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Loitering with Intent: New Evidence for the Role of BRAF Mutations in the Proliferation of Melanocytic Lesions

2004; Elsevier BV; Volume: 123; Issue: 4 Linguagem: Inglês

10.1111/j.0022-202x.2004.23430.x

1523-1747

Keiran S.M. Smalley, Meenhard Herlyn,

Computational Drug Discovery Methods

The discovery that the somatic activating missense mutation T1796A, leading to the substitution of glutamate to valine (V600E—previously known as V599E 1The change of designation from V599E to V600E arose from complications in sequencing the GC-rich exon 1 of the BRAF gene. On the July 24, 2003, the sequence was updated, following the insertion of 3 bp into the coding sequence, resulting in the increase of the length of the BRAF protein by one amino acid. This therefore increased the position of all published mutations by one amino acid and hence V599E became V600E (Richard Wooster, personal communication). More details can be found at http://www.sanger.ac.uk/genetics/CGP/cosmic/News/#200402200928051The change of designation from V599E to V600E arose from complications in sequencing the GC-rich exon 1 of the BRAF gene. On the July 24, 2003, the sequence was updated, following the insertion of 3 bp into the coding sequence, resulting in the increase of the length of the BRAF protein by one amino acid. This therefore increased the position of all published mutations by one amino acid and hence V599E became V600E (Richard Wooster, personal communication). More details can be found at http://www.sanger.ac.uk/genetics/CGP/cosmic/News/#20040220092805) in the kinase domain of BRAF, was found in 66% of human melanomas (Davies et al., 2002Davies H. Bignell G.R. Cox C. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954https://doi.org/10.1038/nature00766Crossref PubMed Scopus (8270) Google Scholar) has prompted much excitement in the melanoma community. Since this time there has been a great flurry of papers attempting to determine what this mutation means, both in terms of clinical significance, as well as the biochemical behavior of melanoma cells. As yet, we only have part of the answer, and it seems that BRAF, although important to this disease, is probably part of a more complex story. In biochemical terms, the effects of aberrant RAF activation in cancer are well characterized. BRAF is a key player in the Ras/Raf/MAP/ERK Kinase (MEK)/Extracellular Signal Related Kinase (ERK) pathway—a mitogen-activated protein (MAP) kinase cascade, whose activation is implicated in a host of cancers through its myriad effects upon cell growth, invasion, and survival (reviewed more extensively inSmalley, 2003Smalley K.S.M. A pivotal role for ERK in malignant melanoma?.Int J Cancer. 2003; 104: 527-532https://doi.org/10.1002/ijc.10978Crossref PubMed Scopus (299) Google Scholar). Recent structural biology studies have shown that the V600E mutation, which accounts for 80% of the reported mutations (Davies et al., 2002Davies H. Bignell G.R. Cox C. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954https://doi.org/10.1038/nature00766Crossref PubMed Scopus (8270) Google Scholar), works by destabilizing the inactive conformation of BRAF, shifting the equilibrium toward the active form of the kinase (Wan et al., 2004Wan P.T. Garnett M.J. Roe S.M. et al.Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.Cell. 2004; 116: 855-867Abstract Full Text Full Text PDF PubMed Scopus (2227) Google Scholar). Once active, BRAF activates MEK, which in turn activates ERK. The effects of increased ERK activity upon melanoma behavior are well known, and include enhanced cell proliferation, altered integrin expression, decreased E-cadherin expression, increased matrix metalloproteinase (MMP) secretion, invasion, and the regulation of the critical melanocyte transcription factor microphthalmia (Mitf) (reviewed inSmalley, 2003Smalley K.S.M. A pivotal role for ERK in malignant melanoma?.Int J Cancer. 2003; 104: 527-532https://doi.org/10.1002/ijc.10978Crossref PubMed Scopus (299) Google Scholar). Proof-of-principle in vitro studies demonstrated both that BRAF is an oncogene in immortalized mouse melanocytes (Wellbrock et al., 2004Wellbrock C. Ogilvie L. Hedley D. V599E B-RAF is an oncogene in melanocytes.Cancer Res. 2004; 64: 2338-2342Crossref PubMed Scopus (310) Google Scholar) and that selective abrogation of V600E BRAF, using RNAi, leads to a complete reversal of the melanoma phenotype—accompanied by reduced MAP kinase activity, reduced growth, and apoptosis (Hingorani et al., 2003Hingorani S.R. Jacobetz M.A. Robertson G.P. Herlyn M. Tuveson D.A. Suppression of BRAF(V599E) in human melanoma abrogates transformation.Cancer Res. 2003; 63: 5198-5202PubMed Google Scholar). From the in vitro data, the BRAF story seems clear, but life is never that simple, and the in vivo data have been a little more confusing. In particular, a number of papers have shown that BRAF mutations are prevalent in unsuspicious benign nevi (although at levels which range from 21% to 80%) (Pollock et al., 2003Pollock P.M. Harper U.L. Hansen K.S. et al.High frequency of BRAF mutations in nevi.Nat Genet. 