Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines

Tanami, Hideaki; Imoto, Issei; Hirasawa, Akira; Yuki, Yasuhiro; Sonoda, Itaru; Inoue, Jun; K, Yasui; Misawa-Furihata, Akiko; Kawakami, Yutaka; Inazawa, Johji

doi:10.1038/sj.onc.1208152

Cited by 88 publications

(72 citation statements)

References 28 publications

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“…Our data suggest that the high biological activity of B-Raf V600E cannot be directly inhibited by compounds interfering with Ras activation or S446 phosphorylation and that the development of therapies for tumours harbouring a BRAF V600E allele should focus on the inhibition of the catalytic centre or expression of B-Raf. However, still to be developed S446 kinase inhibitors might be useful for the treatment of pathologies that are dependent on B-Raf signals but contain BRAF wt alleles, for example, tumours overexpressing B-Raf wt or harbouring activating mutations of upstream activators such as Ras or receptor tyrosine kinases, or polycystic kidney disease, which is characterized by increased B-Raf activation (Tanami et al, 2004;Yamaguchi et al, 2004). Similarly, S446 kinase inhibitors might be useful to attenuate the activity of other clinically relevant non-V600E B-Raf mutants.…”

Section: Regulation Of B-raf Signallingmentioning

confidence: 99%

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

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The BRAF V600E mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf wt ), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5 0 -triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf V600E , although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf V600E was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf wt and B-Raf V600E and suggests that B-Raf V600E cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation. Keywords: Ras; 14-3-3 proteins; b-Catenin; E-cadherin; mammary epithelial cells IntroductionThe Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in control of proliferation and differentiation and, owing to its role as a gatekeeper of this pathway, Raf appears an attractive therapeutic target (O'Neill and Kolch, 2004;Wilhelm et al., 2004). The Raf-kinase family comprises the A-Raf, B-Raf and Raf-1 isoforms in vertebrates as well as D-Raf and LIN-45 in Drosophila and Caenorhabditis, respectively. B-Raf, the major ERK activator in vertebrates, is required for the maintenance of basal ERK activity and displays the most potent transforming activity (Papin et al., 1998;Brummer et al., 2002;Mercer and Pritchard, 2003). All isoforms share three highly conserved regions (CRs; Figure 1a): the N-terminal CR1 contains the Ras-guanine 5 0 -triphosphate (GTP)-binding domain (RBD), which initiates the interaction with Ras-GTP through a conserved arginine residue (R188 in B-Raf) that is required for the recruitment and activation of Raf at the plasma membrane. Consequently, mutation of this residue prevents Ras/Raf interaction and renders D-Raf, B-Raf and Raf-1 unresponsive to most extracellular signals (Hou et al., 1995;Marais et al., 1997;Brummer et al., 2002). The ...

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Section: Regulation Of B-raf Signallingmentioning

confidence: 99%

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

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“…Moreover, two human normal melanocyte cell lines were included as control. Information relative to cell lines are extensively described elsewhere (Daniotti et al, 2004;Tanami et al, 2004).…”

Section: Melanoma Tissues and Cell Culturesmentioning

confidence: 99%

High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells

et al. 2007

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The cAMP-protein kinase A (PKA) pathway is the major signal transduction pathway involved in melanocytestimulating hormone receptor-mediated signaling and melanin production, whereas its role in the control of melanocyte proliferation is still controversial. In this study, we evaluated the effects of selective activation of the different PKA regulatory subunits type 1A (R1A) and type 2B (R2B) on melanocyte proliferation. Immunohistochemistry demonstrated that normal melanocytes lacked R1A protein whereas this subunit was highly expressed in all human melanomas studied (N ¼ 20) and in six human melanoma cell lines. Pharmacological activation of the R2 subunits by the cAMP analogue 8-Cl-cAMP inhibited proliferation and increased caspase-3 activity by 68.77 ± 10.5 and 72 ± 9% respectively, in all cell lines with the exception of the only p53-mutated one. Similar effects were obtained by activating R2 subunits with other analogues and by silencing R1A expression. The antiproliferative and proapoptotic effects of 8-Cl-cAMP were comparable to those observed with commonly used antitumoral drugs. Moreover, 8-Cl-cAMP potentiated the effects of these drugs on both cell proliferation and caspase-3 activity. In conclusion, this study first reports that human melanomas are characterized by a high R1/R2 ratio and that pharmacological and genetic manipulations able to revert this unbalanced expression cause significant antiproliferative and proapoptotic effects in melanoma cells.

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“…Known to be early events and present in benign nevi (Pollock et al, 2003), BRAF mutations may influence tumor progression and give rise to distinct gene expression signatures in melanoma cell lines (Pavey et al, 2004). Gain of chromosome 7q is common in CMM suggesting that BRAF, located on 7q34, is a target for gene amplification (Tanami et al, 2004). Moreover, cyclin D1, a down-stream target of the MAPK pathway and p16INK4A antagonist, is amplified in acral-type CMM in which BRAF and NRAS mutations are infrequent (Takata et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Genomic profiling of malignant melanoma using tiling-resolution arrayCGH

et al. 2007

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Malignant melanoma is an aggressive, heterogeneous disease where new biomarkers for diagnosis and clinical outcome are needed. We searched for chromosomal aberrations that characterize its pathogenesis using 47 different melanoma cell lines and tiling-resolution bacterial artificial chromosome-arrays for comparative genomic hybridization. Major melanoma genes, including BRAF, NRAS, CDKN2A, TP53, CTNNB1, CDK4 and PTEN, were examined for mutations. Distinct copy number alterations were detected, including loss or gain of whole chromosomes but also minute amplifications and homozygous deletions. Most common overlapping regions with losses were mapped to 9p24.3-q13, 10 and 11q14.1-qter, whereas copy number gains were most frequent on chromosomes 1q, 7, 17q and 20q. Amplifications were delineated to oncogenes such as MITF (3p14), CCND1 (11q13), MDM2 (12q15), CCNE1 (19q12) and NOTCH2 (1p12). Frequent findings of homozygous deletions on 9p21 and 10q23 confirmed the importance of CDKN2A and PTEN. Pair-wise comparisons revealed distinct sets of alterations, for example, mutually exclusive mutations in BRAF and NRAS, mutual mutations in BRAF and PTEN, concomitant chromosome 7 gain and 10 loss and concomitant chromosome 15q22.2-q26.3 gain and 20 gain. Moreover, alterations of the various melanoma genes were associated with distinct chromosomal imbalances suggestive of specific genomic programs in melanoma development.

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Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines

Cited by 88 publications

References 28 publications

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells

Genomic profiling of malignant melanoma using tiling-resolution arrayCGH

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