Possible Involvement of BRAFV600E in Altered Gene Expression in Papillary Thyroid Cancer

Abstract

Abstract. Somatic mutations in BRAF, especially BRAFV600E, are frequently identified in papillary thyroid cancer (PTC) tumors. It has been established that expression levels of numbers of genes are characteristically altered in PTC, however, the link between BRAF mutation and gene expression patterns are still elusive. In the present study, we analyzed relative expression levels of the wild type BRAF and BRAFV600E mRNA by using quantitative PCR (qPCR) and cDNA-PCR-RFLP in 19 PTC specimens and adjacent normal thyroid tissues. BRAFV600E mRNA was detected in 17 out of 19 PTC specimens, and the expression levels were valuable among the specimens, suggesting alternative expression of BRAFV600E in each cell and/or alternative population of BRAFV600E-positive clones in the tumor. We then analyzed expression levels of 20 genes by qPCR, and analyzed for possible correlation with expression levels of BRAFV600E mRNA. Expression levels of fibronectin, vimentin and CITED1 (Cbp/p300 interacting protein with glutamic acid and aspartic acid rich carboxyl terminal domain) were positively correlated with those of BRAFV600E, suggesting pathophysiological links between activated BRAF and overexpression of these genes. Among these genes expression of vimentin was decreased by inhibiting BRAF expression in NPA cells that express BRAFV600E by means of siRNA, suggesting activated BRAF positively regulate expression of vimentin. Collectively, our analyses illustrated the possibilities that variable expression of BRAFV600E may modify characters of PTC through its effects on gene expression. PAPillAry thyroid cancer (PTC) is the most common type of thyroid cancer and accounts for more than 80% of all thyroid malignancies [1][2][3]. Somatic mutations in genes involved in the RET-Ras-Raf-MEK-MAPK/ERK signaling pathway, which transmits mitogenic signals from the cell membrane to the nucleus and promotes cell proliferation, have been identified in various cancers [4][5][6]. In PTC, point mutations in the BRAF gene are the most prevalent genetic alteration, with an average prevalence of 44% [7,8]. A thymine to adenine transversion at position 1799 (T1799A), which results in a valine to glutamic acid substitution (V600E) in the BRAF protein, is the most common BRAF mutation in PTC [7,8].It has been known that numbers of genes are characteristically overexpressed in PTC, and these genes have served as possible markers for molecular diagnosis of PTC [9,10]. However the mechanisms involved in overexpression of these genes are not fully understood. In the present study, we determined relative expression levels of the wild type BRAF and BRAFV600E mRNA by using quantitative PCR (qPCR) and cDNA-PCR-RFLP in 19 PTC and adjacent normal thyroid tissues. The results showed that 17 out of 19 PTC specimens expressed BRAFV600E mRNA in variable level. We then analyzed expression level of sets of genes in the tissues and analyzed whether expression levels of BRAFV600E are correlated with the analyzed genes. Our analyses showed that expression levels of BRAFV600...

Gene expression profile of human thyroid cancer in relation to its mutational status

“…Vasko et al proposed an association between CDC42/PAK signaling and the mechanisms of PTC invasion. In opposition to their results, Watanabe et al (2009) performed a QPCR analysis to show a positive correlation between the BRAF V600E mutation and over-expression of vimentin, which was confirmed using NPA cells (inhibition of BRAF expression decreased vimentin levels). These authors also showed the BRAF V600E-dependent upregulation of fibronectin and CITED1, both of which have been identified as PTC markers.…”

Prognostic utility of BRAF mutation in papillary thyroid cancer

“…BRAF mutation is uniquely associated with the over-expression of many classical tumor-promoting molecules in PTC. Examples include VEGF,44 c-MET,41 matrix metalloproteinase,29,42,45,46 nuclear factor kappa B,46 Ki-67,27 prohibitin,47 and vimentin 48. Aberrant promoter methylation of several tumor suppressor and DNA repair genes in association with BRAF mutation was also demonstrated and associated with aggressive pathological characteristics of PTC 49,50.…”

An update on molecular biology of thyroid cancers

“…Following the activation of the MAPK pathway, secondary molecular events occur, such as hypomethylation and genome-wide hypermethylation, which augment the tumorigenic activity of this pathway [52]. In addition, upregulation of several oncogenic proteins, such as chemokines [53,54], nuclear factor B (NF-B) [55], vascular endothelial growth factor A (VEGFA) [56], matrix metalloproteinases (MMPs) [53,55,57], MET [58,59], vimentin [60], prokineticin 1 (PROK1; also known as EG VEGF) [61], prohibitin [62], hypoxia-inducible factor 1␣ (HIF1␣) [63], thrombospondin 1 (TSP1) [64], urokinase plasminogen activator (uPA) and its receptor (uPAR) [65,66] and transforming growth factor ␤1 (TGF␤1) [67,68] drive cancer cell growth, proliferation and survival, together with tumor angiogenesis, invasion and metastasis.…”