Tomomi Kurashige

Postnatal Expression of BRAFV600E Does Not Induce Thyroid Cancer in Mouse Models of Thyroid Papillary Carcinoma

The mutant BRAF (BRAF(V600E)) is the most common genetic alteration in papillary thyroid carcinomas (PTCs). The oncogenicity of this mutation has been shown by some genetically engineered mouse models. However, in these mice, BRAF(V600E) is expressed in all the thyroid cells from the fetal periods, and suppresses thyroid function, thereby leading to TSH elevation, which by itself promotes thyroid tumorigenesis. To overcome these problems, we exploited 2 different approaches, both of which allowed temporally and spatially restricted expression of BRAF(V600E) in the thyroid glands. First, we generated conditional transgenic mice harboring the loxP-neo(R)-loxP-BRAF(V600E)-internal ribosome entry site-green fluorescent protein sequence [Tg(LNL-BRAF(V600E))]. The double transgenic mice (LNL-BRAF(V600E);TPO-Cre) were derived from a high expressor line of Tg(LNL-BRAF(V600E)) mice and TPO-Cre mice; the latter expresses Cre DNA recombinase under the control of thyroid-specific thyroid peroxidase (TPO) promoter and developed PTC-like lesions in early life under normal serum TSH levels due to mosaic recombination. In contrast, injection of adenovirus expressing Cre under the control of another thyroid-specific thyroglobulin (Tg) promoter (Ad-TgP-Cre) into the thyroids of LNL-BRAF(V600E) mice did not induce tumor formation despite detection of BRAF(V600E) and pERK in a small fraction of thyroid cells. Second, postnatal expression of BRAF(V600E) in a small number of thyroid cells was also achieved by injecting the lentivirus expressing loxP-green fluorescent protein-loxP-BRAF(V600E) into the thyroids of TPO-Cre mice; however, no tumor development was again observed. These results suggest that BRAF(V600E) does not appear to induce PTC-like lesions when expressed in a fraction of thyroid cells postnatally under normal TSH concentrations.

Significance of p53-binding protein 1 nuclear foci in uterine cervical lesions: endogenous DNA double strand breaks and genomic instability during carcinogenesis

Matsuda K, Miura S, Kurashige T, Suzuki K, Kondo H, Ihara M, Nakajima H, Masuzaki H & Nakashima M
(2011) Histopathology59, 441–451

Thyrotropin Signaling Confers More Aggressive Features with Higher Genomic Instability on BRAFV600E-Induced Thyroid Tumors in a Mouse Model

BACKGROUND The BRAF(V600E) mutation is the most common genetic alteration in papillary thyroid carcinomas (PTCs). Transgenic mice overexpressing BRAF(V600E) in their thyroids under control of the thyroglobulin promoter (Tg-BRAF2 mice) developed invasive PTCs with high penetrance. However, these mice showed elevated thyrotropin (TSH) levels, which also stimulate the proliferation of thyrocytes and tumorigenesis. The purpose of the present study was to investigate how TSH signaling cooperates with BRAF(V600E) in the process of thyroid carcinogenesis. METHODS We crossed Tg-BRAF2 mice with TSH receptor knockout (TshR(-/-)) mice. Four genetically distinct mice groups-Braf(wt)/TshR(+/-) (group 1), Braf(wt)/TshR(-/-) (group 2), Tg-BRAF2/TshR(+/-) (group 3), and Tg-BRAF2/TshR(-/-) (group 4)--were sacrificed at 12 and 24 weeks of age. We performed histopathological analysis. Genomic instability was evaluated by immunofluorescence for p53-binding protein 1 (53BP1) and γH2AX. Invasiveness and genomic instability were also evaluated using thyroid PCCL3 cells expressing BRAF(V600E). RESULTS Groups 3 and 4 developed distinct neoplasias comparable to human PTCs. Group 3 developed typically larger, more aggressive, invasive tumors compared to group 4. The frequency of 53BP1 and γH2AX foci-indicators of genomic instability--in group 3 was higher than that in group 4. TSH also enhanced invasiveness and genomic instability in PCCL3 cells with BRAF(V600E) expression. CONCLUSIONS These data demonstrate that the TSH signaling confers more aggressive features in BRAF(V600E)-induced thyroid tumors in mice. This might be due, in part, to accelerated genomic instability.

