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Constitutive Activation of the Mitogen-Activated Protein Kinase Signaling Pathway in Acral Melanomas

2005; Elsevier BV; Volume: 125; Issue: 2 Linguagem: Inglês

10.1111/j.0022-202x.2005.23812.x

1523-1747

Minoru Takata, Yasufumi Goto, Nami Ichii, Maki Yamaura, Hiroshi Murata, Hiroshi Koga, Akihide Fujimoto, Toshiaki Saida,

melanin and skin pigmentation

Journal of General Internal MedicineVolume 20, Issue 5 p. 318-322 Free Access Constitutive Activation of the Mitogen-Activated Protein Kinase Signaling Pathway in Acral Melanomas Minoru Takata, Minoru Takata Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorYasufumi Goto, Yasufumi Goto Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan 1Present address: Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, California, USA.Search for more papers by this authorNami Ichii, Nami Ichii Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorMaki Yamaura, Maki Yamaura Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorHiroshi Murata, Hiroshi Murata Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorHiroshi Koga, Hiroshi Koga Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorAkihide Fujimoto, Akihide Fujimoto Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, JapanSearch for more papers by this authorToshiaki Saida, Toshiaki Saida Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this author Minoru Takata, Minoru Takata Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorYasufumi Goto, Yasufumi Goto Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan 1Present address: Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, California, USA.Search for more papers by this authorNami Ichii, Nami Ichii Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorMaki Yamaura, Maki Yamaura Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorHiroshi Murata, Hiroshi Murata Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorHiroshi Koga, Hiroshi Koga Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this authorAkihide Fujimoto, Akihide Fujimoto Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, JapanSearch for more papers by this authorToshiaki Saida, Toshiaki Saida Department of Dermatology, Shinshu University School of Medicine, Matsumoto, JapanSearch for more papers by this author First published: 28 June 2008 https://doi.org/10.1111/j.0022-202X.2005.23812.xCitations: 50 Address correspondence to: Minoru Takata, MD, PhD, Department of Dermatology, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Japan. Email: mtderm@hotmail.com AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract One of the most attractive clinical targets for melanoma is the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we examined MAPK signaling activation in a total of 28 acral melanoma samples, consisting of 13 primary tumors and 15 metastases. In line with the previous reports, NRAS/BRAF mutations were rare; only one metastatic tumor had an NRAS E61R mutation, and one primary tumor and two metastases harbored BRAF V599E mutations. Western blot analyses, however, revealed phosphorylated extracellular signal-regulated kinase (ERK)1/2 proteins in 11 of 14 (78.5%) of the acral melanoma tumors. Furthermore, fluorescence in situ hybridization analyses revealed the prominent amplification of the cyclin D1 (CCND1) gene, which is an important down-stream effecter of the MAPK pathway, in 5 of 21 (23.8%) tumors examined. Interestingly, two of three tumors that were negative for phosphorylated ERK proteins according to western blot harbored CCND1 amplifications, suggesting that the increased gene dosage of CCND1 may exert effects similar to phosphorylated ERK proteins in cell growth. We conclude that, despite the low frequency of BRAF/NRAS mutations, the MAPK signaling pathway is constitutively activated in the majority of acral melanomas. This provides a rational basis to include acral melanomas into the clinical trials with MAPK inhibitors. Abbreviations: CCND1, cyclin D1; ERK, extracellular signal-regulated kinase; FISH, fluorescence in situ hybridization; MAPK, mitogen-activated protein kinase