Genetic Interaction Between NRAS and BRAF Mutations and PTEN/MMAC1 Inactivation in Melanoma

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1 Genetic Interaction Between NRAS and BRAF Mutations and PTEN/MMAC1 Inactivation in Melanoma Hensin Tsao, 1 Vikas Goel, 1 Heng Wu, Guang Yang, and Frank G. Haluska Wellman Laboratories, Department of Dermatology, Division of Oncology, Department of Medicine, Massachusetts General Hospital Melanoma Center, Massachusetts General Hospital, Boston, Massachusetts, USA Extant evidence implicates growth factor signaling in the pathogenesis of many tumor types, including cutaneous melanoma. Recently, reciprocal activating mutations of NRAS and BRAF were found in benign melanocytic nevi and cutaneous melanomas. We had previously reported a similar epistatic relationship between activating NRAS mutations and inactivating PTEN/MMAC1 alterations. We thus hypothesized that BRAF and PTEN/MMAC1 mutations may cooperate to promote melanoma tumorigenesis. Overall, 40 of 47 (85%) melanoma cell lines and 11 of 16 (69%) uncultured melanoma metastases had mutations in NRAS, BRAF, or PTEN/MMAC1. NRAS was exclusively mutated in nine of 47 (19%) cell lines and two of 16 (13%) metastases, whereas BRAF was solely mutated in 28 of 47 (60%) cell lines and nine of 16 (56%) metastases. In the 12 of 15 melanoma cell lines (80%) and two of two melanoma metastases with PTEN alterations, BRAF was also mutated. These findings suggest the existence of possible cooperation between BRAF activation and PTEN loss in melanoma development. Key words: BRAF/melanoma/NRAS/PTEN. J Invest Dermatol 122: , 2004 Sustained growth factor signaling is a critical step in the evolution of cutaneous melanoma. In vitro, basic fibroblast growth factor can form an autocrine stimulatory loop (Meier et al, 2000; Graeven et al, 2001), whereas in vivo, transgenic expression of the receptor tyrosine kinase, RET (Iwamoto et al, 1991; Kato et al, 1998), leads to melanocytic tumors. Post-receptor, activating RAS mutations have also been reported in a significant proportion of cutaneous melanomas (Herlyn and Satyamoorthy, 1996; van Elsas et al, 1996). Two distinct RAS signaling streams the RAS/RAF/MAPK and the RAS/PI3-K/PTEN/AKT pathways have both been shown to be activated in primary melanomas (Cohen et al, 2002; Dhawan et al, 2002; Govindarajan et al, 2003; Satyamoorthy et al, 2003). Moreover, BRAF, which is a component of the RAS/RAF/MAPK signaling cassette, was recently found to be mutated in 60% to 80% of cutaneous melanomas and benign melanocytic nevi (Davies et al, 2002; Pollock et al, 2003). Taken together, the RAS signaling network represents a rich source of oncogenic events. We had previously demonstrated a reciprocal relationship between NRAS activation and PTEN/MMAC1 inactivation in melanoma cell lines and uncultured specimens (Tsao et al, 2000). As RAS directly stimulates PI3-K (Rodriguez-Viciana et al, 1996, 1997) and PTEN attenuates PI3-K signaling (Maehama and Dixon, 1998; Wu et al, 1998), reciprocal NRAS and PTEN/MMAC1 alterations may contribute to the heightened AKT activity observed in melanomas (Dhawan et al, 2002). As loss of PTEN protein expression occurs in 63% of primary melanomas and only 8% of nevi (Tsao et al, 2003), 1 These authors contributed equally to the work. inactivation of PTEN/MMAC1 may be a feature of progression from melanocytic nevi to primary melanoma. Likewise, a similar epistatic relationship between oncogenic alleles of NRAS and BRAF has also been described (Davies et al, 2002; Pollock et al, 2003). As RAS activates RAF leading to MAPK stimulation (Campbell et al, 1998), reciprocal NRAS and BRAF mutations may account for the increased MAPK activity reported for melanoma (Cohen et al, 2002; Govindarajan et al, 2003; Satyamoorthy et al, 2003). The genetic relationship described for NRAS, BRAF, and PTEN/MMAC1 raises the possibility that BRAF activation and PTEN/MMAC1 inactivation cooperate to simulate NRAS