Radiation-Associated Small Cell Neuroendocrine Carcinoma of the Thyroid: A Case Report with Molecular Analyses

BACKGROUND Neuroendocrine tumor (NET) of the thyroid other than medullary carcinoma is extremely rare. We describe here a case of calcitonin-negative small cell neuroendocrine carcinoma (SCNEC), which occurred in a thyroid gland that had previously been irradiated at high dose (60 Gy) for pharyngeal cancer, with molecular analyses for follicular cell origin. PATIENT FINDINGS The tumor cells were small with fine chromatin, inconspicuous nucleoli, and inapparent cytoplasm, and showed neuroendocrine architectures such as palisading, rosettes, and trabeculae. Mitotic figures were numerous exceeding 10 mitoses per 10 high-power fields. The tumor cells invaded into several vessels and metastasized to regional lymph nodes. Immunohistochemically, the tumor cells were strongly positive for neuroendocrine markers and thyroglobulin (Tg), a marker of thyroid follicular cells but negative for calcitonin and carcinoembryonic antigen (CEA). Expression of Tg and thyrotropin receptor (TSHR) were confirmed by quantitative real-time polymerase chain reaction (RT-PCR). Ki-67 labeling index was more than 70% in the tumor cells. Taken together, the tumor was diagnosed as SCNEC of the thyroid. Genetic analyses also revealed microsatellite abnormalities of the phosphatase and tensin homolog (PTEN) gene, suggesting that functional loss of PTEN contributes to carcinogenesis. CONCLUSIONS This is the first report describing a SCNEC of the thyroid with molecular analyses that provide evidence for a follicular epithelial origin.

Aldehyde dehydrogenase activity plays no functional role in stem cell-like properties in anaplastic thyroid cancer cell lines

Recent studies have revealed that aldehyde dehydrogenase (ALDH) is a candidate marker for thyroid cancer stem cells, although its activity is flexible. The goal of this study is to clarify the functional significance of ALDH enzymatic activity on thyroid cancer stem cells properties in anaplastic thyroid cancer cell lines. In vitro sphere formation assay was used to judge the stemness of 4 anaplastic thyroid cancer cell lines (FRO, ACT1, 8505C, and KTC3). Two well-known ALDH inhibitors, N,N-diethylaminobenzaldehyde (DEAB) and disulfiram (DS), were first used. DEAB (50 μM) almost completely suppressed ALDH activity without affecting cell proliferation or spherogenicity. Lack of effect of ALDH suppression on spherogenicity was confirmed using shRNA for ALDH1A3, an ALDH isozyme predominantly expressed in anaplastic thyroid cancer cell lines. In contrast, an ALDH2 inhibitor DS (1 μM) inhibited spherogenicity but did not inhibit ALDH1A3 activity. Based on the recent article from another group reporting the importance of sonic hedgehog (Shh) signaling in ALDH activity and spherogenicity in thyroid cancer, the effects of the Shh inhibitor cyclopamine were also studied. Like DS, cyclopamine (1 μM) decreased spherogenicity but not ALDH activity. Finally, exogenous expression of ALDH1A3 in otherwise ALDH− TPC1 cells (a papillary thyroid cancer cell line) revealed no effect on spherogenicity. In conclusion, we here show no functional role for ALDH activity in thyroid thyroid cancer stem cells properties. That is, ALDH activity and spherogenicity are clearly dissociable. Further understanding of thyroid cancer stem cells biology in thyroid cancers remains necessary for the future development of thyroid thyroid cancer stem cells-targeted therapies.

Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NACs effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NACs biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.

Effect of age on the sensitivity of the rat thyroid gland to ionizing radiation

Exposure to ionizing radiation during childhood is a well-known risk factor for thyroid cancer. Our study evaluated the effect of age on the radiosensitivity of rat thyroid glands. Four-week-old (4W), 7 -week-old (7W), and 8-month-old (8M) male Wistar rats were exposed to 8 Gy of whole-body X-ray irradiation. Thyroids were removed 3–72 h after irradiation, and non-irradiated thyroids served as controls. Ki67-positivity and p53 binding protein 1 (53BP1) focus formation (a DNA damage response) were evaluated via immunohistochemistry. Amounts of proteins involved in DNA damage response (p53, p53 phosphorylated at serine 15, p21), apoptosis (cleaved caspase-3), and autophagy (LC3, p62) were determined via western blotting. mRNA levels of 84 key autophagy-related genes were quantified using polymerase chain reaction arrays. Ki67-positive cells in 4W (with high proliferative activity) and 7W thyroids significantly decreased in number post-irradiation. The number of 53BP1 foci and amount of p53 phosphorylated at serine 15 increased 3 h after irradiation, regardless of age. No increase in apoptosis or in the levels of p53, p21 or cleaved caspase-3 was detected for any ages. Levels of LC3-II and p62 increased in irradiated 4W but not 8M thyroids, whereas expression of several autophagy-related genes was higher in 4W than 8M irradiated thyroids. Irradiation increased the expression of genes encoding pro-apoptotic proteins in both 4W and 8M thyroids. In summary, no apoptosis or p53 accumulation was noted, despite the expression of some pro-apoptotic genes in immature and adult thyroids. Irradiation induced autophagy in immature, but not in adult, rat thyroids.

