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12/18/09
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.
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
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
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.
Figure 2. Two networks (A and B) for the class 2 gene set are shown.
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.
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.
References: