Regulation of 14-3-3sigma expression in human thyroid carcinoma is epigenetically regulated by aberrant cytosine methylation

Increased 14-3-3sigma expression has been observed by immunohistochemistry in papillary and anaplastic tumors, but not follicular thyroid cancers. 14-3-3sigma mRNA expression and methylation status was examined in tumor cell lines and primary thyroid tissues using real-time RT-PCR, bisulfite sequencing and methylation-specific PCR. Most of the 27 CpG's in the gene's CpG island were methylated in normal thyroid, TPC-1, NPA, FTC-238 and 2-7, which did not express 14-3-3sigma. In contrast, they were unmethylated in KAK-1 and anaplastic lines KAT4 and DRO-90. 14-3-3sigma expression was not increased in thyroid carcinomas, the majority of which had a methylated CpG island. In addition, 5-aza-dC treatment increased 14-3-3sigma expression in the FTC-238 and NPA cell lines, which had low baseline expression. We conclude 14-3-3sigma expression in thyroid carcinomas is regulated by CpG island hypermethylation.     

14-3-3 sigma possibly plays a constitutive role in papillary carcinoma, but not in follicular tumor of the thyroid

14-3-3 σ is a negative regulator of the cell cycle and contributes to G2 arrest. Thus far, the lack of its expression due to hypermethylation of the CpG islands has been reported in some carcinomas. In this study, we investigated the expression of 14-3-3 σ in thyroid neoplasms by means of immunohistochemistry as well as Western blot analysis. Normal follicules did not express 14-3-3 σ. In 82 papillary carcinomas, all the cases expressed 14-3-3 σ and its expression was not reduced but even enhanced in the advanced stage and in poorly differentiated types. Furthermore, 21 of the 23 anaplastic carcinomas expressed 14-3-3 σ and its expression level tended to be higher than in papillary carcinoma. On the other hand, none of the 34 follicular carcinomas or 29 follicular adenomas expressed 14-3-3 σ. These results suggest that 14-3-3 σ plays a constitutive role in papillary carcinoma rather than acting as a cell cycle regulator, whereas it is not required for the occurrence and development of follicular tumor.     

Epigenetic regulation of the cell type-specific gene 14-3-3sigma

Epigenetic control participates in processes crucial in mammalian development, such as X-chromosome inactivation, gene imprinting, and cell type-specific gene expression. We provide evidence that the p53-inducible gene 14-3-3sigma is a new example of a gene important to human cancer, where epigenetic mechanisms participate in the control of normal cell type-specific expression, as well as aberrant gene silencing in cancer cells. Like a previously identified cell type-specific gene maspin, 14-3-3sigma is a p53-inducible gene; however, it participates in G2/M arrest in response to DNA-damaging agents. 14-3-3Sigma expression is restricted to certain epithelial cell types, including breast and prostate, whereas expression is absent in nonepithelial tissues such as fibroblasts and lymphocytes. In this report, we show that in normal cells expressing 14-3-3sigma, the 14-3-3sigma CpG island is unmethylated; associated with acetylated histones, unmethylated histone H3 lysine 9; and an accessible chromatin structure. By contrast, normal cells that do not express 14-3-3sigma have a methylated 14-3-3sigma CpG island with hypoacetylated histones, methylated histone H3 lysine 9, and an inaccessible chromatin structure. These findings extend the spectrum of cell type-specific genes controlled, partly, by normal epigenetic mechanisms, and suggest that this subset of genes may represent important targets of epigenetic dysregulation in human cancer.     

Frequent downregulation of 14-3-3 sigma protein and hypermethylation of 14-3-3 sigma gene in salivary gland adenoid cystic carcinoma

14-3-3 sigma:, a target gene of the p53 tumour suppressor protein, has been shown to regulate the cell cycle at the G2/M checkpoint. Recent studies have demonstrated that 14-3-3 sigma is downregulated by hypermethylation of the CpG island in several types of cancer. In this study, we investigated the expression and methylation status of 14-3-3 sigma in human salivary gland adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC). Immunohistochemical analysis revealed that the positive expression rate of 14-3-3 sigma in ACC (one out of 14) was markedly lower than that in MEC (ten out of 10). Since most of the ACCs carried the wild-type p53 protein, downregulation of 14-3-3 sigma in ACC may not be due to the dysfunction of p53 pathway. Microdissection-methylation-specific PCR revealed that frequent hypermethylation of the 14-3-3 sigma gene was observed in ACC when compared to that in MEC. In cultured-ACC cells, we confirmed the downregulation of 14-3-3 sigma via hemimethylation of the gene by sequencing analysis after sodium bisulphite treatment. Furthermore, re-expression of 14-3-3 sigma in the ACC cells was induced by the treatment with DNA demethylating agent, 5-aza-2'-deoxycytidine. Irradiation apparently induced the enhanced expression of 14-3-3 sigma and G2/M arrest in normal salivary gland cells; however, in the ACC cells, neither induction of 14-3-3 sigma nor G2/M arrest was induced by irradiation. These results suggest that downregulation of 14-3-3 sigma might play critical roles in the neoplastic development and radiosensitivity of ACC.     