Studies on Expression of Aldehyde Dehydrogenase in Normal and Cancerous Tissues of Thyroids

Recently published articles have reported the controversial data regarding expression of aldehyde dehydrogenase isozyme 1A1 (ALDH1A1), a potential candidate marker for normal and cancer stem cells (CSCs), in thyroid tissues. These data prompted us to re-evaluate expression of ALDH1A1 in normal and cancerous thyroid tissues by 2 different means. The first method was immunohistochemistry with 2 different anti-ALDH1A1 antibodies from distinct companies. Following validating the integrity of these 2 antibodies by Western blotting with ALDH-expressing and nonexpressing cancer cell lines and immunohistochemistry with breast and colon tissues, we report here significant and comparable expression of ALDH1A1 in both normal and cancerous thyroid tissues with both antibodies. Next, relative expression levels of ALDH isozymes were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), revealing that ALDH1A1 was the most highly expressed isozyme followed by ALDH9A1 and relative expression patterns of isozymes were very similar in normal and cancerous tissues. All these data demonstrate that thyroid cells of normal and cancer origins do express ALDH1A1 and to a lesser extent 9A1. Further study will be necessary to study functional significance of ALDH1A1 in the function and behaviors of thyroid normal and cancer stem cells.

The Nagasaki Atomic Bomb Survivors' Tumor Tissue Bank

1738 www.thelancet.com Vol 386 October 31, 2015 3 Moulder JE, Cohen EP. Renal dysfunction after total body irradiation: dose-eff ect relationship: in regard to Kal and van Kempen-Harteveld. Int J Radiat Oncol Biol Phys 2007; 67: 319. 4 Sera N, Hida A, Imaizumi M, Nakashima E, Akahoshi M. The association between chronic kidney disease and cardiovascular disease risk factors in atomic bomb survivors. Radiat Res 2013; 179: 46–52. 5 Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med 2004; 351: 1296–305. Tumor Tissue Bank for future analysis. Samples from 485 atomic bomb survivors have been collected so far, and the high quality and quantity of DNA and RNA extracted from the samples enable genomic analysis. We have previously reported a higher incidence of multiple primary cancers in atomic bomb survivors, particularly in those who were youngest at the time of and closest to the explosions, and others have reported a higher risk of myelodysplastic syndrome and multiple cancers in atomic bomb survivors. The Nagasaki Atomic Bomb Survivors’ Tumor Tissue Bank will help radiation research, promote worldwide collaboration, and enhance our understanding of the late-onset eff ects of radiation on carcinogenesis at the molecular level. Use of the tissue bank could provide information for the prediction, prevention, and treatment of cancer in both atomic bomb survivors and the individuals affected by the Fukushima nuclear power plant incident.

Mouse models of sporadic thyroid cancer derived from BRAFV600E alone or in combination with PTEN haploinsufficiency under physiologic TSH levels

The BRAFV600E mutation is the most prevalent driver mutation of sporadic papillary thyroid cancers (PTC). It was previously shown that prenatal or postnatal expression of BRAFV600E under elevated TSH levels induced thyroid cancers in several genetically engineered mouse models. In contrast, we found that postnatal expression of BRAFV600E under physiologic TSH levels failed to develop thyroid cancers in conditional transgenic Tg(LNL-BrafV600E) mice injected in the thyroid with adenovirus expressing Cre under control of the thyroglobulin promoter (Ad-TgP-Cre). In this study, we first demonstrated that BrafCA/+ mice carrying a Cre-activated allele of BrafV600E exhibited higher transformation efficiency than Tg(LNL-BrafV600E) mice when crossed with TPO-Cre mice. As a result, most BrafCA/+ mice injected with Ad-TgP-Cre developed thyroid cancers in 1 year. Histologic examination showed follicular or cribriform-like structures with positive TG and PAX staining and no colloid formation. Some tumors also had papillary structure component with lower TG expression. Concomitant PTEN haploinsufficiency in injected BrafCA/+;Ptenf/+ mice induced tumors predominantly exhibiting papillary structures and occasionally undifferentiated solid patterns with normal to low PAX expression and low to absent TG expression. Typical nuclear features of human PTC and extrathyroidal invasion were observed primarily in the latter mice. The percentages of pERK-, Ki67- and TUNEL-positive cells were all higher in the latter. In conclusion, we established novel thyroid cancer mouse models in which postnatal expression of BRAFV600E alone under physiologic TSH levels induces PTC. Simultaneous PTEN haploinsufficiency tends to promote tumor growth and de-differentiation.