Dongfeng Niu

Epigenetic silencing of TTF-1/NKX2-1 through DNA hypermethylation and histone H3 modulation in thyroid carcinomas.

Thyroid transcription factor-1 (TTF-1), also known as NKX2-1, is a homeodomain containing transcriptional factor identified in thyroid, lung and central nervous system. In the thyroid, TTF-1 is essential for thyroid organogenesis and governs thyroid functions by regulating various thyroid-specific genes. We previously demonstrated that most differentiated thyroid neoplasms, including follicular adenomas/carcinomas and papillary carcinomas, express TTF-1 at both protein and mRNA levels. However, certain subtypes of thyroid cancers have shown low or negative expression of TTF-1. The aim of our study was to investigate the function of epigenetic modification in dysregulation of TTF-1 in thyroid carcinoma cells. We evaluated the expression of TTF-1 in primary thyroid tissues (normal thyroid, papillary carcinoma and undifferentiated carcinoma) and in thyroid carcinoma cell lines using immunohistochemistry and RT–PCR. Methylation-specific PCR targeting CpG islands of TTF-1 and chromatin immunoprecipitation (ChIP) for histone H3 lysine 9 (H3-lys9) were applied to clarify the correlation of the TTF-1 expression profile and epigenetic status. We also explored whether epigenetic modifiers, including 5-aza-deoxycytidine, could restore TTF-1 expression in thyroid carcinoma cells. In our current study, immunohistochemistry and RT–PCR showed positive expression of TTF-1 in normal thyroids and papillary carcinomas. Meanwhile, most of the undifferentiated carcinomas and the cell lines lost TTF-1 expression. No methylation in the CpG of TTF-1 promoter was detected in normal thyroids or papillary carcinomas. In contrast, DNA methylation was identified in 60% of the undifferentiated carcinomas (6/10) and 50% of the cell lines (4/8). ChIP assay demonstrated that acetylation of H3-lys9 was positively correlated with TTF-1 expression in thyroid carcinoma cells. Finally, DNA demethylating agents could restore TTF-1 gene expression in the thyroid carcinoma cell lines. Our data suggest that epigenetics is involved with inactivation of TTF-1 in thyroid carcinomas, and provide a possible means of using TTF-1 as a target for differentiation-inducing therapy through epigenetic modification.

Hypoxia-inducible adenosine A2B receptor modulates proliferation of colon carcinoma cells

Extracellular adenosine regulates a wide variety of physiological processes by interacting with 4 adenosine receptor subtypes: A1, A2A, A2B, and A3. However, little is known of their pathophysiological roles in human cancers. In this study, we examined the expression pattern of adenosine receptors in various colorectal tissues and human colon carcinoma cell lines and investigated the biologic functions regarding colon carcinogenesis. Using reverse transcriptase polymerase chain reaction and Western blotting, we found that adenosine receptor A2B (ADORA2B) was consistently up-regulated in colorectal carcinoma tissues and colon cancer cell lines compared with normal colorectal mucosa. In immunohistochemistry, we observed diffuse immunopositivity of ADORA2B in 67% of colorectal adenocarcinomas (39/58), 17% of tubular adenomas (5/30), and 0% of normal colon glands (0/62). During a hypoxic state, there was also a significant induction of ADORA2B expression in the messenger RNA level at 8 hours of incubation and in the protein level at 24 hours of incubation in colon carcinoma cell lines. To examine the function of ADORA2B, we applied an ADORA2B-selective antagonist (MRS1754) to the colon carcinoma cells, which significantly inhibited cell growth in a dose-dependent manner as demonstrated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay. In conclusions, ADORA2B was overexpressed in colorectal carcinomas grown under a hypoxic state, presumably promoting cancer cell growth. Our data suggest that this adenosine receptor is a potential therapeutic target for colorectal cancer.