activation in order to promote melanoma tumorigenesis. To test this hypothesis directly, we screened for BRAF mutations in a series of melanoma samples whose NRAS and PTEN/MMAC1 had been previously characterized and found evidence for possible cooperation between BRAF and PTEN/MMAC1. Results and Discussion Using PCR single strand conformation polymorphism, we evaluated 47 melanoma cell lines and 16 uncultured metastatic melanoma specimens for mutations in exons 11 and 15 of BRAF. Whereas no exon 11 amplicons exhibited altered migration, multiple exon 15 amplicons demonstrated mobility shifts suggestive of sequence variants (Fig 1a). After direct sequencing, we found that 28 of 47 (62%) melanoma lines harbored mutations at valine lines had the canonical T1796A (BRAF V599E ) mutation, whereas two lines harbored the TG AT Copyright r 2004 by The Society for Investigative Dermatology, Inc. 337

2 338 TSAO ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 1 (A) PCR single strand conformation polymorphism. Analysis of BRAF exon 15. Amplified exon 15 fragments containing the normal (V599) and mutant (E599) variants exhibit differential mobilities, as indicated by the upper and lower arrows, respectively. The asterisk marks the presence of the mutant allele. Note that the cell line in lane 3 is homozygous for the E599 mutation (lane 1: Mel-Swift; lane 2: MGH-BO-1; lane 3: SK-Mel 28; lane 4: WM455; lane 5: SK-Mel 63). (B) BRAF genotypes showing loss of normal allele at codon 599. (BRAF V599D ) mutation. The BRAF V599E mutation was also present in nine of 16 (56%) uncultured metastatic melanoma specimens. In three of 28 melanoma cell lines with BRAF V599 mutations, the normal BRAF allele was deleted (Fig 1a,b). Although loss of heterozygosity is typically a signature of a tumor suppressor locus, shedding of the wild-type allele has been described for NRAS (Osaka et al, 1997), KRAS (Sukumar et al, 1991), and HRAS (Saranath et al, 1991). As the BRAF V599 alterations are presumably gain-of-function mutations, the normal allele would represent a hypofunctional competitor whose loss could potentiate the effectiveness of the BRAF V599 variant. Alternatively, Diaz et al (2002), recently found that the presence of a wild-type NRAS allele impedes the formation of murine lymphomas in vivo and suppresses the malignant phenotype in vitro; thus, it is conceivable that the BRAF proto-oncogene may exhibit tumor suppressive properties under certain biologic contexts. The NRAS, BRAF, and PTEN/MMAC1 status for the various cell lines and specimens are shown in Tables I and II. Overall, 40 of 47 (85%) melanoma cell lines and 11 of 16 (69%) uncultured melanoma metastases had mutations in at least one of the three loci. Several observations emerged from our analysis. First, NRAS was solely mutated in nine melanoma cell lines and two uncultured melanoma specimens, although there was one additional cell line that exhibited concurrent NRAS and PTEN/MMAC1 alterations (i.e., HS944; Tsao et al, 2000). Second, 15 cell lines and two uncultured metastases exhibited either PTEN/MMAC1 mutations (11 lines, both metastases) or loss of protein expression (four lines, Table I); epigenetic silencing of PTEN/ MMAC1 has been reported in cutaneous melanoma (Zhou et al, 2000). Of the 63 samples analyzed, BRAF was mutated in 12 of 15 (80%) and two of two (100%) of the PTEN-deficient lines and metastatic specimens, respectively (p ¼ 0.02, two-tailed Fisher exact test). An additional 16 melanoma lines and seven uncultured metastases displayed BRAF mutations without either NRAS or PTEN/ MMAC1 participation. Finally, two melanoma lines showed PTEN/MMAC1 inactivation without BRAF involvement. We report, for the first time that, in a subset of melanomas, BRAF activation accompanies PTEN/MMAC1 inactivation to the mutual exclusion of NRAS mutations. These results suggest that (1) BRAF and PTEN/MMAC1 operate on distinct genetic pathways and could cooperate to promote melanoma tumorigenesis, and (2) a single NRAS mutation, albeit not as common as alterations in either BRAF or PTEN/MMAC1, may be sufficient (model shown in Fig 2). As BRAF and PTEN selectively impact the MAPK and AKT pathways, respectively, activation of both signaling streams may be required for melanoma progression. Cohen et al (2002), found that over 80% of primary cutaneous melanomas, but only 20% of benign nevi, expressed active phosphomapk. Satyamoorthy et al (2003), also reported that vertical growth phase and metastatic melanomas, but not benign nevi, stained intensely for active phosphoerk and that the ERK phosphorylation in melanoma cell lines was abrogated by the MEK inhibitor PD With respect to the AKT pathway, Dhawan et al (2002), found increased staining for active phosphoakt in metastatic melanoma specimens but not benign nevi and observed loss of AKT phosphorylation in melanoma cell lines exposed to PI3-K inhibitors, LY and wortmannin. These data, along with our findings, point to an essential role for ongoing trophic signaling, through both MAPK and AKT pathways. The high rate of BRAF mutations in nevi (Pollock et al, 2003; Uribe et al, 2003) implies that the MAPK pathway is genetically engaged early in melanocytic tumor formation but is insufficient to induce full malignant transformation; other events are clearly necessary for progression. We recently found that 19 of 30 (63%) primary melanomas demonstrated significant decreases in PTEN levels, although only three of 39 melanocytic nevi (8%) exhibited loss of PTEN expression (Tsao et al, 2003); these results parallel other observations that active phosphoakt can be detected in melanomas but not in melanocytic nevi (Dhawan et al, 2002). Thus, unlike BRAF activation, PTEN loss appears to be more specific for melanoma tumorigenesis. Taken together, one hypothesis that emerges is that loss of PTEN cooperates with activation of BRAF in the transition from nevus to melanoma. The high BRAF mutation rate, especially in melanoma, raises the possibility of an effective anti-braf therapy. As

3 122 : 2 FEBRUARY 2004 BRAF AND PTEN/MMAC1 COOPERATE IN MELANOMA 339 Cell line Table I. BRAF, NRAS, and PTEN/MMAC1 mutations in melanoma cell lines NRAS a PTEN/MMAC1 BRAF exon 15 Mutation Effect Mutation Effect Mutation Effect MGH-MC-2 G35A G12D wt WT wt WT Mel-Swift C180A Q61K wt WT wt WT MGH-PO-1 C180A Q61K wt WT wt WT SK Mel-l19 A181G Q61R wt WT wt WT SK Mel-30 C180A Q61K wt WT wt WT Mel Juso A181T Q61L wt WT wt WT K19 C180A Q61K wt WT wt WT HS940 A181G Q61R wt WT wt WT SK Mel 63 C180A Q61K wt WT wt WT HS944 C180A Q61K Del exon2 Frameshift/premature stop wt WT SK Mel 39 wt WT 546insA Frameshift/premature stop T1796A V599E MGH-BO-1 wt WT Del exon2 Frameshift/premature stop T1796A V599E Sk Mel37 wt WT Del exon 2 Frameshift/premature stop T1796A V599E UACC 903 wt WT T226G Tyr 76 Stop T1796A V599E RU wt WT IVS3del þ 1! þ4 Possible splice variant T1796A V599E MM455 wt WT Del exon 6 Frameshift/premature stop T1796A V599E SK Mel 28 wt WT A499G T167A T1796A b 599E SK Mel 131 wt WT IVS5 þ 2T! A Possible splice variant T1796A V599E MEL-11 wt WT wt No protein c T1796A V599E WM1158 wt WT wt No protein c T1796A V599E WM239A wt WT wt No protein c TG AT V599D WM1799 wt WT Failed PCR No protein c T1796A V599E SK Mel 23 wt WT Del gene No predicted product wt WT ML wt WT Del gene No predicted product wt WT MGH-MC-1 wt WT wt WT T1796A V599E K1-Mel wt WT wt WT T1796A V599E MEL-31 wt WT wt WT T1796A V599E WM164 wt WT wt WT T1796A V599E MH-12 wt WT wt WT TG AT V599D MH-2 wt WT wt WT T1796A V599E K-16 wt WT wt WT T1796A V599E A375 wt WT wt WT T1796A b 599E HS939T wt WT wt WT T1796A V599E MH-17 wt WT wt WT T1796A V599E Malme wt WT wt WT T1796A V599E K4 wt WT wt WT T1796A V599E Steele wt WT wt WT T1796A V599E K2 wt WT wt WT T1796A V599E WM115 wt WT wt WT T1796A V599E MM608 wt WT wt WT T1796A b 599E a NRAS, PTEN/MMAC1 status on some cell lines published in Tsao et al (2000). b Both alleles show 599E. c No protein by western.

