Genomic and proteomic analysis in uveal melanoma Zuidervaart, W.

Citation

Zuidervaart, W. (2005, May 25). Genomic and proteomic analysis in uveal melanoma. Retrieved from https://hdl.handle.net/1887/2696

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the_{Institutional Repository of the University of Leiden} Downloaded from: https://hdl.handle.net/1887/2696

I

Despite many ways to effectively treat primary uveal melanoma, little can be done to treat the patient once metastases have been developed. Identification of chromosomal aberrations and altered expression of single genes and proteins in uveal melanoma development has started to provide some insight regarding the behavior of this tumor. In order to be able to treat or prevent metastases, we need more knowledge on the complex genetic interactions and genetic pathways underlying uveal melanoma tumor progression. Therefore, the aim of this thesis is to obtain a better insight into biological pathways underlying uveal melanogenesis by

genomic and proteomic analysis. By making use of these technologies we want to outline the differences between a benign and a malignant cell and between tumors that differ in their metastatic potential.

Known cutaneous melanoma genes and pathways in relation to uveal melanoma

In the first part of this thesis, results of analyses are presented on genes and pathway members known to contribute to cutaneous melanomagenesis. Such molecules may be important for uveal melanomas as well, since both cutaneous and uveal melanomas are melanocytic

neoplasias. In Chapter 2, we described the role of PTEN in uveal melanoma and observed that in contrast to cutaneous melanoma, alterations in this tumor suppressor gene could not be found. In addition to PTEN, the mitogen-activated protein kinase (MAPK) pathway plays an important role in cutaneous melanoma, mainly due to mutations in BRAF (Cohen et al., 2002; Davies et al., 2002). Based on expression data from tumor samples, together with knowledge about the class each sample belongs to, Pavey et al. (2004) were even able to construct a classifier (prediction rule) that, based on gene expression profiles, discriminates between cutaneous melanoma cell lines according to whether they carry mutations in BRAF. Table III (see Chapter 3) provides an overview of published reports on RAS and BRAF mutations, as well as studies on other downstream members of the MAPK pathway, in uveal melanomas. To date, not a single BRAF mutation has been found in a total of 276 primary or secondary uveal melanoma samples (Table III). While we discovered one mutation in our cell line panel (n=11), Calipel et al. (2003) surprisingly observed the common BRAF mutation (V599E) in all three uveal melanoma cell lines used in their study. Taken together, these data suggest that, while a BRAF mutation is not required for uveal melanoma development in vivo, such mutations confer a cellular growth advantage and may be hence selected for, if they occur in cell lines cultured in vitro.

activation of the MAPK pathway that stimulates growth of melanoma cells. Therefore, it would be of great interest to compare these results with the situation seen in uveal melanomas.

G

/

Justification of cell lines

Most of our studies used cell lines derived from uveal melanomas in order to obtain insight into biological pathways underlying uveal melanoma progression. Although cell lines may differ from normal and tumor specimens, we and others have shown that they can act as reliable model systems. For example, expression patterns in cell lines resembled quite well the tumor type they are derived from (De Waard-Siebinga et al., 1995; Ross et al., 2001).

Furthermore, our current comparisons of gene and protein expression of two cell lines derived from metastases have not revealed much differential expression, confirming close similarity between the liver metastases. In addition, the results indicate that maintaining the cell lines in culture for a long time does not influence the expression pattern of the two metastases cell lines. Therefore, extrapolation of our results obtained via cell lines has great potential for further analysis and validation of frozen tumor material directly obtained from uveal melanoma patients.

Macroarray

In Chapters 4 and 5 we performed gene expression profiling using a macroarray technique with nylon filters. By means of this technique, we compared gene expression profiles in different uveal melanoma cell lines and identified potential genes, such as TIMP3, Endothelin 2 (ET2) and Laminin Receptor 1 (LAMR1), involved in uveal melanoma tumor development and progression. Validation of these genes on the primary melanoma samples resulted in two distinct subgroups with specific expression profiles. Besides LAMR1, the histopathologic parameter PAS positive loops revealed a significant association with the expression level of the genes ET2 and Von Hippel Lindau Binding protein 1 (VBP1). The ability to form vascular loops has been reported as an important prognostic parameter in uveal melanoma metastases development (Folberg et al., 2001).