There are great ethnic variations in the incidence and the anatomic distribution of cutaneous melanomas (Pathak et al, 1982). Light-skinned Caucasians have a high incidence of melanomas, and the majority of them occur on the trunk and extremities, suggesting intermittent sun exposure as a major pathogenetic factor (Elwood and Gallagher, 1998). According to a pathological classification of cutaneous melanomas (Clark et al, 1986), most of these melanomas are of the superficial spreading type. In contrast, in non-Caucasian populations such as the Japanese, the overall incidence of melanomas is much lower, and more than half of the melanomas are of the acral lentiginous type that develop in relatively or completely sun-protected sites such as the palms, soles, and nail beds (Ishihara et al, 2001). Although controversy remains as to whether the clinicopathologic variations among cutaneous melanomas reflect inherent biologic differences (McGovern, 1982), recent molecular cytogenetic studies have shown clear differences in the genetic make-up among the different subtypes of melanomas (Bastian et al, 2000, 2003). This may have significant clinical relevance, since potential therapeutic targets might therefore vary among the different melanoma types. One of the most attractive clinical targets for melanoma is the RAS–RAF-mitogen-activated protein kinase (MAPK) signaling pathway (Sebolt-Leopold and Herrera, 2004). The MAPK pathway plays a central role in regulating the growth and survival of cells from a broad spectrum of human tumors (Johnson and Lapadat, 2002) including melanoma (Cohen et al, 2002; Govindarajan et al, 2003). In most Caucasian melanomas, extracellular signal-regulated kinase (ERK)1 and ERK2, downstream effectors of this pathway, were reported to be constitutively activated (Satyamoorthy et al, 2003). The high constitutive ERK activity in melanoma is most likely a consequence of mutations in upstream components. Activating RAS mutations, most of which are at codon 61 of NRAS, have been identified in 9%–15% of melanomas (Albino et al, 1989; van 't Veer et al, 1989; Ball et al, 1994; van Elsas et al, 1996). Furthermore, one of the RAF-family proteins, BRAF, is mutated in 60%–66% of melanomas, with a single substitution (T→A) of glutamate for valine (V599E), being responsible for 92% of the observed mutations (Davies et al, 2002). The incidence of NRAS/BRAF mutations, however, is heavily dependent on the sun exposure pattern of the primary tumor sites. NRAS mutations were most frequently found in tumors from chronically sun-exposed sites, whereas the highest BRAF mutation rates were reported in intermittently sun-exposed sites (van Elsas et al, 1996; Jiveskog et al, 1998; Maldonado et al, 2003). By contrast, both RAS and BRAF mutations were rare in melanomas on glabrous skin and mucous membranes that are unexposed to sun (van Elsas et al, 1996; Jiveskog et al, 1998; Maldonado et al, 2003; Edwards et al, 2004). Thus, whether or not these types of melanomas arising in sun-protected sites show constitutive activation of the MAPK pathway is not known and needs to be investigated. This is important because small-molecule inhibitors such as BAY43-9006 designed to target the MAPK pathway are now available and have entered clinical trials targeting metastatic melanomas (Sebolt-Leopold and Herrera, 2004). This study was aimed at investigating NRAS/BRAF mutations and the MAPK signaling pathway activation in a series of 28 Japanese acral melanomas. We also examined the amplifications of the cyclin D1 gene (CCND1), which is an important down-stream effecter of the MAPK pathway and was reported to be amplified in nearly half of primary acral melanomas (Sauter et al, 2002). Results and Discussion In a total of 28 acral melanoma samples consisting of 13 primary tumors and 15 metastases, one primary tumor and two metastases harbored BRAF V599E mutations (Table I). Although a minor proportion of BRAF mutations other than V599E were detected in exons 11 and 15 (Brose et al, 2002; Davies et al, 2002), we did not detect such mutations. The overall frequency of BRAF mutations in our acral melanoma samples was 