2003; 33: 19-20https://doi.org/10.1038/ng1054Crossref PubMed Scopus (1358) Google Scholar). This raises questions about whether BRAF mutations are acquired at a very early stage in the oncogenic process, or whether they are actually of pathological importance at all. The issue is further complicated by the issue of whether nevi truly constitute a pre-malignant form of melanoma. The data on this subject are far from clear. Whereas nevi are often precursors for a melanocytic malignancy, many primary melanomas can also arise de novo, and in many cases the primary melanoma is never located. It has been further shown that different nevi from the same patients can harbor different BRAF mutations (Kumar et al., 2004Kumar R. Angelini S. Snellman E. Hemminki K. BRAF mutations are common somatic events in melanocytic nevi.J Invest Dermatol. 2004; 122: 342-348Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), and that different nevi from the same patients can harbor either mutant or wild-type BRAF (Loewe et al., 2004Loewe R. Kittler H. Fischer G. Gae I. Wolff K. Petzelbauer P. BRAF kinase gene V599E mutation in growing melanocytic lesions.J Invest Dermatol. 2004; 123: 733-736Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These findings suggest that the different BRAF mutations can arise as the result of distinct somatic events within the same patient (Kumar et al., 2004Kumar R. Angelini S. Snellman E. Hemminki K. BRAF mutations are common somatic events in melanocytic nevi.J Invest Dermatol. 2004; 122: 342-348Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). A number of studies have shown that there is some link between levels of sun-exposure and BRAF mutation status. In particular, non-sun-exposed sites have a much reduced incidence of BRAF mutations compared to sites with at least intermittent sun exposure (seeMaldonado et al., 2003Maldonado J.L. Fridlyand J. Patel H. et al.Determinants of BRAF mutations in primary melanomas.J Natl Cancer Inst. 2003; 95: 1878-1890Crossref PubMed Scopus (531) Google Scholar). It was also found that BRAF mutations, however, are rare in lesions on chronically sun-damaged skin, perhaps hinting that there are distinct genetic pathways that may lead to melanoma development (Maldonado et al., 2003Maldonado J.L. Fridlyand J. Patel H. et al.Determinants of BRAF mutations in primary melanomas.J Natl Cancer Inst. 2003; 95: 1878-1890Crossref PubMed Scopus (531) Google Scholar). A new study in this issue of the JID helps to shed more light on the importance of BRAF in melanoma pathogenesis (Loewe et al., 2004Loewe R. Kittler H. Fischer G. Gae I. Wolff K. Petzelbauer P. BRAF kinase gene V599E mutation in growing melanocytic lesions.J Invest Dermatol. 2004; 123: 733-736Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In this study, the authors retrospectively selected 49 lesions, which did not meet the criteria of malignant melanoma at initial presentation. These lesions were later excised after a 12-month follow-up—because of either increased size or structural changes consistent with malignancy. Samples from these lesions were then sequenced for their BRAF mutation status. The reasoning behind the current study is simple. First-line treatment of early melanoma is the complete surgical removal of the lesion. As benign lesions often display similar structural alterations and the enhanced proliferation characteristics of their malignant counterparts, large numbers of benign lesions are also removed. As yet, the causes of the rapid proliferative and structural changes in these lesions are unknown. One obvious suggestion is that these rapid changes occur following the acquisition of BRAF mutations. Indeed, the primary aim of the current study was to determine whether the BRAF V600E mutation could account for the observed rapid onset of growth in these melanocytic lesions. The results of the study were intriguing. Among growing lesions, 16 (of which 11 were melanoma and five nevi) out of 36 had the V600E (referred to as V599E in this paper) mutation and 4 out of 13 lesions (of which 3 were melanoma and 1 nevi) that had undergone a structural change had the V600E mutation. In the control group of 35 lesions that had not undergone proliferative or structural changes, only 2 had the V600E mutation (both of which were nevi). When statistically analyzed, these results showed that the odds of the presence of a BRAF mutation in rapidly growing lesions were 13 times higher than controls. The odds of finding a BRAF mutation in structurally altered lesions were 7 times higher than controls. These data demonstrate that the rapid growth of melanocytic lesions correlated with the presence