ENRB pathway also leads to increases in MMP2 and activation of focal adhesion kinase (FAK), which lead to enhanced cell proliferation, adhesion, migration and MMP-dependent invasion. Furthermore, VE-cadherin, a non-classical cadherin predominantly expressed in endothelial cells (Dejana 1999), is known to interact with EphA2, facilitating downstream signalling with PI3K and FAK, that could play significant roles in the vasculogenic mimicry process (Hendrix et al., 2003). VE-cadherin has been reported to be dramatically

overexpressed in aggressive human cutaneous and uveal melanoma cells. Its downregulation results in the complete inability of invasive melanoma cells to form vasculogenic-like

networks in 3-D cultures (Bittner et al., 2000; Hendrix et al., 2001; Hess et al., 2001; Seftor et al., 2001; Seftor et al., 2002).

The filter macroarray technique has proven to be a strong instrument to compare gene expression profiles of cell lines of different origin, but careful attention should be paid to the quantitative value of this filter method with regard to genes with low expression levels, where its validation cannot easily be performed. Despite more advanced gene expression tools have become available (cDNA microarrays), the required specialized and expensive equipment and software as well as the volume and subsequent required validation of generated data, often puts the technique out of reach of many researchers. Therefore, low-density "macroarrays" are still used today and could be valuable in exploring one area of biological interest.

Microarray

Microarray technology is an extremely useful tool to analyze the expression of an entire genome at once. In Chapter 6 we used microarray analysis to unravel differences in gene expression profiles during uveal melanoma development. Because of this powerful approach, in combination with in vitro assays, we were able to demonstrate a role for two members of the p21-activated kinase (PAK) family, PAK1 and PAK7 in invasiveness of uveal melanoma. In regard to Chapter 3, it is of particular interest that constitutively active PAK allows

anchorage-independent MAPK signalling (Howe et al., 2000). PAKs can also directly phosphorylate and promote the activity of the kinases RAF and MEK, leading to increased ERK1/2 activity (Frost et al., 1997, King et al, 1998), and contribute to the adhesion

Recently, two studies have identified ZD1839 as a potent inhibitor of PAK1 (Barnes et al., 2003; Yang et al., 2004), with inhibition of PAK1 activity in exponentially growing cancer cells resulting in attenuation of transformed cell phenotypes. ZD1839 therefore may have the potential to affect the metastatic ability of uveal melanoma by deregulating the PAK1

pathway, and thus warrants investigation as a potential therapy for this highly invasive tumor. In chapter 7 we furthermore show an association between activation of the Wnt signalling pathway and invasiveness of uveal melanomas. Similar to cutaneous melanoma, one of the primary mediators of this effect is Wnt5a. The expression levels of a number of downstream targets of Wnt signalling, including MMP7 and β-catenin, are increased in more invasive

uveal melanoma cell lines and primary tumors. Uveal melanomas with a high proportion of cells expressing β-catenin were associated with significantly decreased overall survival. These

data point to the importance of Wnt signalling in conferring a metastatic phenotype in uveal melanoma and indicate that components of this pathway may be useful markers of prognosis as well as therapeutic targets.

For a tumor cell to metastasize from the primary tumor to other organs, it must locally

degrade ECM components that are the physical barriers for cell migration. Degradation of the basement membrane has been correlated with the metastatic potential of tumor cells (Liotta et al., 1988; Meyer et al., 1998; Engbring et al., 2003). The key enzymes responsible for ECM breakdown are matrix metalloproteinases (MMPs). We observed increased expression of MMP7 with invasiveness in chapter 7. Although little is known about the role of MMP7 in uveal melanoma, another member of the MMP’s, MMP2, was found to be elevated in uveal melanoma in a previous report (Elshaw et al., 2001). MMP2 is a key mediator of invasion, metastasis, tumor angiogenesis and tumor cell vascular mimicry (Seftor et al., 2001; Hess et al., 2003), and has been identified as an independent prognostic factor for survival in

