We analyzed RASSF1A and NORE1A methylation and BRAF mutation in 89 thyroid tumors, 42 non-neoplastic thyroid tissues and three thyroid tumor cell lines using polymerase chain reaction (PCR), methylation-specific PCR, Western blotting and DNA sequencing in order to study thyroid tumor pathogenesis and progression. RASSF1A promoter methylation was present in all three thyroid cell lines and in 27/78 (35%) of benign and malignant thyroid tumors. We showed for the first time that there was generally good agreement between RASSF1A methylation status and RASSF1A protein expression. We also examined for the first time NORE1A promoter region methylation in thyroid cell lines and primary tumors and showed that two of three thyroid cell lines were methylated in the NORE1A promoter region, while all primary thyroid tumors analyzed (n=51) were unmethylated. BRAF mutation was present in 38% of papillary thyroid carcinomas (PTC), including 20% of PTC with a follicular variant pattern and 67% of the tall cell variant of PTC. Hyalinizing trabecular tumors (n=23), which had nuclear features similar to PTC, did not have BRAF mutations, indicating that the presence of BRAF mutations can help to separate these two tumor types. Phospho-MEK expression was increased in the NPA cell line, which had a BRAF mutation, supporting the importance of the BRAF pathway alterations in PTC pathogenesis. These results indicate that RASSF1A epigenetic changes are an early event in thyroid tumor pathogenesis and progression and that NORE1A methylation is uncommon in primary thyroid tumors. BRAF mutation occurs later in thyroid tumor progression and is restricted mainly to PTC and anaplastic thyroid carcinoma.

To identify proteins involved in ovarian clear cell carcinoma (OCCCa), shotgun proteomics analysis was applied using formalin-fixed and paraffin-embedded samples of ovarian carcinoma. Analysis of 1521 proteins revealed that 52 were differentially expressed between four OCCCa and 12 non-OCCCa samples. Of the highly expressed proteins in OCCCa, we focused on left-right determination factor (LEFTY), a novel member of the transforming growth factor-β superfamily. In 143 cases of ovarian epithelial carcinoma including 99 OCCCas and 44 non-OCCCas, LEFTY expression at both mRNA and protein levels was significantly higher in OCCCas compared with non-OCCCas, with the mRNA expression of LEFTY1 being predominant compared to that of LEFTY2. OCCCa cells stably overexpressing LEFTY1 showed reduced cell proliferation, along with decreased pSmad2 expression, and also either displayed an activated p53/p21waf1 pathway or increased p27kip1 expression, directly or indirectly. Moreover, the treatment of stable cell lines with cisplatin led to increased apoptotic cells, together with the inhibition of protein expression of a pSmad2-mediated X-linked inhibitor of apoptosis and a decreased bcl2/bax ratio. Blocking LEFTY1 expression with a specific short hairpin RNA inhibited cisplatin-induced apoptosis, probably through the increased expression of both XIAP and bcl2, but not bax. In clinical samples, a significantly higher number of apoptotic cells and lower Ki-67 labeling indices were observed in OCCCas with a high LEFTY score relative to those with a low score. These findings suggest that LEFTY may be an excellent OCCCa-specific molecular marker, which has anti-tumor effects in altering cell proliferation and cellular susceptibility to apoptosis.

The development of papillary thyroid carcinoma is influenced by many factors including genetic alterations, growth factors, and physical agents such as radiation. Arachidonic acid and its derivatives including prostaglandins (PG) and thromboxane along with the enzymes involved in their synthesis have been shown to influence the growth of various tumors. We analyzed the immunoreactivity for cyclooxygenase-2 (COX-2) and mRNA expression levels of the enzymes COX-2, thromboxane A 2 (TXA 2 ) synthase, and PGI 2 synthase by RT-PCR in papillary carcinomas and matching normal tissues to determine the role of these enzymes in the development of papillary thyroid carcinomas. A papillary thyroid carcinoma cell line TPC-1 was also studied in vitro to determine the role of the specific COX-2 inhibitor NS-398 on COX-2 and vascular endothelial growth factor-A, since COX-2 also has a role in regulating tumor angiogenesis. RT-PCR analysis showed significant increases in TXA 2 synthase mRNA levels in papillary thyroid carcinomas compared to normal thyroid tissues. Although COX-2 mRNA levels were generally increased in papillary carcinomas, the differences were not statistically significant. There were no significant differences in PGI 2 synthase mRNA levels. COX-2 protein expression was greater in papillary carcinoma compared to normal thyroid tissues; however, the levels were quite variable. In vitro studies with a COX-2 inhibitor, NS-398, showed inhibition of tumor growth along with increased levels of COX-2 and vascular endothelial growth factor-A mRNA expression. These results indicate that specific enzyme levels in the PG synthesis pathway such as TXA 2 synthase are increased in papillary thyroid carcinomas. COX-2 also has a role in papillary thyroid growth, since a specific inhibitor of COX-2 regulates papillary thyroid carcinoma cell proliferation. These results implicate several enzymes in the synthesis of prostanoids as regulators of thyroid papillary carcinoma proliferation and suggest that increased levels of expression of these enzymes may play a role in the pathogenesis of these tumors.

Distinct molecular events may occur in relatively early stages of tumorigenesis of endometriosis-associated OEMCas and OCCCas.

Hepatocyte nuclear factor-1β (HNF-1β) is a transcriptional factor that has an important role in endometriosis-ovarian clear cell carcinoma (OCCC) sequence by modulating cell kinetics and glucose metabolism. However, little is known about the detailed molecular mechanisms that govern its regulation and function. Herein, we focus on upstream and downstream regulatory factors of HNF-1β in OCCCs. In clinical samples, HNF-1β expression was positively correlated with the active form of NF-κB/p65 in OCCCs, and closely linked with a low nuclear grade and non-solid architecture. In cell lines, transfection of p65 resulted in increased HNF-1β mRNA and protein expression in TOV-21G cells (OCCC cell line with endogenous HNF-1β expression), in line with activation of the promoter, probably through interacting with the basic transcriptional machinery. Suppression of endogenous HNF-1β expression by siRNA increased apoptosis in TOV-21G cells, while treatment of Hec251 cells (endometrial carcinoma cell line with extremely low endogenous HNF-1β expression) stably overexpressing exogenous HNF-1β with doxorubicin abrogated apoptosis of the cells, along with increased ratio of bcl-2 relative to bax. Moreover, overexpression of HNF-1β led to upregulation of bcl-2 expression at the transcriptional level in TOV-21G cells, which provided evidence for a positive correlation between HNF-1β and bcl-2 expression in OCCCs. These data, therefore, suggest that association between HNF-1β and NF-κB signaling may participate in cell survival by alteration of apoptotic events, particularly in mitochondria-mediated pathways, through upregulation of bcl-2 expression in OCCCs.Laboratory Investigation (2015) 95, 962-972;