10.7%, which is similar to the 15% (6 of 39) incidence of primary acral melanomas reported previously (Maldonado et al, 2003). Although a much higher incidence of BRAF mutations was reported in metastatic than in primary lesions (Houben et al, 2004; Shinozaki et al, 2004), only two of 15 (13.3%) acral melanoma metastases had mutations. NRAS mutations were also rare; a CAA (Glu) to CGA (Arg) mutation at codon 61 was detected in only one metastasis (Table I). The result is consistent with the previous reports showing rare NRAS mutations in melanomas arising from the mucous membrane or unexposed skin (van Elsas et al, 1996; Jiveskog et al, 1998). Table I. NRAS/BRAF mutations, ERK phosphorylation, and CCND1 amplification in acral melanomas Case no. Sex Age Primary site Tumor sample NRAS BRAF Phospho-ERK CCND1 amplification Western blot Immunohistochemistry 1 M 51 Sole Primary Wild type Wild type + − − 2 M 82 Sole Primary Wild type Wild type − − + 3 M 81 Finger nail Primary Wild type Wild type − − − 4 F 61 Toe Primary Wild type V599E + − − 5 M 69 Sole Primary Wild type Wild type NE − + 6 F 38 Finger nail Primary Wild type Wild type NE − − 7 F 74 Sole Primary Wild type Wild type NE − + 8 F 86 Sole Primary Wild type Wild type NE − NE 9 M 77 Finger Primary Wild type Wild type NE + NE 10 M 75 Toe Primary Wild type Wild type NE − − 11 F 78 Sole Primary Wild type Wild type NE − − 12 F 80 Toe nail Primary Wild type Wild type NE − − 13 M 72 Sole Primary Wild type Wild type NE + NE 14 M 79 Palm Metastasis Wild type Wild type + − − 15 M 80 Sole Metastasis E61R Wild type + + − 16 F 69 Sole Metastasis Wild type Wild type + + − 17 F 75 Finger Metastasis Wild type Wild type + + NE 18 M 57 Sole Metastasis Wild type Wild type + + − 19 M 35 Sole Metastasis Wild type Wild type − − + 20 M 74 Finger nail Metastasis Wild type Wild type + − + 21 F 76 Sole Metastasis Wild type Wild type + − − 22 F 81 Toe Metastasis Wild type Wild type + + − 23 M 77 Sole Metastasis Wild type Wild type + − − 24 M 75 Sole Metastasis Wild type V599E NE + NE 25 F 44 Sole Metastasis Wild type V599E NE − − 26 M 73 Sole Metastasis Wild type Wild type NE − NE 27 M 77 Sole Metastasis Wild type Wild type NE + − 28 F 76 Finger Metastasis Wild type Wild type NE − NE NE, not examined; ERK, extracellular signal-regulated kinase; CCND1, cyclin D1. Despite the low frequency of NRAS/BRAF mutations, western blot analyses revealed phosphorylated-ERK1/2 proteins in 11/14 (78.5%) of the acral melanoma tumors (Table I and Fig 1). This result indicates that the MAPK signaling pathway is constitutively activated in the majority of acral melanomas without NRAS/BRAF mutations. Similar results have already been reported in uveal melanomas. Uveal melanomas had neither NRAS nor BRAF mutations, but constitutive activation of the MAPK pathway was, respectively, demonstrated in 50% (20 of 40) and 86% (36 of 42) of samples with western blot (Rimoldi et al, 2003) and immunohistochemistry (Weber et al, 2003), suggesting that neither NRAS nor BRAF mutations may be a prerequisite for the activation of the MAPK signaling pathway. Several alternative ways of activation of MAPK signaling have recently been demonstrated, including autocrine growth factor stimulation (Satyamoorthy et al, 2003), epigenetic inactivation of RAS association domain family protein 1 (RASSF1A) (Spugnardi et al, 2003), downregulation of an ERK signaling inhibitor SPRY2 (Tsavachidou et al, 2004), loss or reduction of RAF kinase inhibitory protein (PKIP) (Schuierer et al, 2004), and overexpression of wild-type BRAF in part as a result of gene amplification (Tanami et al, 2004). Specific pathways leading to constitutive activation of ERK without BRAF mutations in acral melanomas remain to be established. Figure 1Open in figure viewerPowerPoint Analysis of the status of mitogen-activated protein kinase (MAPK) signaling pathway proteins in acral melanoma samples in vivo. Protein extracts from the indicated tumor samples were analyzed by western blotting using anti-p42/p44 MAPK (extracellular signal-regulated kinase (ERK)1/2) and anti-phosphorylated p42/p44 MAPK (phosphorylated ERK1/2) antibodies. β-actin