Functional retinoid and thyroid hormone receptors in human thyroid-carcinoma cell lines and tissues

Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [³H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and supershift assays using a DR2 (“direct repeat” 2) RA response element demonstrated DNA-binding of RARα, RARγ, RXRα and RXRβ in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRα1, TRα2, and TRβ. Northern-blot analysis showed low expression of RXRβ mRNA in FTC-133 and of TRα1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARα, RARβ, RARγ, RXRα, and RXRβ in the 4 cell lines and in human thyroid-carcinoma samples. RARβ mRNA was reduced in FTC-238 cells and RXRβ mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding. Int. J. Cancer 76:368–376, 1998.© 1998 Wiley-Liss, Inc.     

miR-143-3p靶向KRAS阻滞PI3K/Akt信号通路抑制分化型甲状腺癌细胞增殖、侵袭和迁移

目的:探讨miR-143-3p对分化型甲状腺癌细胞增殖、侵袭和迁移的影响及其作用机制。方法:采用qRT-PCR检测miR-143-3p在30例分化型甲状腺癌组织和对应的癌旁组织,以及人分化型甲状腺癌细胞（TCP-1、FTC-133、SW579、BCPAP）和甲状腺滤泡上皮正常细胞（Nthy-ori3-1）中的表达水平。将miR-143-3p mimic和miR-143-3p mimic+pcDNA3.1-KRAS转染于FTC-133细胞中,采用CCK-8检测FTC-133细胞增殖活力;Transwell检测FTC-133细胞侵袭和迁移能力。双荧光素酶报告基因验证miR-143-3p和KRAS的靶向关系。Western blot检测蛋白的表达水平。结果:miR-143-3p在分化型甲状腺癌组织中的表达水平明显低于对应的癌旁组织。miR-143-3p在分化型甲状腺癌细胞系中的表达水平低于甲状腺滤泡上皮正常细胞的表达,尤其在FTC-133细胞中表达最低。过表达miR-143-3p显著抑制了FTC-133细胞增殖、侵袭和迁移能力。此外,双荧光素酶报告基因证实,KRAS为miR-143-3p的靶基因。进一步,过表达KRAS通过激活PI3K/Akt信号通路缓解了仅过表达miR-143-3p对FTC-133细胞增殖、侵袭和迁移能力的抑制作用。结论:过表达miR-143-3p通过靶向KRAS且阻滞PI3K/Akt信号通路,进而抑制分化型甲状腺癌细胞恶性生物学行为。     

长链非编码RNA RP11-385J1.2通过靶向微小RNA-370-3p调控甲状腺癌细胞增殖、侵袭及凋亡的作用机制

目的探讨长链非编码RNA(lncRNA)RP11-385J1.2对甲状腺癌细胞增殖、侵袭和凋亡的影响。方法采用实时荧光聚合酶链反应(PCR)检测人甲状腺癌细胞株SW579、FTC-133、TPC-1、K1和正常甲状腺细胞Nthyori 3-1中RP11-385J1.2和微小RNA(miRNA)-370-3p的表达情况。采用抑制剂si-RP11-385J1.2和(或)anti-miRNA-370-3p转染SW579细胞,噻唑蓝(MTT)法检测细胞增殖,Transwell实验检测细胞侵袭,流式细胞术检测细胞凋亡,蛋白质印记法(Western blot)检测凋亡相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2)和Bax的表达情况,双荧光素酶报告系统验证RP11-385J1.2和miRNA-370-3p的靶向关系。结果人甲状腺癌细胞株SW579、FTC-133、TPC-1、K1中RP11-385J1.2的表达水平均高于正常甲状腺细胞Nthy-ori 3-1,miRNA-370-3p的表达水平均低于正常甲状腺细胞Nthy-ori 3-1,差异均有统计学意义(P﹤0.05)。敲减RP11-385J1.2可抑制SW579细胞增殖和侵袭并促进细胞凋亡,RP11-385J1.2能够靶向负调控miRNA-370-3p的表达,下调miRNA-370-3p的表达可逆转敲减RP11-385J1.2对SW579细胞增殖、侵袭和凋亡的影响。结论lncRNA RP11-385J1.2通过靶向下调miRNA-370-3p的表达促进甲状腺癌SW579细胞增殖和侵袭并抑制细胞凋亡。RP11-385J1.2和miRNA-370-3p是甲状腺癌的潜在分子靶点。     