Global histone modification of histone H3 in colorectal cancer and its precursor lesions

Chromatin remodeling through histone modification is an important mechanism of epigenetic gene dysregulation in human cancers. However, little is known about global alteration of histone status during tumorigenesis and cancer progression. Histone H3 status was examined in benign and malignant colorectal tumors by immunohistochemistry and Western blotting. For immunohistochemical evaluation, 4 anti-histone H3 antibodies, specific to dimethylation at lysine 4 (H3K4me2), acetylation at lysine 9 (H3K9ac), dimethylation at lysine 9 (H3K9me2), and trimethylation at lysine 27 (H3K27me3), were used. On immunohistochemistry, H3K4me2, H3K9ac, and H3K27me3 showed no significant changes between normal and colorectal tumors. On the other hand, the global level of H3K9me2 was distinctly higher in neoplastic cells (adenoma and adenocarcinoma) than in normal glandular cells. In addition, it was significantly higher in adenocarcinoma than in adenoma. Correspondingly, Western blotting confirmed that H3K9me2 expression was significantly higher in adenocarcinomas than in normal colorectal mucosa. No alteration of H3K9me2 was observed with tumor differentiation and with the histological subtypes of colorectal cancers. These results suggest that aberration of the global H3K9me2 level is an important epigenetic event in colorectal tumorigenesis and carcinogenesis involved with gene regulation in neoplastic cells through chromatin remodeling.

Differential Expression of Aquaporins and Its Diagnostic Utility in Thyroid Cancer

Background Aquaporin3 (AQP3) and Aquaporin4 (AQP4) play a major role in transcellular and transepithelial water movement as water channel membrane proteins. Little is known of their expression and significance in human thyroid tissues. Thus, we examined the expression of AQP3 and AQP4 in normal, hyperplastic and neoplastic thyroid tissues in conjunction with human thyroid cancer cell lines. Methods and Results Immunohistochemical analyses demonstrated AQP3 in the cytoplasmic membrane of normal C cells, but not in follicular cells. In contrast, AQP4 was not found in C cells but was identified in normal follicular cells. AQP4 was positive in 92% of Graves’ disease thyroids and 97% of multinodular goiters, and we failed to demonstrate AQP3 in these hyperplastic tissues. In neoplastic thyroid lesions, we observed AQP3 in 91% of medullary thyroid carcinomas but in no other follicular cell tumors. AQP4 was demonstrated in 100% of follicular adenomas, 90% of follicular carcinomas, and 85% of papillary carcinomas, while it was negative in all medullary carcinomas and undifferentiated carcinomas. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed AQP3 mRNA expression only in medullary carcinomas and AQP4 mRNA expression in follicular cell-derived tumors except for undifferentiated carcinomas. In thyroid cancer cell lines, using RT-PCR and western blotting, AQP3 mRNA and protein were only identified in the TT cell line (human medullary carcinoma cell line) and AQP4 in the other cell lines. In addition, AQP3 mRNA expression was up-regulated by FBS and calcium administration in both a dose and time dependent manner in TT cells. Conclusion The differential expressions of AQP3 and AQP4 may reflect the biological nature and/or function of normal, hyperplastic, and neoplastic thyroid cells and additionally may have value in determining differential diagnoses of thyroid tumors.

Expression of aquaporin3 in human neoplastic tissues

Niu D, Kondo T, Nakazawa T, Yamane T, Mochizuki K, Kawasaki T, Matsuzaki T, Takata K & Katoh R 
(2012) Histopathology 61, 543–551

RET/PTC rearrangements arising from a small population of papillary thyroid carcinoma cells, possible candidate for passenger mutation

RET rearrangements (RET/PTC) is a major genetic alteration in papillary thyroid carcinomas. However, the prevalence of RET/PTC differs considerably among investigators, and its impact on cancer progression has been controversial. In the current study, we applied interphase fluorescence in situ hybridization (FISH) to touch imprint cytology of 14 papillary thyroid carcinomas along with reverse-transcription polymerase chain reaction (RT-PCR) analysis. FISH DNA probes included RET locus, and PCR primers were designed targeting RET/PTC1 or RET/PTC3. Split FISH signals of RET was observed in 78.6% (11/14) of tumors. Proportions of tumor cells having split RET signals ranged from 1.8% to 19.6% (mean 9.7%) in those 11 tumors. In RT-PCR analysis, RET/PTC was found in 28.6% (4/14) of tumors. Among tumors with split RET signals, 36.4% (4/11) of tumors exhibited detectable messenger RNA of RET/PTC1 or RET/PTC3. The remaining seven tumors with split RET signals had no RET/PTCs amplicon. In conclusion, the current study disclosed that RET/PTCs occur in a small population of tumor cells in papillary thyroid carcinomas. Even though RET/PTC is a specific genetic event in the carcinomas, our results suggested the possibility of RET/PTC as “passenger” abnormalities rather than “driver” oncogenic mutation during thyroid cancer progression, warranting further studies on mechanisms and implication of RET gene instability.

