673

BRAF

Mice with thyroid-specific expression of oncogenic BRAF (Tg-Braf) develop papillary thyroid cancers (PTCs) that are locally invasive and have well-defined foci of poorly differentiated thyroid carcinoma (PDTC). To investigate the PTC-PDTC progression, we performed a microarray analysis using RNA from paired samples of PDTC and PTC collected from the same animals by laser capture microdissection. Analysis of eight paired samples revealed a profound deregulation of genes involved in cell adhesion and intracellular junctions, with changes consistent with an epithelial-mesenchymal transition (EMT). This was confirmed by immunohistochemistry, as vimentin expression was increased and E-cadherin lost in PDTC compared with adjacent PTC. Moreover, PDTC stained positively for phospho-Smad2, suggesting a role for transforming growth factor (TGF)beta in mediating this process. Accordingly, TGFbeta-induced EMT in primary cultures of thyroid cells from Tg-Braf mice, whereas wild-type thyroid cells retained their epithelial features. TGFbeta-induced Smad2 phosphorylation, transcriptional activity and induction of EMT required mitogen-activated protein kinase (MAPK) pathway activation in Tg-Braf thyrocytes. Hence, tumor initiation by oncogenic BRAF renders thyroid cells susceptible to TGFbeta-induced EMT, through a MAPK-dependent process.

In vitro studies in melanoma indicate that up-regulation of the transcriptional repressor Snail occurs with a concomitant decrease of its target E-cadherin, both hallmarks of epithelial-mesenchymal transition-an association not established in vivo. We sought to elucidate the relationship between BRAF, Snail, E-cadherin, and established histopathologic prognosticators in primary cutaneous melanoma. Archived annotated samples with a diagnosis of primary cutaneous melanoma were retrieved (n = 68 cases; 34 BRAF mutant and 34 BRAF wild type) and immunohistochemically stained for Snail and E-cadherin protein expression. A semiquantitative scoring system was used. Multivariate logistic analysis was used to control confounders of BRAF. Snail expression was significantly associated only with ulceration (42% versus 13%; P = .02). E-cadherin expression was present in 26% of BRAF mutant and 71% of BRAF wild-type cases (P = .0003). Loss of E-cadherin expression was associated with female sex (60% versus 34%; P = .05), BRAF mutation (74% versus 29%; P = .0003), thickness greater than or equal to 1 mm (68% versus 32%; P = .004), mitosis (63% versus 25%; P = .007), and ulceration (75% versus 44%; P = .05). BRAF mutation was associated with male sex (60% versus 30%; P = .02), Breslow thickness (P = .007), thickness greater than or equal to 1 mm (68% versus 29%; P = .002), and ulceration (75% versus 42%; P = .02). Snail expression did not correlate with loss of E-cadherin expression (47% versus 53%; P = .79). After controlling for potential confounding, BRAF mutation was associated with loss of E-cadherin (adjusted odds ratio, 8.332; 95% confidence interval, 2.257-30.757; P = .0015) and Breslow thickness greater than 1 mm (adjusted odds ratio, 7.360; 95% confidence interval, 1.534-35.318; P = .0126). Our findings, indicating that mutant BRAF represses E-cadherin expression, implicating a catalytic role for BRAF in epithelial-mesenchymal transition.CI - Published by Elsevier Inc.