Biological significance of aminopeptidase N/CD13 in thyroid carcinomas

Aminopeptidase N (APN)/CD13 is a transmembrane ectopeptidase expressed on a wide variety of cells. However, the precise function of APN/CD13 in tumor cells and the relationship of APN/CD13 to thyroid cancer remain unclear. In our study, we quantified the expression of APN/CD13 and additionally dipeptidyl peptidase IV (DPIV)/CD26 in thyroid carcinoma cell lines and in tissues of patients with thyroid carcinomas. Undifferentiated anaplastic thyroid carcinomas expressed more APN/CD13 than differentiated thyroid carcinomas. DPIV/CD26 showed an opposite expression pattern. We detected higher levels of DPIV/CD26 in follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas than in undifferentiated anaplastic thyroid carcinomas. In the undifferentiated thyroid carcinoma cell line 1736, APN/CD13 mRNA expression could be increased by epidermal growth factor, basic fibroblast growth factor, interleukin-6, and tumor necrosis factor alpha. FTC-133 cells stably transfected with an expression vector for APN-enhanced green fluorescent protein showed a higher migration rate than FTC-133 cells transfected with the enhanced green fluorescent protein-control plasmid. Overexpression of APN/CD13 in stably transfected cells is associated with down-regulation of N-myc down-regulated gene (NDRG)-1, melanoma-associated antigen ME491/CD63, and DPIV/CD26 gene expression. Inhibition of APN/CD13 mRNA expression by small interfering RNA induced NDRG-1, ME491/CD63, and DPIV/CD26 mRNA expression in cells of the undifferentiated thyroid carcinoma cell line C643. We conclude that APN/CD13-associated down-regulation of NDRG-1, ME491/CD63, and DPIV/CD26 in thyroid carcinoma cells is an important step of tumor progression to more malignant phenotypes, and we underline the important role of APN/CD13 as mediator in a multimolecular process regulating cell migration.     

Decreased expression of 14-3-3sigma in neuroendocrine tumors is independent of origin and malignant potential

We recently reported that 14-3-3sigma is frequently inactivated in small cell lung cancer (SCLC) and a part of large cell carcinomas. Subsequent studies revealed that the large cell carcinomas could be morphologically categorized as large cell neuroendocrine carcinomas (LCNEC). The present study therefore examines 14-3-3sigma expression in a spectrum of neuroendocrine lung tumors, which had varied p53 status, proliferative activity and clinical aggressiveness. The expression of 14-3-3sigma was decreased in all four categories of the spectrum, (5 out of 5 typical carcinoids, 2 out of 2 atypical carcinoids, 5 out of 7 LCNECs and 15 out of 18 SCLCs). In sharp contrast, the level of 14-3-3sigma expression in 75 non-small cell lung cancers (NSCLCs) was the same as that in normal lung tissue, with only one exception. The expression status of neuroendocrine tumors and NSCLCs was not affected by p53 status, but dense promoter hypermethylation of the 14-3-3sigma gene was specifically observed in neuroendocrine tumors, suggesting that methylation plays a regulatory role in 14-3-3sigma expression in vivo as well as in vitro. Furthermore, the expression was not only down-regulated in pulmonary neuroendocrine tumors, but also in neuroendocrine tumors arising from various other organs, through examination of 123 non-pulmonary tumors. Since various carcinogenic machineries are involved in the neuroendocrine tumors, a reduced expression of 14-3-3sigma might be required for the development of neuroendocrine tumors. Constitutive 14-3-3sigma expression was distributed exclusively in putative stem cells of the normal lung, namely the basal cells of the bronchus, and type II pneumocytes. Notably, 14-3-3sigma expression was up-regulated during the regeneration of type II pneumocytes, suggesting that 14-3-3sigma plays a biological role when a regenerative and/or differentiating drive is activated, facilitating exit from stem cells.     