serves as loading control. Phosphorylated-ERK1/2 proteins were detected in 11 of 14 samples (78.5%). It was noteworthy that there was a marked discrepancy in the results detecting phosphorylated-ERK proteins between the western blot and immunohistochemistry (Table I). Six of 11 tissues containing phosphorylated-ERK proteins demonstrated by western blotting failed to exhibit immunoreactivity in the immunohistochemistry (Fig 2). The most common problem encountered in the application of phosphorylation-specific antibodies to fixed tissue is a false-negative reaction. This can be because of the inaccessibility of the antigen to antibody, although we performed antigen retrieval as recommended by the manufacturer. Another possibility is the loss of phosphorylation sites during fixation (Mandell, 2003). Our results raise the possible objections to the use of immunohistochemistry for the detection of phosphorylated proteins in vivo unless appropriate positive and negative controls are included. Figure 2Open in figure viewerPowerPoint Immunohistochemical staining for phosphorylated-p44/42 mitogen-activated protein kinase (MAPK) proteins in acral melanomas. Paraffin-embedded tissue sections were stained with phospho-p44/42 MAPK antibody. (A) Nuclear and cytoplasmic expression of phosphorylated-p44/42 MAPK in case 17. (B) No staining in the case 14 tumor, although western blot detected the expression of phosphorylated-p44/42 MAPK in this tumor. Scale bar: 50 μm. In three of 14 frozen samples, western blotting did not reveal phosphorylated-ERK proteins, suggesting that constitutive activation of the MAPK pathway is not taking place in these tumors (Table I and Fig 1). Interestingly, fluorescence in situ hybridization (FISH) analyses revealed the prominent amplification of CCND1 in two of three phosphorylated-ERK-negative cases (cases 2 and 19) (Fig 3). A primary tumor of case 5 also showed CCND1 amplification. Although in vivo tissue sample for western blotting was not available in this case, we established a cell line (SMYM-PRGP) from this primary tumor. SMYM-PRGP also harbors CCND1 amplification, and shows no expression of phosphorylated-ERK proteins by western blotting (H. Murata and M. Takata, unpublished observations). Thus, most of the acral melanoma tumors that lacked any evidence of constitutive MAPK pathway activation seemed to harbor CCND1 amplifications. Cyclin D1 protein is one of the important down-stream effectors of the MAPK pathway, and the CCND1 promoter acts as a sensor for growth signals conveyed via the MAPK cascade (Sherr, 2000). The importance of cyclin D1 in the cell growth of melanomas was demonstrated using anti-sense treatment targeted at cyclin D1 in melanoma cell lines (Sauter et al, 2002). It is possible, therefore, that the increased gene dosage of CCND1 may exert effects similar to phosphoryled-ERK proteins and promote the growth of melanoma cells in these cases. Figure 3Open in figure viewerPowerPoint Fluorescence in situ hybridization (FISH) showing clustered cyclin D1 (CCND1) amplification in a phosphorylated-p44/42 mitogen-activated protein kinase (MAPK)-negative acral melanoma metastasis (A) and no CCND1 amplification in a phosphorylated-p44/42 MAPK-positive metastasis (B). Dual-color FISH was performed on paraffinsections. Red signals, CCND1 probe; green signals, CEP 11 probe. (A) Case 19; (B) case 22. Because of the high incidence of NRAS/BRAF oncogenic mutations in melanomas in the Caucasian population, clinical trails with MAPK-pathway inhibitors for metastatic melanoma have already started. A favorable response was seen in a phase I trial of the RAF inhibitor BAY43-9006 against melanoma in combination with paclitaxel or carboplatin (Sebolt-Leopold and Herrera, 2004). The results from this study, showing a high frequency of MAPK-pathway activations in acral melanomas, provide a rational basis to include this type of melanoma in such clinical trails with MAPK inhibitors. Materials and Methods Tissues A total of 28 cases of acral melanoma (13 primary tumors and 15 metastases) were included in this study (Table I). According to their histology, all the primary tumors