Ron Tongbai

12/18/09

Research Summary: Transcriptional Regulatory Network Analysis of Uveal

Melanoma

Abstract:
Uveal melanoma is the most common form of intraocular melanoma in adults and
metastasis to the liver and lungs is associated with a worse prognosis (1). A study by
Onken et al utilized microarrays to characterize the gene expression profiles of tumors
which metastasized and compared them with the gene expression profiles of tumors
which did not metastasize. The study identified specific genetic signatures which
discriminated between tumors that do and do not metastasize. The study provides an
important insight into the molecular pathways and biological processes that govern the
establishment and growth of these metastases. Promoter region sequences from a 600 bp
region (-500 bp upstream to +100 bp downstream) were obtained for each gene in each of
the gene lists using the ProSpector free web-based promoter annotation tool (4). The
promoter regions of each of the gene signatures were analyzed for matches to
approximately 300 position weight matrices (TFBS) using the MatInspector module of
the GEMS LauncheR 4.1 (Genomatix, Munich, Germany). A regulatory profile was
generated based on the methods described above. Each of the gene lists were analyzed to
generate p-values representing the degree of statistical enrichment for each of the
approximately 300 TFBS in the promoter regions of these genes as described in the
methods.

Background: Uveal melanoma is the most common form of intraocular melanoma in

adults and metastasis to the liver and lungs is associated with a worse prognosis (1). A
study by Onken et al (2) utilized microarrays to characterize the gene expression profiles
of tumors which metastasized and compared them with the gene expression profiles of
tumors which did not metastasize. The study identified specific genetic signatures which
discriminated between tumors that do and do not metastasize. The study provides an
important insight into the molecular pathways and biological processes that govern the
establishment and growth of these metastases.

Data:

The data analyzed including the gene expression profiles from Onken et al (2) as well as
gene expression profiles that were generated in-house based on structural chromosomal
differences between tumors. The data from Onken et al were divided into 6 unique gene
lists based on the following 2 criteria:
• First, 34,382 probe sets were filtered to exclude those with a median significance
level P > .05 resulting in 8750 significant genes. These 8750 genes were filtered
for a mean expression difference > 1.5 fold, t test P< .05 to yield 1605 genes.
 • Second, 34,382 probe sets filtered to exclude those with a median signaficance
level P > .005 resulting in 3075 highly significant genes. Median expression for
the 3075 genes were compared between class 1 and class 2 using a signal to noise
algorithm and genes were filtered for a signal-to-noise score > 0.25 and a
significance level p < 0.01, yielding 62 highly discriminating genes

These genes were subdivided into 6 groups as follows:

 Class 1 Gene List: Of the 62 genes, these 42 genes were upregulated in class 1
tumors and downregulated in class 2 tumors
 Class 2 Gene List: Of the 62 genes, these 20 genes were upregulated in class 2
tumors and downregulated in class 1 tumors
 Class 1 Low Stringent Gene List: Of the 1605 genes, 662 were upregulated in
class 1 tumors
 Class 2 Low Stringent Gene List: Of the 1605 genes, 943 were upregulated in
class 2 tumors
 Class 1 3fold Gene List: same conditions as above, but with a mean difference >
3 fold, t test P <.05 -> 64 genes upregulated in class 1 tumors
 Class 2 3fold Gene List: same conditions as above but with a mean difference >
3 fold, t test P < .05 -> 86 genes upregulated in class 2 tumors

The data that was generated in-house was subdivided into 2 unique gene lists which were
generated by taking the 173 unique genes that were found to be upregulated in
chromosome 3 loss tumors compared to chromosome 6p gain tumors (will be referred to
as chrom3_up gene list in this paper) and 133 unique genes that were found to be
downregulated in chromosome 3 loss tumors compared to chromosome 6p gain tumors
(will be refered to as chrom3_down gene list in this paper).

