Mitsutake N, Knauf JA, Mitsutake S, Mesa Jr C, Zhang L, Fagin JA.. Conditional BRAFV600E expression induces DNA synthesis, apoptosis, dedifferentiation, and chromosomal instability in thyroid PCCL3 cells. Cancer Res 65: 2465-2473

University of Cincinnati, Cincinnati, Ohio, United States
Cancer Research (Impact Factor: 9.33). 04/2005; 65(6):2465-73. DOI: 10.1158/0008-5472.CAN-04-3314
Source: PubMed


The activating mutation BRAF(T1796A) is the most prevalent genetic alteration in papillary thyroid carcinomas (PTC). It is associated with advanced PTCs, suggesting that this oncoprotein confers thyroid cancers with more aggressive properties. BRAF(T1796A) is also observed in thyroid micropapillary carcinomas and may thus be an early event in tumor development. To explore its biological consequences, we established doxycycline-inducible BRAF(V600E)-expressing clonal lines derived from well-differentiated rat thyroid PCCL3 cells. Expression of BRAF(V600E) did not induce growth in the absence of thyrotropin despite increasing DNA synthesis, which is likely explained because of a concomitant increase in apoptosis. Thyrotropin-dependent cell growth and DNA synthesis were reduced by BRAF(V600E) because of decreased thyrotropin responsiveness associated with inhibition of thyrotropin receptor gene expression. These results are similar to those obtained following conditional expression of RET/PTC. However, in contrast to RET/PTC, BRAF activation did not impair key activation steps distal to the thyrotropin receptor, such as forskolin-induced adenylyl cyclase activity or cyclic AMP-induced DNA synthesis. We reported previously that acute RET/PTC expression in PCCL3 cells did not induce genomic instability. By contrast, induction of BRAF(V600E) expression increased the frequency of micronuclei by both clastogenic and aneugenic events. These data indicate that BRAF(V600E) expression confers thyroid cells with little growth advantage because of concomitant activation of DNA synthesis and apoptosis. However, in contrast to RET/PTC, BRAF(V600E) may facilitate the acquisition of secondary genetic events through induction of genomic instability, which may account for its aggressive properties.