Epigenetic inactivation of 14-3-3 sigma in oral carcinoma: association with p16(INK4a) silencing and human papillomavirus negativity

In vitro studies have identified 14-3-3sigma as a regulator of senescence in human keratinocytes. To assess its contribution to squamous neoplasia, we have analyzed genetic and epigenetic changes in this gene in squamous cell carcinomas (SCCs) and dysplastic lesions of the oral cavity. No mutations were detected in the coding sequence of 14-3-3sigma in 20 oral carcinomas, and there was loss of heterozygosity in only 7 of 40 informative cases. In contrast to the absence of genetic change, aberrant methylation within 14-3-3sigma was detected in 32 of 92 squamous cell carcinomas and in 3 of 6 oral dysplasias and was associated with reduced or absent expression at both mRNA and protein levels. Methylation was not detected in matched, normal epithelial tissue controls. Carcinomas in which 14-3-3sigma was methylated were significantly more likely to lack DNA sequences from human papillomavirus and to have coincident methylation of p16(INK4a) than cases that expressed 14-3-3sigma. Methylation was detected in SCC, both wild-type and mutant for p53, but was more commonly detected in cancers with wild-type p53. These results implicate coincident epigenetic abrogation of function in both sigma and p16(INK4a) in a subset of SCCs of the oral cavity.     

Over-expression of 14-3-3sigma in budding colorectal cancer cells modulates cell migration in the presence of tenascin-C

Epigenetic silencing of the 14-3-3sigma gene by CpG hypermethylation has been reported in many kinds of cancers, but has been considered inapplicable in the colorectal variety. The expression of 14-3-3sigma in colorectal cancer is located primarily in the invasive area. The interaction between tumor cells and the extracellular matrix (ECM) is involved in tumor invasion. In the current study, we investigated the correlation between 14-3-3sigma expression and the ECM, focusing especially on the presence of tenascin-C (TNC) at the invasive area of colorectal cancers. Correlations between the immunohistochemical expression of 14-3-3sigma and TNC, as well as other clinicopathological factors, were evaluated in 123 colorectal carcinoma tissues. 14-3-3sigma expression was frequently observed in budding tumor cells in the invasive area and expression was significantly correlated with budding formation (p=0.001), pTNM classification (p=0.001) and stromal TNC expression (p=0.004). Using colorectal cancer cell lines and ECMs, the up-regulation of 14-3-3sigma mRNA levels was investigated by semi-quantitative RT-PCR. TNC surrounding the tumor cells increased 14-3-3sigma mRNA expression 1.8- to 2.2-fold in HCT116 cells. The effect of 14-3-3sigma over-expression on tumor cell migration was investigated using an agarose-cell droplet migration assay. Over-expression of 14-3-3sigma up-regulated HCT116 cell migration on TNC (p<0.001). We concluded that the expression of 14-3-3sigma in the invasive area modulates tumor cell migration in certain types of colorectal cancer and thus facilitates tumor progression.     

Epigenetic silencing of 14-3-3sigma in cancer

The 14-3-3sigma gene is a direct target of the p53 tumor suppressor and its product inhibits cell cycle progression. Recently, a proteomic analysis revealed that 14-3-3sigma regulates additional cellular processes relevant to carcinogenesis, as migration and MAP-kinase signalling. The expression of 14-3-3sigma is down-regulated by CpG methylation in several types of human cancer, among them prostate, lung, breast and several types of skin cancer. The epigenetic inactivation of 14-3-3sigma occurs at an early stage of tumor development and may allow evasion from senescence and promote genomic instability. In the future the detection of CpG methylation of 14-3-3sigma may be used for diagnostic and prognostic purposes.     

胃癌14-3-3sigma mRNA的表达及其临床意义

[目的]探讨胃癌14-3-3sigma mRNA的表达及其与胃癌生物行为学关系。[方法]用半定量RT-PCR检测胃癌患者肿瘤组织和远癌组织标本中的14-3-3sigma mRNA。[结果]39．6％（21／54）（的胃癌组织中的14-3-3sigma mRNA出现低表达，但14-3-3sigma mRNA的低表达与患者的性别、年龄以及胃癌的病理分型、分期以及淋巴结转移均无显著性相关。[结论]14-3-3sigma mRNA的低表达与胃癌的发生相关。