• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Invest Dermatol. Author manuscript; available in PMC Nov 25, 2008.
Published in final edited form as:
PMCID: PMC2586668
NIHMSID: NIHMS76652

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

Abstract

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.

Keywords: BRAF, melanoma, NRAS, PTEN

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), 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 va-line599– 26 lines had the canonical T1796A (BRAFV599E) mutation, whereas two lines harbored the TG1796-97AT(BRAFV599D) mutation. The BRAFV599E mutation was also present in nine of 16 (56%) uncultured metastatic melanoma specimens.

Figure 1
(A) PCR–single strand conformation polymorphism

In three of 28 melanoma cell lines with BRAFV599 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 BRAFV599 alterations are presumably gain-of-function mutations, the normal allele would represent a hypo-functional competitor whose loss could potentiate the effectiveness of the BRAFV599 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 andII.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 = 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.

Table I
BRAF, NRAS, and PTEN/MMAC1 mutations in melanoma cell lines
Table II
BRAF, NRAS, and PTEN/MMAC1 mutationsa in melanoma metastases

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.

Figure 2
Model of various genetic interactions

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 PD98059. 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, LY294002 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 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 μL reaction containing 1 μL of DNA, 1.5 mM MgCl2 and 40 ng of each primer under standard conditions with or without 0.25 μL of [α32P]deoxycytidine triphosphate (NEN, Boston, Massachusetts). The samples were initially denatured at 95°C 5 min, than amplified for 35 cycles of 95°C 1 min, 62°C 1.5 min, 72°C 1.5 min, and final extension at 72°C for 10 min. The PCR product was confirmed on 1.2% agarose gel. Ten microliters of PCR product was then mixed with 10 μL 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 95°C 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 4°C 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 μg 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 Biosciences, Piscataway, New Jersey), the blot was developed with chemiluminescent substrate.

Acknowledgments

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.)

References

  • Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ. Increasing complexity of Ras signaling. Oncogene. 1998;17:1395–1413. [PubMed]
  • Cohen C, Zavala-Pompa A, Sequeira JH, et al. Mitogen-activated protein kinase activation is an early event in melanoma progression. Clin Cancer Res. 2002;8:3728–3733. [PubMed]
  • Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–954. [PubMed]
  • 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. Cancer Res. 2002;62:7335–7342. [PubMed]
  • Diaz R, Ahn D, Lopez-Barcons L, et al. The N-ras proto-oncogene can suppress the malignant phenotype in the presence or absence of its oncogene. Cancer Res. 2002;62:4514–4518. [PubMed]
  • van Elsas A, Zerp SF, van der Flier S, et al. Relevance of ultraviolet-induced Nras oncogene point mutations in development of primary human cutaneous melanoma. Am J Pathol. 1996;149:883–893. [PMC free article] [PubMed]
  • Govindarajan B, Bai X, Cohen C, et al. Malignant transformation of melanocytes to melanoma by constitutive activation of mitogen-activated protein kinase kinase (MAPKK) signaling. J Biol Chem. 2003;278:9790–9795. [PubMed]
  • Graeven U, Rodeck U, Karpinski S, Jost M, Philippou S, Schmiegel W. Modulation of angiogenesis and tumorigenicity of human melanocytic cells by vascular endothelial growth factor and basic fibroblast growth factor. Cancer Res. 2001;61:7282–7290. [PubMed]
  • Herlyn M, Satyamoorthy K. Activated ras: Yet another player in melanoma? Am J Pathol. 1996;149:739–744. [PMC free article] [PubMed]
  • Iwamoto T, Takahashi M, Ito M, et al. Aberrant melanogenesis and melanocytic tumour development in transgenic mice that carry a metallothionein/ret fusion gene. EMBO J. 1991;10:3167–3175. [PMC free article] [PubMed]
  • Kato M, Takahashi M, Akhand AA, et al. Transgenic mouse model for skin malignant melanoma. Oncogene. 1998;17:1885–1888. [PubMed]
  • Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998;273:13375–13378. [PubMed]
  • Meier F, Nesbit M, Hsu MY, et al. Human melanoma progression in skin reconstructs: Biological significance of bFGF. Am J Pathol. 2000;156:193–200. [PMC free article] [PubMed]
  • Osaka M, Matsuo S, Koh T, Sugiyama T. Loss of heterozygosity at the N-ras locus in 7,12-dimethylbenz[a] anthracene-induced rat leukemia. Mol Carcinog. 1997;18:206–212. [PubMed]
  • Pollock PM, Harper UL, Hansen KS, et al. High frequency of BRAF mutations in nevi. Nat Genet. 2003;33:19–20. [PubMed]
  • Rodriguez-Viciana P, Marte BM, Warne PH, Downward J. Phosphatidylinositol 3′ kinase: One of the effectors of Ras. Philos Trans R Soc London B Biol Sci. 1996;351:225–231. discussion 231–222. [PubMed]
  • Rodriguez-Viciana P, Warne PH, Khwaja A, et al. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell. 1997;89:457–467. [PubMed]
  • Saranath D, Chang SE, Bhoite LT, et al. High frequency mutation in codons 12 and 61 of H-ras oncogene in chewing tobacco-related human oral carcinoma in India. Br J Cancer. 1991;63:573–578. [PMC free article] [PubMed]
  • Satyamoorthy K, Li G, Gerrero MR, et al. Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res. 2003;63:756–759. [PubMed]
  • Sukumar S, Armstrong B, Bruyntjes JP, Leav I, Bosland MC. Frequent activation of the Ki-ras oncogene at codon 12 in N-methyl-N-nitrosourea-induced rat prostate adenocarcinomas and neurogenic sarcomas. Mol Carcinog. 1991;4:362–368. [PubMed]
  • Tsao H, Zhang X, Benoit E, Haluska FG. Identification of PTEN/MMAC1 alterations in uncultured melanomas and melanoma cell lines. Oncogene. 1998;16:3397–3402. [PubMed]
  • Tsao H, Zhang X, Fowlkes K, Haluska FG. Relative reciprocity of NRAS and PTEN/MMAC1 alterations in cutaneous melanoma cell lines. Cancer Res. 2000;60:1800–1804. [PubMed]
  • Tsao H, Mihm MC, Sheehan C. PTEN expression in normal skin, acquired melanocytic nevi and cutaneous melanoma. J Am Acad Dermatol. 2003;49:865–872. [PubMed]
  • Uribe P, Wistuba II, Gonzalez S. BRAF mutation: A frequent event in benign, atypical, and malignant melanocytic lesions of the skin. Am J Dermatopathol. 2003;25:365–370. [PubMed]
  • Wu X, Senechal K, Neshat MS, Whang YE, Sawyers CL. The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Natl Acad Sci USA. 1998;95:15587–15591. [PMC free article] [PubMed]
  • Zhou XP, Gimm O, Hampel H, Niemann T, Walker MJ, Eng C. Epigenetic PTEN silencing in malignant melanomas without PTEN mutation. Am J Pathol. 2000;157:1123–1128. [PMC free article] [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Gene
    Gene
    Gene links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene
    HomoloGene links
  • MedGen
    MedGen
    Related information in MedGen
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...