Methods:

The methods utilized in this study will be similar to the methods in a previous study
which examined the promoter composition of gene lists representing 5 different
molecular subtypes of breast cancer known to be associated with distinct clinical
outcomes (3). Please see reference for a more detailed description of the methods.
Briefly, promoter region sequences from a 600 bp region (-500 bp upstream to +100 bp
downstream) were obtained for each gene in each of the gene lists using the ProSpector
free web-based promoter annotation tool (4). The promoter regions of each of the gene
lists were analyzed for matches to approximately 300 position weight matrices (TFBS)
using the MatInspector module of the GEMS LauncheR 4.1 (Genomatix, Munich,
Germany). A p-value for each of the approximate 300 matrices or TFBS for a gene list
were calculated by comparing the number of matches per 1-kb promoter region in each of
the gene lists against the number of matches per 1-kb promoter region in a reference
background model using a complemented Poisson distribution in the Perl math library.
The reference background model was derived from extracting all unique RefSeq IDs
(24,704) from the UCSC genome browser (5) and mapping them to 15,318 IDs using
Prospector followed by promoter TFBS annotation using MatInspector as described
above. A Perl script was used to calculate p-values for each of the approximately 300
position weight matrices for each of the gene lists- this resulted in what will be referred to
as a regulatory profile that is specific for that particular gene list. Hierarchical clustering
was performed with the Expander tool (6). Network analysis will be done with the
Ingenuity software package.

Results:

For each of the 8 gene lists, a regulatory profile was generated based on the methods
described above. Briefly, each of the gene lists were analyzed to generate p-values
representing the degree of statistical enrichment for each of the approximately 300 TFBS
in the promoter regions of these genes as described in the methods. The tables contain all
the TFBS with a p-value < 0.1 for each of the gene lists.

Table 1:
A. Class 1 3fold Gene List
Table: Class 1
TF gene p-Value EF
M00624[DBP] 10 0.000143 2.604
M00701[SMAD-3] 8 0.003 3.583
M00750[HMG_IY] 9 0.007 1.969
M01109[SZF1-1] 7 0.012 2.04
M00432[TTF1] 7 0.015 2.92
M00257[RREB-1] 12 0.016 1.961
M00655[PEA3] 12 0.017 1.238
M00800[AP-2] 17 0.025 1.734
M00253[cap] 7 0.028 2.588
M00619[Alx-4] 3 0.03 4.556
M00133[Tst-1] 5 0.033 2.315
M00117[C/EBPbeta] 5 0.033 2.249
M01004[Helios_A] 6 0.041 2.123
M00237[AhR:Arnt] 8 0.043 1.687
M00252[TATA] 4 0.043 2.203
M00083[MZF1] 6 0.057 2.382
M00821[Nrf2] 6 0.067 2.225
M00414[AREB6] 5 0.072 1.561
M00987[FOXP1] 11 0.075 1.847
M00960[PR,_GR] 5 0.078 2.326
M00062[IRF-1] 7 0.086 2.044
M00646[LF-A1] 4 0.088 1.826
M01068[UF1H3BETA] 17 0.089 1.332
M00979[PAX6] 7 0.094 1.942
M00959[ER] 7 0.099 1.952

B. Class 2 3fold Gene List

Table: Class 2
TF gene p-Value EF
M00803[E2F] 16 0.002 1.649
M00277[Lmo2_complex] 7 0.004 2.308
M00514[ATF4] 6 0.008 2.437
M00143[Pax-5] 9 0.011 2.559
M00695[ETF] 13 0.011 1.708
M01081[Zec] 3 0.016 1.637
M00221[SREBP-1] 6 0.017 3.249
M00449[Zic2] 6 0.019 3.187
M00333[ZF5] 6 0.02 1.873
M00497[STAT3] 7 0.021 2.891
M00017[ATF] 8 0.024 2.301
M00716[ZF5] 10 0.027 1.497
M00189[AP-2] 8 0.034 1.363
M00237[AhR:Arnt] 6 0.035 2.215
M00938[E2F-1] 7 0.041 1.756
M00649[MAZ] 7 0.046 1.367
M00256[NRSF] 6 0.046 1.785
M00962[AR] 4 0.052 3.085
M00414[AREB6] 5 0.059 2.731
M00134[HNF-4] 4 0.06 2.1
M00332[Whn] 4 0.062 2.055
M00285[TCF11] 2 0.076 1.338
M00650[MTF-1] 6 0.08 1.955
M00624[DBP] 4 0.094 1.823
M00940[E2F-1] 4 0.096 2.567
M00800[AP-2] 8 0.099 1.428
M00744[POU1F1] 4 0.1 2.01
M01003[Helios_A] 2 0.1 1.902
M00005[AP-4] 4 0.104 1.786

