Constitutive activation of the ERK pathway in melanoma and skin melanocytes in Grey horses

BMC Cancer

Anna Golovko¹  Leif Andersson³  Giorgia Egidy⁶  Mats J Olsson⁴  Gerli Pielberg¹  Monika Seltenhammer²  Saima Imran¹  Pedro Sousa¹  Elisabeth Sundström¹  Cécile Campagne⁵  Lin Jiang⁷ 

[1]Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden

[2]Department of Forensic Sciences, Medical University of Vienna, Sensengasse 2, 1090 Vienna, Austria

[3]Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Box 597, SE-751 24 Uppsala, Sweden

[4]Present address: ParkCell AB, Uppsala Science Park, Uppsala, Sweden

[5]Ecole Nationale Vétérinaire d’Alfort, Maisons-Alfort, France

[6]Unité de Recherche Associée 2578, Centre National de la Recherche Scientifique, Paris, France

[7]Present address: Institute of Beijing Animal Science and Veterinary, Chinese Academy of Agricultural Science, Beijing 100194, China

关键词: Melanocytes;    STX17;    ERK pathway;    Grey horse;    Melanoma;   

Others  :  1118055
DOI  :  10.1186/1471-2407-14-857

 received in 2013-05-07, accepted in 2014-10-27,  发布年份 2014

【 摘 要 】

Background

Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses.

Methods

Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting.

Results

We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses.

Conclusions

These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this pathway during melanomagenesis. These findings are consistent with the universal importance of the ERK pathway in melanomagenesis and may have valuable implications for human melanoma research.