4 340 TSAO ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Table II. BRAF, NRAS, and PTEN/MMAC1 mutations a in melanoma metastases NRAS PTEN/MMAC1 BRAF Exon 15 Cutaneous melanoma specimens Mutation Effect Mutation Effect Mutation Effect 30 A181G Q61R wt WT wt WT 7 A181G Q61R wt WT wt WT 31 (KM16) wt WT ROH HD T1796A V599E 33 (KM17) wt WT 1591 Dup Frameshift T1796A V599E (nt ) 271 stop 6 wt WT wt WT T1796A V599E 8 wt WT wt WT T1796A V599E 16 wt WT wt WT T1796A V599E 22 wt WT wt WT T1796A V599E 24 wt WT wt WT T1796A V599E 25 wt WT wt WT T1796A V599E 28 wt WT wt WT T1796A V599E 4 wt WT wt WT wt WT 15 wt WT wt WT wt WT 12 wt WT wt WT wt WT 9 wt WT wt WT wt WT 10 wt WT wt WT wt WT a PTEN/MMAC1 andnras genetic status reported in Tsao et al (1998, 2000). ROH: retention of heterozygosity. our data document a significant rate of concurrent mutations in the RAS/PI3-VK/PTEN/AKT pathway, monotherapy targeted at BRAF alone may be ineffectual. Further studies will obviously be necessary in order to optimize rationale drug design. In summary, we have identified a potential genetic interaction between three components of the RAS signaling network NRAS, BRAF, and PTEN/MMAC1. Based on the known cellular functions of these respective gene products, the pattern of mutations suggest that the MAPK and AKT pathways are frequently activated in parallel, by genetic means, to promote melanoma development. Materials and Methods Cell lines and DNA The human melanoma cell lines, culture conditions, uncultured melanoma specimens, and their NRAS and PTEN/MMAC1 status have been described previously (Tsao et al, 2000). All studies were done in accordance with a protocol approved by the IRB at the Massachusetts General Hospital. Polymerase chain reaction (PCR) single strand conformation polymorphism Primer sequences for the exons 11 and 15 of BRAF are published (Davies et al, 2002). Amplification was carried out in 50 ml reaction containing 1 ml of DNA, 1.5 mm MgCl 2 and 40 ng of each primer under standard conditions with or without 0.25 ml of[a 32 P]deoxycytidine triphosphate (NEN, Boston, Massachusetts). The samples were initially denatured at 951C 5 min, than amplified for 35 cycles of 951C 1 min, 621C 1.5 min, 721C 1.5 min, and final extension at 721C for 10 min. The PCR product was confirmed on 1.2% agarose gel. Ten microliters of PCR product was then mixed with 10 ml of the gel loading buffer (95% formamide, 10 mm EDTA, 0.02% bromophenol blue and 0.02% xylene cyanol FF) and the samples were denatured at 951C for 5 min and chilled immediately for 5 min. The sample was loaded on to 6% acrylamide gel containing 10% glycerol, 1 TBE ph 8.0. The gel was run in 0.6 TBE at 5 6 W at 41C for 5 to 6 h. The gel was either stained with a silver staining kit (DNA Silver Staining Kit, Amersham/Pharmacia, Uppsala, Sweden) or exposed to X-ray film (Biomax-MR, Kodak, Rochester, New York), if radioactivity was used. DNA fragments showing mobility shifts were than prepared by PCR under the same condition, purified using Qiaquick PCR purification kit per manufacturer s protocol (Qiagen Inc., Valencia, California) and submitted to Massachusetts General Hospital sequencing core facility for automated sequencing. Western blotting Whole cell lysate was prepared from the melanoma cell lines using RIPA buffer (150 mm NaCl, 50 mm Tris ph 8.0, 1% nonidet P40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 5 mg per ml each of aprotinin and leupeptin and 1 mm of phenylmethylsulfonyl fluoride) containing the cocktail of protease inhibitors (Roche Molecular Biology). Fifty micrograms of protein were loaded on to sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred on to nitrocellulose paper. The blot was incubated with an anti-pten monoclonal antibody at 1:1000 (A2B1, Santa Cruz Biotechnology Inc., Santa Cruz, California). After washing, incubating with secondary antibody (sheep anti-mouse horseradish peroxidase; Amersham

5 122 : 2 FEBRUARY 2004 BRAF AND PTEN/MMAC1 COOPERATE IN MELANOMA 341 Figure 2 Model of various genetic interactions. (A) Normal NRAS stimulates BRAF and PI3-K thereby activating downstream effectors MAPK and AKT, respectively. (B) An NRAS mutation activates both pathways. (C) Activation of BRAF accompanied by concurrent loss of PTEN cooperate to trigger signaling in both pathways. (D) An isolated BRAF mutation unaccompanied by PTEN loss may still engage the AKT signaling through unknown mechanisms. Biosciences, Piscataway, New Jersey), the blot was developed with chemiluminescent substrate. This work was funded in part through grants from the American Cancer Society, Dermatology Foundation, the American Skin Cancer (to H.T.) and the National Institutes of Health (to H.T. and F.G.H.) DOI: /j X x Manuscript received September 17, 2003; revised October 21, 2003; accepted for publication October 28, 2003 Address correspondence to: Hensin Tsao, Wellman Center, Department of Dermatology, Massachusetts General Hospital Melanoma Center, Massachusetts General Hospital, Boston, MA 02114, USA. References Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ: Increasing complexity of Ras signaling. Oncogene 17: , 1998 Cohen C, Zavala-Pompa A, Sequeira JH, et al: Mitogen-activated protein kinase activation is an early event in melanoma progression. Clin Cancer Res 8: , 2002 Davies H, Bignell GR, Cox C, et al: Mutations of the BRAF gene in human cancer. Nature 417: , 2002 Dhawan P, Singh AB, Ellis DL, Richmond A: Constitutive activation of Akt/protein kinase B in melanoma leads to up-regulation of nuclear factor-kappab and tumor progression. 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