C. Class 1 Low Stringent Gene List

Table: All genes upregulated in class 1 tumors
TF gene p-Value EF
M00498[STAT4] 90 0.000474 1.413
M00287[NF-Y] 113 0.000828 1.392
M00196[Sp1] 254 0.000851 1.192
M00652[Nrf-1] 149 0.001 1.249
M00216[TATA] 50 0.002 1.547
M00237[AhR:Arnt] 84 0.003 1.3
M00024[E2F] 34 0.01 1.445
M00133[Tst-1] 42 0.012 1.427
M00115[Tax/CREB] 49 0.012 1.452
M00148[SRY] 134 0.013 1.239
M00291[Freac-3] 61 0.017 1.315
M00135[Oct-1] 59 0.018 1.307
M00145[Brn-2] 43 0.02 1.332
M00129[HFH-1] 68 0.021 1.301
M00099[S8] 48 0.022 1.227
M00418[TGIF] 48 0.023 1.363
M00797[HIF-1] 126 0.023 1.219
M00658[PU.1] 66 0.024 1.136
M00360[Pax-3] 58 0.024 1.209
M00716[ZF5] 195 0.028 1.224
M01066[BLIMP1] 79 0.028 1.286
M00979[PAX6] 65 0.035 1.323
M00152[SRF] 27 0.036 1.341
M00695[ETF] 203 0.037 1.118
M00650[MTF-1] 93 0.042 1.271
M00235[AhR:Arnt] 57 0.044 1.27
M00117[C/EBPbeta] 38 0.045 1.254
M00243[Egr-1] 117 0.047 1.121
M00807[EGR] 149 0.055 1.16
M00017[ATF] 97 0.06 1.169
M00441[GBF] 55 0.065 1.29
M00493[STAT5A] 55 0.066 1.254
M00322[c-Myc:Max] 113 0.068 1.088
M00938[E2F-1] 101 0.072 1.062
M00514[ATF4] 70 0.072 1.192
M00619[Alx-4] 14 0.075 1.56
M00456[FAC1] 58 0.078 1.224
M00189[AP-2] 160 0.081 1.143
M00062[IRF-1] 58 0.083 1.243
M00915[AP-2] 150 0.086 1.146
M01020[TBX5] 32 0.088 1.341
M00485[Nkx2-2] 42 0.09 1.112
M00042[Sox-5] 51 0.099 1.265