of a V600E BRAF mutation. In this study, there was no correlation seen between the BRAF mutation status and the site of the original lesion (whether site of chronic sun exposure, intermittent sun exposure or non-exposure). Although the study showed that melanomas had a higher incidence of BRAF mutations than nevi, the levels of mutation were still higher in growing versus dormant nevi. These findings led the authors to ask two highly relevant questions: “Are nevi that harbor the V600E mutations the precursor lesions of melanoma?” and “Is the BRAF mutation responsible for the sudden onset of growth in melanocytic lesions?” As yet, these two questions are rather difficult to answer. The fact that rapidly growing nevi are more likely to have the V600E mutation, but dormant nevi do not, suggests there is some link between mutational status and malignancy. The assertion that the V600E mutation is contributing to enhanced nevus growth is certainly backed by the available in vitro data (Hingorani et al., 2003Hingorani S.R. Jacobetz M.A. Robertson G.P. Herlyn M. Tuveson D.A. Suppression of BRAF(V599E) in human melanoma abrogates transformation.Cancer Res. 2003; 63: 5198-5202PubMed Google Scholar;Wellbrock et al., 2004Wellbrock C. Ogilvie L. Hedley D. V599E B-RAF is an oncogene in melanocytes.Cancer Res. 2004; 64: 2338-2342Crossref PubMed Scopus (310) Google Scholar). The finding, however, that BRAF V600E is also found in 20%–80% of histologically normal nevi would dispute this (Pollock et al., 2003Pollock P.M. Harper U.L. Hansen K.S. et al.High frequency of BRAF mutations in nevi.Nat Genet. 2003; 33: 19-20https://doi.org/10.1038/ng1054Crossref PubMed Scopus (1358) Google Scholar). But again, this may not be the whole story. Primary human cells are notoriously hard to transform, and require mutational activation/inactivation in at least four cellular control circuits. Typically these can include: growth stimulation through activation of the Ras/Raf/MEK/ERK pathway, loss of growth control via the E2F/pRB pathway, loss of apoptosis through p53 inactivation/mutation, and telomere maintenance through activation of telomerase. The mutation and activation of V600E BRAF alone would be unable to transform a primary human melanocyte and would instead lead to irreversible cell-cycle arrest and senescence. Indeed, it has been suggested that the melanocytes that make up nevi are senescent. Adding weight to this argument is the fact that only melanocytes that were null for the cell cycle inhibitors p16INK4A and p19ARF were transformed by V600E BRAF in vitro (Wellbrock et al., 2004Wellbrock C. Ogilvie L. Hedley D. V599E B-RAF is an oncogene in melanocytes.Cancer Res. 2004; 64: 2338-2342Crossref PubMed Scopus (310) Google Scholar). This seems to suggest that BRAF, like other oncogenes, may also require a genetic “second hit”, possibly in the cell-cycle regulation pathway, in order to be transforming. This then begs the question, what is the genetic hit that is required for melanoma? The prime candidate for genetic inactivation in cancer is the p53 pathway, but this is known to be structurally normal in melanoma; however, it is currently unknown whether the p53 pathway is functionally active in this disease. What is more likely to be important are the other three known melanoma susceptibility genes, INK4A, CDK4, and ARF, which are all known to regulate senescence. Indeed, this idea seems to be born out by a very recent work, in which genetic analysis of 41 early passage melanoma biopsy cell lines revealed that p16/ARF loss accompanied by the V600E BRAF mutation was the most prevalent genetic profile (Daniotti et al., 2004Daniotti M. Oggionni M. Ranzani T. et al.BRAF alterations are associated with complex mutational profiles in malignant melanoma.Oncogene. 2004Crossref Scopus (166) Google Scholar). The importance of BRAF mutations in melanoma progression is undoubted; very recent studies have also shown that there is a definite link between BRAF mutational status and clinical outcome, with BRAF mutations in melanoma metastases, but not the primary tumor, being correlated with poor outcome (Houben et al., 2004Houben R. Becker J.C. Kappel A. et al.Constitutive activation of the Ras-Raf signaling pathway in metastatic melanoma is associated with poor prognosis.J Carcinog. 2004; 3: 6https://doi.org/10.1186/1477-3163-3-6Crossref PubMed Scopus (255) Google Scholar). Preliminary results from clinical trials suggest that RAF-kinase inhibitors, such as BAY 43-9006, may be of clinical benefit to melanoma patients, particularly when used in combination with other drugs. It now seems that the tide is finally turning in the treatment of melanoma. Understanding more about the role of BRAF mutations in this disease will play an important role in guiding our future understanding of the underlying pathology and may lead to much enhanced treatment for this currently deadly disease.