Polyclonal origin of hormone-producing cell populations evaluated as a direct in situ demonstration in EGFP/BALB/C chimeric mice

We report the first demonstration of the embryonal patch patterns of endocrine organs and the polyclonality of hormone-producing cell populations using chimeric mice produced by aggregation of C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb transgenic mice and BALB/C mice. Confocal laser scanning microscopy (CLSM) analysis for enhanced green fluorescent protein (EGFP) and immunohistochemistry with anti-EGFP antibody revealed that all endocrine organs of chimeric mice had a mosaic appearance of EGFP-positive patches and EGFP-negative patches. The patches composed of EGFP-positive cells were distinctive in their size and shape. The pituitary patches were large and irregular, representing a geographical pattern. In contrast, parathyroid, pancreatic islet, and adrenal medulla patches were small and demarcated, representing an island-like pattern. Thyroid follicles and adrenal cortex cords showed a mixture of monophenotypia and polyphenotypia, indicating polyclonal embryonic origin. Furthermore, we studied the tissue clonality of hormone-producing cell populations in the pituitary, thyroid, and pancreatic islets using a combination method of CLSM for EGFP and immunohistochemistry for hormones. All the pituitary cell populations of GH, prolactin, TSH, FSH, LH, and ACTH, the calcitonin-producing cell population in the thyroid, and the insulin- and glucagon-producing cell populations in pancreatic islets had mosaic patterns in EGFP expression in the chimeric mice, suggesting polyclonal embryonic origin. In conclusion, the different patch patterns of the endocrine organs could contribute to the understanding of embryonic development and organization of endocrine organs. Furthermore, we clearly demonstrate that all hormone-producing cell populations are of polyclonal embryonic origin, derived from more than two progenitor cells.

Involvement of centrosomes in nuclear irregularity of thyroid carcinoma cells.

Nuclear irregularities including nuclear pseudoinclusions and nuclear grooves are characteristic of papillary thyroid carcinoma cells and are regarded as important diagnostic clues in histopathology. We observed nuclear features of thyroid carcinoma cell lines (KTC-1 and TPC-1) in various culture conditions and performed immunocytochemical examinations for cytoskeleton molecules to clarify the morphogenesis of thyroid carcinoma nuclei. We found that nuclear irregularities presenting as bean-like nuclei (BLNs) and donut-like nuclei (DLNs) appeared in cells from confluent cultures, but not in cells from sparse cultures. On immunocytofluorescence analyses, clusters of γ-tubulin, representing a centrosome, frequently localized at the indentation of BLNs or in the hole of DLNs of thyroid carcinoma cells. In conclusion, we suggest that cell-to-cell contact may affect nuclear changes such as BLNs and DLNs in cancer cell lines and that centrosomes may be involved in the morphogenetic process of these nuclear changes.

Neuroendocrine ductal carcinoma in situ, comedo type, of the breast detected by screening mammography: a potentially pre-invasive counterpart of high grade neuroendocrine tumours

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Giant oesophageal liposarcoma mimicking spindle cell liposarcoma and containing eosinophilic cells with rhabdomyoblastic differentiation

An-83-year-old Japanese man presenting with chest discomfort and vomiting was admitted to our hospital. Upper gastrointestinal endoscopy and radiological examinations showed a giant submucosal tumour in the thoracic oesophagus. Consecutive subtotal oesophagectomy showed a tumour with a maximum diameter of 12 cm, protruding into the oesophageal lumen. The patient remains stable and disease-free 18 months after tumour resection. The tumour was well demarcated with a glistening, gelatinous, white-yellowish cut surface. Microscopically, the tumour was primarily composed of spindle cells and mature adipocytes, sparsely intermingled with lipoblasts (figure 1A,B). Throughout most of the tumour, spindle cells had inconspicuous nuclear atypia, …