D. Class 2 3fold Gene List

Table: All genes upregulated in class 2 tumors
TF gene p-Value EF
M00695[ETF] 357 0.000000000
1.69E-14 1.317
M00716[ZF5] 319 0.0000000000161 1.342
M00803[E2F] 438 0225 1.268
M00652[Nrf-1] 247 0.000000443 1.388
M00976[AHRHIF] 192 0.00000191 1.376
M00800[AP-2] 256 0.00000241 1.284
M00469[AP-2alpha] 173 0.0000123 1.365
M00778[AhR] 112 0.0000852 1.405
M01072[HIC1] 275 0.00009 1.224
M00196[Sp1] 375 0.000116 1.179
M00938[E2F-1] 187 0.000116 1.317
M00189[AP-2] 251 0.0002 1.201
M00915[AP-2] 235 0.000473 1.203
M00322[c-Myc:Max] 193 0.000954 1.245
M00287[NF-Y] 142 0.003 1.172
M00237[AhR:Arnt] 125 0.004 1.296
M00333[ZF5] 143 0.004 1.254
M00797[HIF-1] 184 0.004 1.193
M00062[IRF-1] 90 0.005 1.292
M00650[MTF-1] 138 0.006 1.263
M00430[E2F-1] 35 0.009 1.474
M00807[EGR] 219 0.013 1.142
M01104[MOVO-B] 237 0.016 1.113
M00453[IRF-7] 84 0.021 1.281
M00285[TCF11] 66 0.027 1.24
M00243[Egr-1] 174 0.03 1.117
M00033[p300] 81 0.034 1.152
M00251[XBP-1] 106 0.036 1.205
M00148[SRY] 175 0.04 1.084
M00004[c-Myb] 99 0.042 1.184
M00377[Pax-4] 53 0.042 1.198
M00116[C/EBPalpha] 68 0.044 1.248
M00441[GBF] 74 0.057 1.162
M00649[MAZ] 205 0.058 1.124
M00373[Pax-4] 152 0.058 1.166
M00997[DEC] 68 0.06 1.198
M01029[TFE] 23 0.075 1.384
M00801[CREB] 104 0.079 1.165
M00017[ATF] 133 0.082 1.074
M00256[NRSF] 131 0.083 1.094
M00055[N-Myc] 129 0.091 1.175
M00531[NERF1a] 114 0.092 1.146
M00706[TFII-I] 133 0.096 1.13
E. Chrom3_Up Gene list
Table: Chrom3 up
TF gene p-Value EF
M00803[E2F] 96 5.08E-05 1.252
M00716[ZF5] 80 1.23E-04 1.516
M00695[ETF] 79 1.57E-04 1.314
M00322[c-Myc:Max] 50 0.004 1.454
M00649[MAZ] 49 0.006 1.211
M00109[C/EBPbeta] 20 0.006 1.452
M00189[AP-2] 59 0.007 1.272
M00414[AREB6] 24 0.008 1.659
M01068[UF1H3BETA] 73 0.009 1.267
M00333[ZF5] 35 0.01 1.383
M00256[NRSF] 40 0.012 1.506
M01032[HNF4] 60 0.015 1.256
M00915[AP-2] 55 0.017 1.269
M00243[Egr-1] 49 0.018 1.418
M00777[STAT] 19 0.019 1.682
M01033[HNF4] 98 0.02 1.128
M00449[Zic2] 25 0.022 1.681
M00632[GATA-4] 15 0.024 1.473
M00193[NF-1] 17 0.024 1.567
M00800[AP-2] 58 0.024 1.311
M00938[E2F-1] 43 0.026 1.365
M00974[SMAD] 16 0.027 1.661
M00802[Pit-1] 25 0.027 1.399
M00285[TCF11] 19 0.029 1.609
M00721[CACCC-binding_factor] 29 0.032 1.34
M00116[C/EBPalpha] 18 0.035 1.488
M00062[IRF-1] 24 0.037 1.552
M00237[AhR:Arnt] 31 0.039 1.448
M00720[CAC-binding_protein] 41 0.041 1.417
M00011[Evi-1] 14 0.042 1.628
M00419[MEIS1] 16 0.045 1.655
M00277[Lmo2_complex] 34 0.047 1.419
M00701[SMAD-3] 15 0.047 1.488
M01047[AP-2alphaA] 22 0.048 1.384

F. Chrom3_down Gene List

Table: Chrom3 Down
TF gene p-Value EF
M00403[aMEF-2] 16 0.008 2.013
M00999[AIRE] 13 0.009 1.754
M01124[Oct-4_(POU5F1)] 17 0.015 1.555
M00097[Pax-6] 12 0.024 1.532
M01081[Zec] 14 0.027 1.389
M00499[STAT5A] 17 0.03 1.573
M00138[Oct-1] 26 0.032 1.491
M00042[Sox-5] 13 0.033 1.398
M00423[FOXJ2] 17 0.033 1.67
M00420[MEIS1A:HOXA9] 11 0.035 1.775
M00672[TEF] 17 0.037 1.48
M00478[Cdc5] 12 0.039 1.623
M00256[NRSF] 29 0.042 1.569
M00725[HP1_site_factor] 11 0.045 1.752
M00292[Freac-4] 15 0.046 1.629
M00991[CDX] 18 0.051 1.442
M00472[FOXO4] 17 0.053 1.583
M00456[FAC1] 16 0.059 1.465
M00086[Ik-1] 11 0.063 1.792
M00377[Pax-4] 11 0.064 1.61
M00744[POU1F1] 17 0.071 1.553
M00987[FOXP1] 26 0.075 1.389
M00115[Tax/CREB] 12 0.075 1.542
M00037[NF-E2] 13 0.079 1.636
M00191[ER] 14 0.08 1.602
M00136[Oct-1] 9 0.084 1.29
M00720[CAC-
binding_protein] 28 0.085 1.39
M00935[NF-AT] 11 0.087 1.674
M00729[Cdx-2] 10 0.09 1.318
M00291[Freac-3] 15 0.09 1.403
M00201[C/EBP] 11 0.092 1.636
M00250[Gfi-1] 11 0.093 1.489
M00493[STAT5A] 14 0.099 1.384