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    • "The BcPAP and TPC1 cell lines, derived from PTCs, carry either of these genetic modifications (BRAF V600E and RTE/PTC1 respectively) and both exclusively express podoplanin. Recently published data, with induced expression of BRAF V600E or RET/PTC, suggest that there are differences in the oncogenic strength and the molecular events, as well as differences in the genes affected by these two genetic changes [44], [50], [51], [52]. Therefore, taking into account that BcPAP line is derived, in fact, from poorly differentiated cancer, and has stronger oncogenic potential, which can not only initiate development of papillary tumors, but is also required to maintain and promote their progression, we choose for our study the TPC1 cell line, which is derived from well differentiated conventional PTC [53], [54]. "
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    ABSTRACT: Podoplanin (PDPN), a mucin-type transmembrane glycoprotein specific to the lymphatic system is expressed in a variety of human cancers, and is regarded as a factor promoting tumor progression. The purpose of this study was to elucidate the molecular role of PDPN in the biology of thyroid cancer cells. PDPN expression was evaluated in primary thyroid carcinomas and thyroid carcinoma cell lines by RT-qPCR, Western blotting, IF and IHC. To examine the role of podoplanin in determining a cell's malignant potential (cellular migration, invasion, proliferation, adhesion, motility, apoptosis), a thyroid cancer cell line with silenced PDPN expression was used. We observed that PDPN was solely expressed in the cancer cells of 40% of papillary thyroid carcinoma (PTC) tissues. Moreover, PDPN mRNA and protein were highly expressed in PTC-derived TPC1 and BcPAP cell lines but were not detected in follicular thyroid cancer derived cell lines. PDPN knock-down significantly decreased cellular invasion, and modestly reduced cell migration, while proliferation and adhesion were not affected. Our results demonstrate that PDPN mediates the invasive properties of cells derived from papillary thyroid carcinomas, suggesting that podoplanin might promote PTC progression.
    PLoS ONE 05/2014; 9(5):e96541. DOI:10.1371/journal.pone.0096541 · 3.23 Impact Factor
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    • "BRAF regulates the expression of various genes that may modify PTC features, such as fibronectin, vimentin, CITED1, vimentin, prohibitin, sodium iodide symporter, and thyroperoxidase [12–16,23]. In addition, BRAF V600E may be involved in aberrant methylation of important tumor suppressor genes, up-regulation of metalloprotease expression, up-regulation of micro-RNAs and chromosomal instability [24] [25] [26] [27] [28]. The presence of BRAF V600E was not related to BRAF mRNA levels or to any clinical or pathological feature in our cases, perhaps because the relatively small number of cases investigated hindered the statistical analysis. "
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    ABSTRACT: Literature has consistently shown associations of BRAFV600E mutation with papillary thyroid cancer clinical features. However, the clinical utility of BRAF expression has not been clinically explored so far. We studied 67 thyroid nodules (32 benign nodules and 35 PTC cases). BRAF mRNA expression levels measured by a quantitative real-time PCR and a PCR-RFLP were used to identify BRAFV600E mutation. BRAF mRNA expression was significantly higher in malignant (198.2±373.9 AU) than in benign (4.1±6.9 AU) nodules (p<0.0001). BRAF expression identified malignancy with a sensitivity of 80.6%, specificity of 77.1%, positive predictive value of 75.8%, and negative predictive value of 81.8%. A cut-point of 4.712, identified by the ROC curve, was able to sort out malignant nodules with an accuracy of 78.8%. Although we did not find any correlation between the presence of BRAF V600E mutation and clinical or tumor features such as age (p=0.309), gender (p=0.5453), ethnicity (p=0.9820), tumor size (p=1.000), multifocality (p=0.2530) or mRNA levels (p=0.7510), the study power for BRAF expression and diagnosis (99%; FPRP=0.85) indicated that data is noteworthy despite the relative small number of patients investigated. We concluded that BRAF mRNA expression may help to identify PTC among thyroid nodules independently of the presence of BRAFV600E mutation.
    Pathology - Research and Practice 07/2012; 208(8):489-92. DOI:10.1016/j.prp.2012.05.013 · 1.40 Impact Factor
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    • "Indeed, transgenic mice overexpressing BRAFV600E in thyroid cells develop invasive PTCs with some tall-cell features and poorly differentiated areas [7]. In addition, conditional expression of BRAFV600E in the rat normal thyroid PCCL3 cells induced invasion and chromosomal instability, which was not observed in the same system with RET/PTC oncoprotein [8], [9]. "
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    ABSTRACT: The aim of the present study was to investigate chromosomal aberrations in sporadic Japanese papillary thyroid carcinomas (PTCs), concomitant with the analysis of oncogene mutational status. Twenty-five PTCs (11 with BRAF(V600E), 4 with RET/PTC1, and 10 without mutation in HRAS, KRAS, NRAS, BRAF, RET/PTC1, or RET/PTC3) were analyzed using Genome-Wide Human SNP Array 6.0 which allows us to detect copy number alteration (CNA) and uniparental disomy (UPD), also referred to as copy neutral loss of heterozygosity, in a single experiment. The Japanese PTCs showed relatively stable karyotypes. Seven cases (28%) showed CNA(s), and 6 (24%) showed UPD(s). Interestingly, CNA and UPD were rarely overlapped in the same tumor; the only one advanced case showed both CNA and UPD with a highly complex karyotype. Thirteen (52%) showed neither CNA nor UPD. Regarding CNA, deletions tended to be more frequent than amplifications. The most frequent and recurrent region was the deletion in chromosome 22; however, it was found in only 4 cases (16%). The degree of genomic instability did not depend on the oncogene status. However, in oncogene-positive cases (BRAF(V600E) and RET/PTC1), tumors with CNA/UPD were less frequent (5/15, 33%), whereas tumors with CNA/UPD were more frequent in oncogene-negative cases (7/10, 70%), suggesting that chromosomal aberrations may play a role in the development of PTC, especially in oncogene-negative tumors. These data suggest that Japanese PTCs may be classified into three distinct groups: CNA(+), UPD(+), and no chromosomal aberrations. BRAF(V600E) mutational status did not correlate with any parameters of chromosomal defects.
    PLoS ONE 04/2012; 7(4):e36063. DOI:10.1371/journal.pone.0036063 · 3.23 Impact Factor
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