radiation-induced apoptosis in cells of both cell lines and potentiated the induction of terminal growth arrest accompanied by the expression of senescence-associated β-galactosidase. The antitumor effect of Gleevec® is potentiated in adjunctive therapy with radiation not only due to inhibition of proliferative cell growth with transient cell cycle arrest and apoptosis, but also due to the terminal growth arrest associated with senescence, suggesting that tumor cell senescence is a mechanism for tumor targeting therapy in combination with ionizing radiation.

Major Factors Affecting Incidence of Childhood Thyroid Cancer in Belarus after the Chernobyl Accident: Do Nitrates in Drinking Water Play a Role?

One of the major health consequences of the Chernobyl Nuclear Power Plant accident in 1986 was a dramatic increase in incidence of thyroid cancer among those who were aged less than 18 years at the time of the accident. This increase has been directly linked in several analytic epidemiological studies to iodine-131 (131I) thyroid doses received from the accident. However, there remains limited understanding of factors that modify the 131I-related risk. Focusing on post-Chernobyl pediatric thyroid cancer in Belarus, we reviewed evidence of the effects of radiation, thyroid screening, and iodine deficiency on regional differences in incidence rates of thyroid cancer. We also reviewed current evidence on content of nitrate in groundwater and thyroid cancer risk drawing attention to high levels of nitrates in open well water in several contaminated regions of Belarus, i.e. Gomel and Brest, related to the usage of nitrogen fertilizers. In this hypothesis generating study, based on ecological data and biological plausibility, we suggest that nitrate pollution may modify the radiation-related risk of thyroid cancer contributing to regional differences in rates of pediatric thyroid cancer in Belarus. Analytic epidemiological studies designed to evaluate joint effect of nitrate content in groundwater and radiation present a promising avenue of research and may provide useful insights into etiology of thyroid cancer.