Conditional Activation of RET/PTC3 and BRAFV600E in Thyroid Cells Is Associated with Gene Expression Profiles that Predict a Preferential Role of BRAF in Extracellular Matrix Remodeling

Abstract

Papillary thyroid cancers (PTC) are associated with nonoverlapping mutations of genes coding for mitogen-activated protein kinase signaling effectors (i.e., the TK receptors RET or NTRK and the signaling proteins RAS and BRAF). We examined the pattern of gene expression after activation of these oncoproteins in thyroid PCCL3 cells, with the goal of identifying pathways or gene subsets that may account for the phenotypic differences observed in human cancers. We hybridized cDNA from cells treated with or without doxycycline to induce expression of BRAF V600E , RET/PTC3, or RET/PTC3 with small interfering RNA-mediated knockdown of BRAF, respectively, to slides arrayed with a rat 70-mer oligonucleotide library consisting of 27,342 oligos. Among the RET/PTC3-induced genes, 2,552 did not require BRAF as they were similarly regulated by RET/PTC3 with or without BRAF knockdown and not by expression of BRAF V600E . Immune response and IFN-related genes were highly represented in this group. About 24% of RET/PTC3-regulated genes were BRAF dependent, as they were similarly modified by RET/ PTC3 and BRAF V600E but not in cells expressing RET/PTC3 with knockdown of BRAF. A gene cluster coding for components of the mitochondrial electron transport chain pathway was down-regulated in this group, potentially altering regulation of cell viability. Metalloproteinases were also preferentially induced by BRAF, particularly matrix metalloproteinase 3 (MMP3), MMP9, and MMP13. Accordingly, conditional expression of BRAF was associated with markedly increased invasion into Matrigel compared with cells expressing RET/PTC3. The preferential induction of MMPs by BRAF could explain in part the more invasive behavior of thyroid cancers with BRAF mutations. (Cancer Res 2006; 66(13): 6521-9)

Targeting BRAFV600E in thyroid carcinoma: therapeutic implications

“…This suggests that the effect of BRAF V600E on tumor aggressiveness, if any, is mediated via increased metastatic potential and not via increased proliferation of the neoplastic cells. This is supported by the recent finding that B-Raf V600E preferentially induces metalloproteinase expression (42). In our present study, we did not detect any association between the BRAF V600E mutation and disease stage at presentation or tendency to give lymph node metastasis.…”

“…This suggests that alternative molecular lesions upstream of B-Raf, e.g., mutant RAS or RET/PTC translocations (44), or autocrine growth factor signaling (45), can sustain a high level of stimulation of B-Raf activity, even in the absence of constitutively activating V600E mutational events, leading to comparable stimulation of downstream kinases (MEK/ ERK) and proliferation rate. Specifically, Mitsutake et al have shown that B-Raf mediates RET/PTC-induced mitogen-activated protein kinase activation in thyroid cells (13), whereas Mesa et al showed that expression of a substantial percentage of RET/PTC3-regulated genes was BRaf-dependent (42). As RET/PTC, RAS, and BRAF mutational activation have been shown to be frequently present yet mutually exclusive in thyroid carcinomas (2), the constitutive proliferative/antiapoptotic activation by all these events is eventually mediated via B-Raf (13), which suggests that, irrespective of its mutational status, B-Raf is a valid therapeutic target for this broader spectrum of molecularly defined thyroid carcinomas.…”

CD44 Proteolysis Increases CREB Phosphorylation and Sustains Proliferation of Thyroid Cancer Cells

“…The fact that extracellular shedding of ectoCD44 accompanies the generation of CD44-ICD and that RET and BRAF blockade impairs CD44-CTF accumulation indicates that metalloprotease-mediated cleavage is one level at which CD44-ICD generation is stimulated in cells expressing RET/PTC and BRAF. Accordingly, RET/PTC and BRAF upregulate the transcription of several metalloproteases (44). CD44 cleavage can be triggered by binding to different extracellular ligands, including low molecular weight hyaluronan acid (45).…”

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.…”