Figure 1. Hierarchical clustering was performed on the 8 regulatory profiles to not only
look for similarities and differences between each of the regulatory profiles based on
differences in statistical enrichment of TFBS (A) but to also look for clusters of TFBS
that could potentially be co-regulated (B).

Ingenuity network analysis allows us to understand the impact of a set of genes on well-
characterized pathways. We can combine the genes in each gene list with the
transcription factor gene cognate for the most significantly enriched TFBS for that gene
list. This in essence creates a composite list of the regulated and regulator which will be
referred to as a gene set. We can then input these sets of genes into the Ingenuity network
analysis which overlays a set of genes onto a global molecular network based on
information in the Ingenuity Pathways knowledge base. Each edge represents a biological
relationship between 2 genes and all edges are supported by at least 1 reference from
literature, from a textbook, or from canonical information stored in Ingenuity knowledge
base. For each of these gene sets, a table indicating the top networks for that gene set was
created and a few select networks are shown. For Ingenuity network analysis, only genes
from class 1 gene list and class 2 gene lists were used. The rest of the tables and figures
are derived from data generated by the Ingenuity network analysis.

Table 2. Ingenuity network analysis of class 2 gene set.

Figure 2. Two networks (A and B) for the class 2 gene set are shown.

Table 3. Ingenuity network analysis of class 1 gene set.

Figure 3. Three networks (A,B, and C) for the class 1 gene set are shown.

Table 4: The networks generated by the Ingenuity Network Analysis for each of the
classes are compared. Genes that are common in both the class 1 and class 2 gene sets are
highlighted blue. Genes that are specific to only class 1 are highlighted red and genes that
are specific to only class 2 are highlighted yellow.

Table: Network Comparisons

Gene Sets Network Class 1 Class 2

Gene Signatures Network 1 CEBPB, SMAD3, NFkB, MAPK,
+ Regulators Akt, Erk, TFAP2A, ERBB3,
Jnk, SPP1, ARNT, DBP,
insulin, ERBB3,
Network 2 Beta-estradiol, HNF4A
Network 3 TP53, TNF, progesterone, TP73,
ENPP2, dihydrotestosterone
Stat3, ERK, NFkB, ATF1,
ATF4, Creb, Akt, SREBF1,
MAPK, insulin, SHC1,
ARNT, PAX5, PDGFBB
TFAP2A, TP53, TP73, SP1,
SP3, TGFB1
Table 5. Canonical Pathways are core pathway established for a given molecule in the
cell in which molecular interactions occur in a linear and stepwise manner. Highly
significant canonical pathways for each of the gene lists are generated below.
Table: Top Canonical Pathways
Name p-value Ratio
Class 2 Genes + Regulators
ERK/MAPK Signaling 8.71E-04 4/188 (0.021)
p53 Signaling 1.39E-03 3/87 (0.034)
Huntington's Disease Signaling 1.59E-03 4/232 (0.017)
Estrogen Receptor Signaling 2.38E-03 3/121 (0.025)
Oncostatin M Signaling 4.00E-03 2/38 (0.053)
Class 1 Genes + Regulators
Aryl Hydrocarbon Receptor Signaling 1.02E-02 3/155 (0.019)
Glutathione Metabolism 1.15E-02 2/99 (0.02)
VDR/RXR Activation 2.61E-02 2/80 (0.025)
Xenobiotic Metabolism Signaling 4.09E-02 3/251 (0.012)
Riboflavin Metabolism 4.80E-02 1/49 (0.02)
Class 2 Genes
Estrogen Receptor Signaling 3.98E-04 3/121 (0.025)
p53 Signaling 6.54E-03 2/87 (0.023)
Neuregulin Signaling 6.68E-03 2/98 (0.02)
Huntington's Disease Signaling 3.17E-02 2/232 (0.009)
Glucocorticoid Receptor Signaling 4.49E-02 2/276 (0.007)
Class 1 Genes
Glutathione Metabolism 6.72E-03 2/99 (0.02)
Riboflavin Metabolism 3.65E-02 1/49 (0.02)
Metabolism of Xenobiotics by Cytochrome P450 3.85E-02 2/208 (0.01)
Pantothenate and CoA Biosynthesis 3.89E-02 1/63 (0.016)
Aryl Hydrocarbon Receptor Signaling 4.54E-02 2/155 (0.013)