【 授权许可】

   
2014 Jiang et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Smalley KS: A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? Int J Cancer 2003, 104(5):527-532.
• [2]Fecher LA, Amaravadi RK, Flaherty KT: The MAPK pathway in melanoma. Curr Opin Oncol 2008, 20(2):183-189.
• [3]Hearing V, Leong S: From Melanocytes to Melanoma: The Progression to Malignancy. Totowa, NJ: Humana Press; 2006.
• [4]Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, et al.: Mutations of the BRAF gene in human cancer. Nature 2002, 417(6892):949-954.
• [5]Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O’Brien JM, Simpson EM, Barsh GS, Bastian BC: Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009, 457(7229):599-602.
• [6]Curtin JA, Busam K, Pinkel D, Bastian BC: Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol 2006, 24(26):4340-4346.
• [7]Jiang X, Zhou J, Yuen NK, Corless CL, Heinrich MC, Fletcher JA, Demetri GD, Widlund HR, Fisher DE, Hodi FS: Imatinib targeting of KIT-mutant oncoprotein in melanoma. Clin Cancer Res 2008, 14(23):7726-7732.
• [8]Monsel G, Ortonne N, Bagot M, Bensussan A, Dumaz N: c-Kit mutants require hypoxia-inducible factor 1alpha to transform melanocytes. Oncogene 2010, 29(2):227-236.
• [9]Nikolaev SI, Rimoldi D, Iseli C, Valsesia A, Robyr D, Gehrig C, Harshman K, Guipponi M, Bukach O, Zoete V, Michielin O, Muehlethaler K, Speiser D, Beckmann JS, Xenarios I, Halazonetis TD, Jongeneel CV, Stevenson BJ, Antonarakis SE: Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma. Nat Genet 2012, 44(2):133-139.
• [10]Tanami H, Imoto I, Hirasawa A, Yuki Y, Sonoda I, Inoue J, Yasui K, Misawa-Furihata A, Kawakami Y, Inazawa J: Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines. Oncogene 2004, 23(54):8796-8804.
• [11]Panka DJ, Atkins MB, Mier JW: Targeting the mitogen-activated protein kinase pathway in the treatment of malignant melanoma. Clin Cancer Res 2006, 12(72):2371s-2375s.
• [12]Tsavachidou D, Coleman ML, Athanasiadis G, Li S, Licht JD, Olson MF, Weber BL: SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant. Cancer Res 2004, 64(16):5556-5559.
• [13]Sutton RH, Coleman GT: Melanoma and the Graying Horse. Barton, Australia: RIRDC Research Paper Series; 1997:1-34.
• [14]Smith SH, Goldschmidt MH, McManus PM: A comparative review of melanocytic neoplasms. Vet Pathol 2002, 39(6):651-678.
• [15]Gorham S, Robl M: Melanoma in the gray horse: the darker side of equine aging. Vet Med 1986, 11:1191-1204.
• [16]Seltenhammer MH, Heere-Ress E, Brandt S, Druml T, Jansen B, Pehamberger H, Niebauer GW: Comparative histopathology of grey-horse-melanoma and human malignant melanoma. Pigment Cell Res 2004, 17(6):674-681.
• [17]Rosengren Pielberg G, Golovko A, Sundstrom E, Curik I, Lennartsson J, Seltenhammer MH, Druml T, Binns M, Fitzsimmons C, Lindgren G, Sandberg K, Baumung R, Vetterlein M, Strömberg S, Grabherr M, Wade C, Lindblad-Toh K, Pontén F, Heldin CH, Sölkner J, Andersson L: A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse. Nat Genet 2008, 40(8):1004-1009.
• [18]Sundstrom E, Komisarczuk AZ, Jiang L, Golovko A, Navratilova P, Rinkwitz S, Becker TS, Andersson L: Identification of a melanocyte-specific, microphthalmia-associated transcription factor-dependent regulatory element in the intronic duplication causing hair greying and melanoma in horses. Pigment Cell Melanoma Res 2012, 25(1):28-36.
• [19]Sundstrom E, Imsland F, Mikko S, Wade C, Sigurdsson S, Pielberg GR, Golovko A, Curik I, Seltenhammer MH, Sölkner J, Lindblad-Toh K, Andersson L: Copy number expansion of the STX17 duplication in melanoma tissue from Grey horses. BMC Genomics 2012, 13:365. BioMed Central Full Text
• [20]Eskandarpour M, Kiaii S, Zhu C, Castro J, Sakko AJ, Hansson J: Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells. Int J Cancer 2005, 115(1):65-73.
• [21]Johnson JP, Demmer-Dieckmann M, Meo T, Hadam MR, Riethmuller G: Surface antigens of human melanoma cells defined by monoclonal antibodies. I. Biochemical characterization of two antigens found on cell lines and fresh tumours of diverse tissue origin. Eur J Immunol 1981, 11(10):825-831.
• [22]Liao SK, Dent PB, McCulloch PB: Characterization of human maligant melanoma cell lines. I. Morphology and growth characteristics in culture. J Natl Cancer Inst 1975, 54(5):1037-1044.
• [23]Campagne C, Jule S, Bernex F, Estrada M, Aubin-Houzelstein G, Panthier JJ, Egidy G: RACK1, a clue to the diagnosis of cutaneous melanomas in horses. BMC Vet Res 2012, 8:95. BioMed Central Full Text
• [24]Seltenhammer MH, Sundström E, Meisslitzer-Ruppitsch C, Cejka P, Kosiuk J, Neumüller J, Almeder M, Majdic O, Steinberger P, Losert UM, Stöckl J, Andersson L, Sölkner J, Vetterlein M, Golovko A: Establishment and characterization of a primary and a metastatic melanoma cell line from Grey horses. In Vitro Cell Dev Biol Anim 2014, 50(1):56-65.
• [25]Haase B, Brooks SA, Schlumbaum A, Azor PJ, Bailey E, Alaeddine F, Mevissen M, Burger D, Poncet PA, Rieder S, Leeb T: Allelic heterogeneity at the equine KIT locus in dominant white (W) horses. PLoS Genet 2007, 3(11):e195.
• [26]Marklund S, Moller M, Sandberg K, Andersson L: Close association between sequence polymorphism in the KIT gene and the roan coat color in horses. Mamm Genome 1999, 10(3):283-288.
• [27]Garnett MJ, Marais R: Guilty as charged: B-RAF is a human oncogene. Cancer Cell 2004, 6(4):313-319.
• [28]Marquette A, Andre J, Bagot M, Bensussan A, Dumaz N: ERK and PDE4 cooperate to induce RAF isoform switching in melanoma. Nat Struct Mol Biol 2011, 18(5):584-591.
• [29]Calipel A, Mouriaux F, Glotin AL, Malecaze F, Faussat AM, Mascarelli F: Extracellular signal-regulated kinase-dependent proliferation is mediated through the protein kinase A/B-Raf pathway in human uveal melanoma cells. J Biol Chem 2006, 281(14):9238-9250.
• [30]Wellbrock C, Marais R: Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation. J Cell Biol 2005, 170(5):703-708.
• [31]Cohen C, Zavala-Pompa A, Sequeira JH, Shoji M, Sexton DG, Cotsonis G, Cerimele F, Govindarajan B, Macaron N, Arbiser JL: Mitogen-actived protein kinase activation is an early event in melanoma progression. Clin Cancer Res 2002, 8(12):3728-3733.
• [32]Govindarajan B, Bai X, Cohen C, Zhong H, Kilroy S, Louis G, Moses M, Arbiser JL: Malignant transformation of melanocytes to melanoma by constitutive activation of mitogen-activated protein kinase kinase (MAPKK) signaling. J Biol Chem 2003, 278(11):9790-9795.