were of the acral lentiginous type. Paraffin-embedded tissues of these cases were retrieved from the archives of the Department of Dermatology, Shinshu University Hospital, Matsumoto, Japan, and the Department of Dermatology, Kanazawa University Hospital, Kanazawa, Japan. Fresh-frozen tissues were also available for 14 tumors. This study was conducted according to the Declaration of Helsinki Principles, and written informed consents were obtained from the patients. The medical ethical committee of the Shinshu University School of Medicine approved this study. Mutation analyses DNA was extracted from 10 μm paraffin-embedded tissue sections using a MagneSil Genomic, Fixed Tissue System (Promega, Madison, Wisconsin). To detect the NRAS/BRAF mutations, NRAS exon 2 and BRAF exons 11 and 15 were PCR amplified as described previously (Davies et al, 2002; Cruz et al, 2003). Because of the frequent dropouts not yielding desired PCR products, we also used the nested-PCR approach for the amplification of NRAS exon 2 (Houben et al, 2004). PCR amplicons were purified with QIAquick (Qiagen, Valencia, California), and directly sequenced using Big Dye Terminator sequencing chemistry (Applied Biosystems, Foster City, California) and an ABI automated sequencer (Applied Biosystems). When a low mutant peak was observed overlapping with a wild-type peak at nucleotide positions of the reported mutation sites, the samples were also sequenced in reverse direction to confirm the presence or absence of the mutation. Western blotting Fresh-frozen tumor samples were lysed in a Tris-HCl buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany), NP-40, Na3VO4, dithiothreitol, EGTA, NaF, and β-glycerophosphate (Wako Kagaku Industry, Osaka, Japan). Protein concentrations were determined by Coomassie brilliant blue staining. Lysates were subjected to SDS-PAGE under non-reducing conditions. The separated proteins were transferred on to a PVDF membrane (Bio-Rad Laboratories, Hercules, California). We detected the phosphorylated-p44/42 MAPK protein and p44/42 MAPK protein using a PhosphoPlus p44/42 Kinase Antibody Kit (Cell Signaling Technology, Beverly, Massachusetts). Mouse monoclonal anti-β-actin antibody (Abcam, Cambridge, Massachusetts) was used as loading control. Immunoreactivity was detected using a FluorImager 575 (Bio-Rad Laboratories). Immunohistochemistry Four micron thick paraffin-embedded tissue sections were stained with phospho-p44/42 MAP kinase (Thy202/Try204) antibody #9101 using a SignalStain Phospho-p44/42 IHC Detection Kit (Cell Signaling Technology). Prior to staining, antigen unmasking was performed by boiling the slides for 10 min in 0.01 M sodium citrate buffer (pH 6.0). Pre-diluted negative control was included in the kit. Nuclear staining in >10% of tumor cells was regarded as positive. FISH Amplification of the CCND1 was examined in the paraffin-embedded tissue sections with a dual-color FISH technique. A dual-color probe mixture consisting of a Spectrum Orange LSI Cyclin D1 probe (band region 11q13) and Spectrum Green CEP 11 (band region 11p–11.11–q11, locus D11Z1) was purchased from Vysis (Downers Grove, Illinois). The staining was performed following the manufacturer's protocol. Briefly, paraffin-embedded tissues were cut into 4 μm sections and mounted on silanized slides (DAKO, Carpinteria, California). After deparaffinization, pre-treatment was performed using a Paraffin Pretreatment Reagent Kit (Vysis). Hybridization was carried out for 15 h at 37°C, and then slides were washed once in post-hybridization washing buffer (2 × SSC, 0.3% NP-40) at room temperature, followed by a rinse in post-hybridization washing buffer at 72°C for 2 min. Sections were then counterstained with 4,6-diamino-2-phenylindole (Vysis). FISH signals were scored with a fluorescent microscope (Carl Zeiss, OberKochen, Germany) equipped with a triple band-pass filter. Three-color images were captured by using a digital imaging analysis system. Copy numbers of CEP11 (green) and CD1 signals (orange) were, respectively, counted for more than 50 non-overlapping tumor cells. 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