Discussion

AP2 is enriched predominantly in the class 2 gene signature. AP-2 found on the
short arm of chromosome 6 near the HLA locus and it is implicated in the regulation of
cell proliferation, differentiation, apoptosis, and carcinogenesis. AP2 known to play a
role in metastasis of cutaneous melanoma. Studies by Bar-Eli et al have shown
progression of cutaneous melanoma associated with loss of expression of AP-2, resulting
in the overexpression of MCAM/MUC18 and MMP-2, and lack of expression of c-KIT.
Additionally, they found AP2 regulates additional genes involved in melanoma
development and progression, including E-cadherin, p21, HER2, Bcl-2, FAS/APO-1,
IGF-R-1, and VEGF (7). However, the role of AP2 in uveal melanoma is not well known

Table 6. Enrichment of AP2 in each of the gene lists (highlighted yellow). POM_up
refers to Class 2 Low Stringent Gene List and POM_down refers to Class 1 Low
Stringent Gene List. 62_class1 refers to Class 1 Gene List and 62_class2 refers to Class 2
Gene List.
Table: Transcriptional Regulation by AP-2
TF pom_down pom_up 62_class1 62_class2 pom_3fold_uppom_3fold_down
M00189[AP-2] 0.081 0.0002 0.552 0.034 0.424 0.411
M00915[AP-2] 0.086 0.000473 0.529 0.462 0.384 0.875
M00800[AP-2] 0.558 0.00000241 0.025 0.099 0.333 0.83
M00469[AP-2alpha] 0.124 0.0000123 0.16 0.163 0.799 0.459
M01047[AP-2alphaA] 0.217 0.318 0.726 0.37 0.355 0.422
M01045[AP-2alphaA] 0.239 0.255 0.519 0.176 0.626 0.994
M00468[AP-2rep] 0.852 0.987 0.186 0.823 0.951 0.412

ATF has also been found to be significantly enriched in the class 2 tumor gene lists
(see above tables and figures). ATF has also been implicated in cutaneous melanoma
progression, particularly the transition of melanoma cells from radial growth phase to
vertical growth phase is associated with overexpression of CREB and ATF1.
Additionally, Anti-ATF-1 inhibited tumorigenicity and metastatic potential of cutaneous
melanoma cells in nude mice (7). However, the role of ATF in uveal melanoma has never
been well described.
According to Tables 4 and 5, the MapK/ERK signaling pathway seems to play a role
in both class 1 and class 2 phenotypes. A study by Zuidervaart et al (8) suggests that
activation of MAPK pathway is commonly involved in the development of uveal
melanoma, but rarely occurs through mutation of BRAF or RAS. According to Table 5,
estrogen receptor signaling could potentially play an important role in the class 2
phenotype. Tamoxifen has long been a mainstay of treatment for cutaneous melanoma.
The role of tamoxifen in uveal melanoma has never been well studied, although a small
study by Macneil et al found that tamoxifen inhibited ocular melanoma cell attachment to
matrix proteins (9) and could therefore potentially inhibit metastatic spread.

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