cutaneous melanoma (Vaisanen et al.,1998). PI3K activation has been linked to MMP2 expression in uveal melanoma, with specific inhibition of PI3K leading to a decrease in MMP2 activity (Hess et al., 2003). In addition, in transfected melanoma cells expressing reduced levels of laminin receptor, reduced MICS invasion was associated with decreased MMP2 expression and activity (Givant-Horwitz et al., 2004). The local balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is believed to play a major role in ECM remodeling during development and in diseases such as cancer. In support of the in chapter 4 of this thesis reported association of reduced TIMP3 expression with uveal

melanoma invasiveness (Van der Velden et al., 2003), the TIMP3 levels as revealed in chapter 6 were decreased as low as 7.1-fold in the more invasive group of cell lines. Figure 9.1

Figure 9.1 Interrelation between pathways.

Proteomic analysis

During the last decade, proteomic analysis methods have been established as powerful new tools for the investigation of a variety of biological phenomena. Conventional

two-dimensional gel electrophoresis method is still the most commonly used technique, and allows analysis of over 1000 different proteins in one single experiment. It has a high

resolution and a relatively high sensitivity. One of the greatest strengths of 2D-PAGE analysis is its capability to study protein modifications, including posttranslational modifications (such as phosphorylation (Chapter 8) and glycosylation), and changes in expression levels.

In Chapter 8, we described how 2D-PAGE allowed us to identify protein expression

differences, in a model of a primary uveal melanoma compared to two cell lines derived from metastases of that primary tumor. A panel of these proteins were identified by mass

spectrometry and has led to the discovery of interesting proteins possibly involved in uveal melanoma progression. Most of these proteins have not been related to uveal melanoma development before. Further studies will be undertaken to validate these proteins in primary uveal melanoma specimen by e.g. Western blot analysis and immunostaining experiments. Although we identified interesting molecules, 2D-PAGE analysis still needs time-consuming manual interventions and reproducibility cannot always be achieved. In addition, hydrophobic membrane proteins, very large and small proteins, and proteins revealing extreme isoelectric point values, are difficult to resolve. The use of nano-high-performance liquid

chromatography (HPLC) method could overcome certain problems encountered by the 2D-PAGE method. Nano-HPLC is a more flexible method, and allows the combination of

different separation techniques. Samples could be tagged and modified before, in between, or after a single separation step, and sequences of multiple runs could be easily automated. Nevertheless, the 2D-PAGE method will still be the choice for differential expression

research. To refine the 2D-PAGE method, several additional procedures have been developed. The challenge of analysing a complicated proteome may be addressed by the use of very narrow-range immobilized pH gradients (IPGs) (Görg et al., 2000). These provide a means to ‘zoom-in’ on, and expand, crowded areas of broad-range IPG 2-D gels for better resolution and separation (Wildgruber et al., 2000). Another important step in the 2D-PAGE method is the choice of staining of the 2-D gels depending on the purpose of the experiment. Coomassie Blue is insensitive, having detection limit of about 1 µg, but is widely used because the

staining procedure is relatively simple. Silver staining is more sensitive and had a detection limit of about 1 ng. However, different proteins stain with different avidities and the total sensitivity range of silver staining is limited, making it less suitable for quantitation of proteins. Fey (1997) localized yeast proteins by autoradiography, which requires no fixing and staining, leading to an improved overall sensitivity. Thus, proteins which cannot be visualized with Coomassie or silver staining are readily detected.

To overcome another limitation of the conventional proteomic approaches, the detection of protein-protein interactions, an activity based protein profiling (ABPP) strategy has been developed. This approach employs chemical probes that covalently label the active sites of enzyme superfamilies in a manner that provides a direct readout of changes in catalytic activity and permits identification of active enzymes directly from complex proteomes (Liu et al., 1999). Using this method, Jessani et al. (2002) could generate molecular profiles that classify human breast cancer and cutaneous melanoma cell lines into subtypes, based on higher-order cellular properties, including tissue of origin and state of invasiveness.

