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Endocrine Care: Valentina De Falco, Riccardo Giannini, Anna Tamburrino, Clara Ugolini, Cristiana Lupi, Efisio Puxeddu, Massimo Santoro, and Fulvio Basolo Functional Characterization of the Novel T599I-VKSRdel BRAF Mutation in a Follicular Variant Papillary Thyroid Carcinoma J Clin Endocrinol Metab 2008; 93: 4398-4402 [Abstract] [Full text] [PDF]

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Impaired BRAF activity mutant, Gly474Arg, in anaplastic thyroid carcinomas

Ginesa M Garcia - Rostan, Angela M. Costa, Gregorio Fernandez-Ballester, Manuel F. Fresno, Jose Cameselle-Teijeiro,   (3 November 2008)

Impaired BRAF activity mutant, Gly474Arg, in anaplastic thyroid carcinomas 3 November 2008
Ginesa M Garcia - Rostan, Senior Research Scientist IPATIMUP, Angela M. Costa, Gregorio Fernandez-Ballester, Manuel F. Fresno, Jose Cameselle-Teijeiro, Send letter to journal: Re: Impaired BRAF activity mutant, Gly474Arg, in anaplastic thyroid carcinomas ginesarostan{at}telefonica.net Ginesa M Garcia - Rostan, et al.	We have read with great interest the paper by De Falco et al. (1) in which the authors characterize the mutant G474R at exon-11 of BRAF and demonstrate that it behaves similarly to BRAFD594V (2) and the kinase-dead BRAFK483M mutant (1, 2), knocking-down BRAF catalytic activity and downstream MEK/ERK signalling. On the basis of these findings and the observation that BRAFG474R is able to drive TSH-independent proliferation the authors suggest that BRAFG474R mutants may contribute to thyroid tumorigenesis in a kinase-independent manner, through pathways alternative to the MAPK one. We believe these results are potentially of great interest in that BRAFG474R may significantly impact on the rationale of future treatment modalities for patients with deadly thyroid cancers bearing that mutation. Indeed, we have found the BRAFG474R mutation in 4 of 50 anaplastic thyroid carcinomas (ATC) (8%) investigated for the existence of mutations at the activation loop, the glicine-rich loop and the AKT-binding sites of BRAF by means of PCR-SSCP and direct sequence (see conditions at ref. 3). Despite the activation loop was wild type, the four ATCs disclosed increased cellular proliferation rates (4) with high mitotic indexes. In agreement with De Falco in vitro observations none of the aforementioned four ATCs expressed phospho-p44/42 MAPK (Thr202/Tyr204) [Cell Signaling, Beverly MA], which specifically recognizes the dually phosphorylated and active forms of ERK1 and ERK2. This finding would support the existence of impaired BRAF kinase activity in vivo and the abrogation of intracellular MEK/ERK signalling in thyroid tumours with BRAFG474R loss-of-function point mutations. The structural-conformational changes behind the missense G474R mutation severely compromise the ability of BRAF to phosphorylate MEK in vivo. The residue Gly474 is located in a beta strand, within a beta-sheet of the BRAF kinase N-lobe, supporting the glycine-rich P-loop. The mutant G474R introduces a bulky residue that strongly clashes with neighbours (Val482, Val528) and buries a positive charge into a small hydrophobic ATP-binding pocket formed by Ile457, Val459, Val480, Val482, and Val528. As a consequence of the mutation the beta strand needs to change its conformation, which could in turn avoid the beta-sheet formation and produce a broken or distorted P-loop. Since the P-loop is pivotal for ATP coordination and phosphotransfer reactions, it is expected that G474R will give rise to an inactive BRAF kinase unable to phosphorylate the downstream substrate in the signalling pathway. Our results in combination with the data recently reported by De Falco et al. provide strong evidence for the functional significance of particular residues within the BRAF kinase domain. The BRAFG474R mutation, by preventing the N lobe from folding into a well ordered beta-sheet structure, efficiently downregulates BRAF catalytic activity. As a consequence, clinical trials involving the inhibitor of threonine/serine kinases Sorafenib (BAY 43-9006) or kinase-specific inhibitors targeting constitutively active BRAF will prove inefficient therapeutic strategies in patients with BRAFG474R. References 1. De Falco V, Giannini R, Tamburrino A, Ugolini C, Lupi C, Puxeddu E, Santoro M, Basolo F 2008 Functional characterization of the novel T599I-VKSRdel-BRAF mutation in a follicular variant papillary thyroid carcinoma. J Clin Endocrin Metab doi:10.1210/jc.2008-0887 2. Wan PTC, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Cancer Genome Project, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R 2004 Mechanism of activation of the RAF-ERK signalling pathway by oncogenic mutations of B-RAF. Cell 116:855-867 3. Costa AM, Herrero A, Fresno MF, Heymann J, Alvarez JA, Cameselle-Teijeiro J, Garcia-Rostan G 2008 BRAF mutation associated with other genetic events identifies a subset of aggressive papillary thyroid carcinoma. Clin Endocrinol, 68:618-634 4. Tallin G, García-Rostán G, Herrero A, Zelterman D, Viale G, Bosari S, Carcangiu ML 1999 Downregulation of p27 and Ki67/Mib-1 labelling index support the classification of thyroid carcinoma into prognostically relevant categories. Am J Surg Pathol 23: 678-685