C

From the clinical perspective, it would be of particular interest to determine which subset of patients will have the highest risk to develop distant metastasis. As enucleation of the tumor-containing eye does not appear to prevent metastases, it is clear that micrometastases have spread prior to recognition of the presence of the primary tumor. Therefore, one of the most valuable outcomes of the current investigation would be the application of data from the uveal melanoma molecular and biological profile to develop new targets for therapeutic intervention such as development of treatment strategies to prevent activation of dormant metastases. Although recent technological improvements did provide us with new insights in the complex system of uveal melanoma development, translation of these data into clinical practice is far from realistic yet.

monosomy is an important prognostic finding (Tschentscher et al., 2003). Array-based high resolution comparative genomic hybridization (CGH) analysis now enables genome-wide screening of chromosomal imbalances (Solinas-Toldo 1997, Pinkel 1998) and would prosper the identification of less prominent chromosome changes relevant to uveal melanoma. Especially, a combination of cDNA and CGH microarray methods with tissue microarray technology, that allows analysis of hundreds of small tissue cores on one single paraffin section (Kononen et al., 1998), enables rapid identification of putative amplification target genes as well as analysis of their clinical significance (Heiskanen et al., 2001).

Integration of results of genomic and proteomic analyses could be expanded with

metabolomic analysis, the study of metabolite profiles in biological samples such as blood plasma, which could identify potential markers directly in serum of patients.

R

Adam L, Vadlamudi R, Mandal M, Chernoff J, Kumar R. Regulation of microfilament reorganization and invasiveness of breast cancer cells by kinase dead p21-activated kinase-1. J Biol Chem

2000;275(16):12041-50.

Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 1990;348:730–732.

Bagnato A, Rosano L, Spinella F, Di Castro V, Tecce R, Natali PG.Endothelin B receptor blockade inhibits dynamics of cell interactions and communications in melanoma cell progression.Cancer Res. 2004;64:1436-43.

Barnes CJ, Bagheri-Yarmand R, Mandal M, et al. Suppression of epidermal growth factor receptor, mitogen-activated protein kinase, and Pak1 pathways and invasiveness of human cutaneous squamous cancer cells by the tyrosine kinase inhibitor ZD1839 (Iressa). Mol Cancer Ther. 2003;2:345-51.

Bittner M, Meltzer P, Chen Y, Jiang Y, Seftor E, Hendrix M, Radmacher M, Simon R, Yakhini Z, Ben-Dor A, Sampas N, Dougherty E, Wang E, Marincola F, Gooden C, Lueders J, Glatfelter A, Pollock P, Carpten J, Gillanders E, Leja D, Dietrich K, Beaudry C, Berens M, Alberts D, Sondak V. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature. 2000;406:536-40.

Calipel A, Lefevre G, Pouponnot C, Mouriaux F, Eychene A, Mascarelli F(2003) Mutation of B-Raf in human choroidal melanoma cells mediates cell proliferation and transformation through the MEK/ERK pathway. J Biol Chem 278:42409–42418.

Carter JH, Douglass LE, Deddens JA, Colligan BM, Bhatt TR, Pemberton JO, Konicek S, Hom J, Marshall M, Graff JR. Pak-1 expression increases with progression of colorectal carcinomas to metastasis. Clin Cancer Res 2004;10(10):3448-56.

Cheung VG, Conlin LK, Weber TM, Arcaro M, Jen KY, Morley M, Spielman RS. Natural variation in human gene expression assessed in lymphoblastoid cells. Nat Genet. 2003;33:422-5.

Cohen C, Zavala-Pompa A, Sequeira JH, Shoji M, Sexton DG, Cotsonis G, Cerimele F, Govindarajan B, Macaron N, Arbister JL. Mitogen -activated protein kinase activation is an early event in melanoma progression. Clin Cancer Res. 2002;8:3728-3733.

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, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA. Mutations of the BRAF gene in human cancer. Nature. 2002 ;417:949-54.

Dejana E, Bazzoni G, Lampugnani MG. Vascular endothelial (VE)-cadherin: only an intercellular glue? Exp Cell Res. 1999 Oct 10;252(1):13-9.

De Waard-Siebinga I, Kool J, Jager MJ. HLA antigen expression on uveal melanoma cells in vivo and in vitro. Hum Immunol. 1995;44:111-7.

Eblen ST, Slack JK, Weber MJ, Catling AD. Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes. Mol Cell Biol. 2002;22:6023-33. Elshaw SR, Sisley K, Cross N, Murray AK, MacNeil SM, Wagner M, Nichols CE, Rennie IG. A comparison of

ocular melanocyte and uveal melanoma cell invasion and the implication of alpha1beta1, alpha4beta1 and alpha6beta1 integrins. Br J Ophthalmol 2001;85(6):732-8.

Engbring JA, Kleinman HK. The basement membrane matrix in malignancy. J Pathol 2003;200(4):465-70. Fey SJ. Proteome analysis of Saccharomyces cerevisiae: A methodological outline Electrophoresis 1997, 18,

1361-72

Folberg R, Chen X, Boldt HC, Pe'er J, Brown CK, Woolson RF, Maniotis AJ. Microcirculation patterns other than loops and networks in choroidal and ciliary body melanomas. Ophthalmology. 2001;108:996–1001. Frost JA, Steen H, Shapiro P, et al. Cross-cascade activation of ERKs and ternary complex factors by Rho family

proteins. Embo J. 1997;16:6426-38.

Görg A, Obermaier C, Boguth G. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000; 21: 1037-53

Heiskanen M, Kononen J, Barlund M, Torhorst J, Sauter G, Kallioniemi A, Kallioniemi O. CGH, cDNA and tissue microarray analyses implicate FGFR2 amplification in a small subset of breast tumors. Anal Cell Pathol. 2001;22:229-34.

Hendrix MJ, Seftor EA, Meltzer PS, Gardner LM, Hess AR, Kirschmann DA, Schatteman GC, Seftor

RE.Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry. Proc Natl Acad Sci U S A. 2001;98:8018-23.

Hendrix MJ, Seftor EA, Hess AR, Seftor RE. Molecular plasticity of human melanoma cells. Oncogene. 2003 May 19;22(20):3070-5.

Hess AR, Seftor EA, Gardner LM, Carles-Kinch K, Schneider GB, Seftor RE, Kinch MS, Hendrix MJ. Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2). Cancer Res. 2001;61:3250-5.

Hess AR, Seftor EA, Seftor RE, Hendrix MJ. Phosphoinositide 3-kinase regulates membrane Type 1-matrix metalloproteinase (MMP) and MMP-2 activity during melanoma cell vasculogenic mimicry. Cancer Res 2003;63(16):4757-62.

Howe AK, Juliano RL. Regulation of anchorage-dependent signal transduction by protein kinase A and p21-activated kinase. Nat Cell Biol. 2000;2:593-600.

Hyman E, Kauraniemi P, Hautaniemi S, Wolf M, Mousses S, Rozenblum E, Ringner M, Sauter G, Monni O, Elkahloun A, Kallioniemi OP, Kallioniemi A. Impact of DNA amplification on gene expression patterns in breast cancer. Cancer Res. 2002;62:6240-5.

Jessani N, Liu Y, Humphrey M, Cravatt BF. Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc Natl Acad Sci U S A. 2002;99:10335-40.

Jung ID, Lee J, Lee KB, Park CG, Kim YK, Seo DW, Park D, Lee HW, Han JW, Lee HY. Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells. Eur J Biochem 2004;271(8):1557-65.

King AJ, Sun H, Diaz B, et al. The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338. Nature. 1998;396:180-3.

Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Milhatsch MJ, Sauter G, Kallioniemi OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998; 4:844–847

Liotta LA, Wewer U, Rao NC, Schiffmann E, Stracke M, Guirguis R, Thorgeirsson U, Muschel R, Sobel M. Biochemical mechanisms of tumor invasion and metastases. Adv Exp Med Biol 1988;233:161-9. Liu Y, Patricelli MP, Cravatt BF. Activity-based protein profiling: the serine hydrolases.

Proc Natl Acad Sci U S A. 1999;96:14694-9.

Meyer T, Hart IR. Mechanisms of tumour metastasis. Eur J Cancer 1998;34(2):214-21.

Pavey S, Johansson P, Packer L, Taylor J, Stark M, Pollock PM, Walker GJ, Boyle GM, Harper U, Cozzi SJ, Hansen K, Yudt L, Schmidt C, Hersey P, Ellem KA, O'Rourke MG, Parsons PG, Meltzer P, Ringner M, Hayward NK. Microarray expression profiling in melanoma reveals a BRAF mutation signature. Oncogene. 2004;23:4060-7.

Pinkel D, Segraves R, Sudar D, Clark S, Poole I, Kowbel D, Collins C, Kuo WL, Chen C, Zhai Y, Dairkee SH, Ljung BM, Gray JW, Albertson DG. High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat Genet. 1998;20:207-11.

Reid K, Turnley AM, Maxwell GD, Kurihara Y, Kurihara H, Bartlett PF, Murphy M. Multiple roles for endothelin in melanocyte development: regulation of progenitor number and stimulation of differentiation. Development. 1996;122:3911-3919.

Ross DT, Perou CM. A comparison of gene expression signatures from breast tumors and breast tissue derived cell lines. Dis Markers. 2001;17:99-109.

Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 1990;348:732-735.

Seftor EA, Meltzer PS, Kirschmann DA, Pe'er J, Maniotis AJ, Trent JM, Folberg R, Hendrix MJ. Molecular determinants of human uveal melanoma invasion and metastasis. Clin Exp Metastasis. 2002;19(3):233-46.

Slack-Davis JK, Eblen ST, Zecevic M, et al. PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation. J Cell Biol. 2003;162:281-91.

Smith SL, Damato BE, Scholes AG, Nunn J, Field JK, Heighway J. Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival. Br J Cancer. 2002;87:1308-1313.

Solinas-Toldo S, Lampel S, Stilgenbauer S, Nickolenko J, Benner A, Dohner H, Cremer T, Lichter P. Matrix-based comparative genomic hybridization: biochips to screen for genomic imbalances.Genes

Chromosomes Cancer. 1997;20:399-407.

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;23:8796-804.

Tschentscher F, Hüsing J, Hölter T, Kruse E, Dresen IG, Jöckel KH, Anastassiou G, Schilling H, Bornfeld N, Horsthemke B, Lohnmann DR, Zeschnigk M. Tumor classification based on gene expression profiling shows that uveal melanomas with and without monosomy 3 represent two distinct entities. Cancer Res. 2003 May 15;63(10):2578-84

Vadlamudi RK, Kumar R. P21-activated kinases in human cancer. Cancer Metastasis Rev 2003;22(4):385-93. Vaisanen A, Kallioinen M, Taskinen PJ, Turpeenniemi-Hujanen T. Prognostic value of MMP-2 immunoreactive

protein (72 kD type IV collagenase) in primary skin melanoma. J Pathol 1998;186:51-8.

Van der Velden PA, Zuidervaart W, Hurks MH, Pavey S, Ksander BR, Krijgsman E, Frants RR, Tensen CP, Willemze R, Jager MJ, Gruis NA. Expression profiling reveals that methylation of TIMP3 is involved in uveal melanoma development. Int J Cancer 2003;106(4):472-9.

Wildgruber R, Harder A, Obermaier C. Towards higher resolution: two-dimensional electrophoresis of

Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients. Electrophoresis 2000; 21: 2610-6

Yan H, Yuan W, Velculescu VE, Vogelstein B, Kinzler KW. Allelic variation in human gene expression. Science. 2002;297:1143.