BACKGROUND
the majority of sporadic forms of colorectal cancer are characterized by deregulation of wnt/β-catenin signaling resulting in increased transcriptional activity of the protein β-catenin.
in colon cells, a protein complex consisting of the protein adenomatous polyposis coli , the scaffolding protein axin, casein kinase  <dig>  and glycogen synthase kinase 3β  tightly controls the transcriptional activity of the β-catenin protein by targeting it for proteasomal degradation.
stimulation of colon cells with wnt proteins results in the disruption of the apc protein complex and stabilization of the β-catenin protein.
β-catenin is then transported into the nucleus where it recruits cofactors of the tcf/lef family to activate the transcription of its target genes  <cit> .
in colorectal tumor cells, the tumor suppressor gene apc is frequently affected by loss of heterozygosity  as well as mutations, resulting in inactivation of the apc protein complex targeting β-catenin for degradation  <cit> .
consequently, β-catenin activity is deregulated and promotes aberrant transcription of its target genes  <cit> .
β-catenin target genes have been implicated in regulating different cellular processes including proliferation , stem cell fate , survival , differentiation , migration , and angiogenesis   <cit> .
while these β-catenin functions play an important role in embryonic development and tissue homeostasis, they can also contribute to the initiation and progression of colon cancer.
in particular, deregulation of genes involved in proliferation and migration is frequently observed in colorectal carcinomas  <cit> .
genes encoding members of the basic helix-loop-helix  family of proteins, like ascl <dig>  hath <dig>  and itf <dig>  represent one of the largest groups of genes on the list of previously published β-catenin target genes  <cit> .
members of the bhlh protein family are characterized by the presence of a basic dna binding domain and a hlh protein-protein interaction domain.
based on their primary sequence, bhlh proteins are grouped into seven classes and are known to form homo- and heterodimers to regulate their functions  <cit> .
for example, the class i bhlh protein itf-2b encoded by the gene itf <dig> is known to interact with class ii and class v bhlh proteins  <cit> .
the colon carcinoma cell lines, dld <dig> and sw <dig>  are frequently used as model systems to study colorectal carcinogenesis in general and β-catenin function in particular.
to identify target genes that are commonly regulated by β-catenin, we chose these non-isogenic colorectal cancer cell lines for our experiments.
while dld <dig> cells are derived from a microsatellite instable  tumor, sw <dig> tumor cells are characterized by chromosomal instability   <cit> .
in both cell lines the apc gene is affected by loh on chromosome  <dig> and contains inactivating mutations in the remaining apc allele  <cit> .
in contrast, the msi colon carcinoma cell line ls174t –that was used for validation purposes– expresses wild-type apc and a mutated β-catenin protein  <cit> .
here, we analyzed the gene expression profiles of dld <dig> and sw <dig> cells after down regulation of β-catenin expression and compared the potential β-catenin target genes that are similarly regulated in dld <dig> and sw <dig> cells with a comparable data set published for ls174t cells in the gene expression omnibus  database as well as confirmed β-catenin target genes found in the pubmed database.
we also identified  <dig> kyoto encyclopedia of genes and genomes  pathways that are regulated similarly in dld <dig> and sw <dig> cells.
in addition, one kegg pathway was regulated in all three cell lines.
based on the large number of genes that are regulated in a similar way in dld <dig>  sw <dig>  and ls174t cells as well as the large overlap with previously published β-catenin target genes, we conclude that dld <dig> and sw <dig> are good model systems to study β-catenin target genes and signaling pathways.
furthermore, we were able to confirm a large number of previously published β-catenin target genes and identified many new potential β-catenin target genes.
therefore, this data is a useful resource for further studies to elucidate the role of β-catenin signaling in colorectal cancer.
RESULTS
identification of β-catenin target genes in dld <dig> and sw <dig> colon carcinoma cells
to test the efficacy of the β-catenin sirna used for our dna microarray experiments, dld <dig> and sw <dig> cells were treated with this β-catenin sirna and β-catenin protein expression was analyzed using immuno detection.
since we observed the strongest down regulation of β-catenin protein after sirna treatment for  <dig> hrs, cells were treated for this period of time.
compared to dld <dig> and sw <dig> cells treated with control sirna , treatment with β-catenin sirna  attenuated β-catenin protein expression levels in both cell lines .
similarly, treatment of dld <dig> and sw <dig> cells with β-catenin sirna strongly reduced the activity of the β-catenin dependent 8×topflash reporter gene construct, confirming the efficacy of the β-catenin sirna .
to identify commonly regulated β-catenin target genes in colorectal cancer cell lines, we analyzed the expression of β-catenin target genes in dld <dig> and sw <dig> cells after sirna treatment of these cells using dna microarrays.
treatment of dld <dig> and sw <dig> cells with sirna targeting β-catenin resulted in differential expression of  <dig> and  <dig> genes, respectively .
the intersecting set comprised  <dig> genes, which are regulated in the same direction in both cell lines .
we compared the lists of potential β-catenin target genes obtained for dld <dig> and sw <dig> cells to a previously published data set containing differentially expressed genes after treatment of the colon carcinoma cell line ls174t with β-catenin shrna.
out of the  <dig> differentially expressed genes in ls174t cells ,  <dig> genes and  <dig> genes were regulated similarly in dld <dig> and sw <dig> cells, respectively.
furthermore, we identified  <dig> genes that are regulated in the same direction in all three cell lines .
out of the  <dig> genes that are up or down regulated in dld <dig>  sw <dig>  and ls174t cells,  <dig> genes  have previously been described as β-catenin target genes  as well as the fold change  for each gene).
in addition, we identified the genes abcb <dig>  cd <dig>  fgf <dig>  and hef <dig>  as potential β-catenin target genes in dld <dig> and sw <dig> cells  as well as the fold change  for each gene).
based on a pubmed search, we identified  <dig> β-catenin target genes.
nusse et al.
have previously reported  <dig> of these genes on their web site.
.
the gene and protein names follow the naming convention of the swiss-prot database .
a regulation of a given target gene by β-catenin had to be confirmed by chromatin immune precipitation , electrophoretic mobility shift assay , or reporter gene assays  with mutated tcf binding sites to be classified as “direct”.
based on a literature search in the pubmed database, we identified  <dig> genes that are regulated in a β-catenin dependent manner in addition to the  <dig> β-catenin target genes published by roel nusse.
<dig> out of these  <dig> genes are directly regulated by β-catenin as demonstrated by chip, emsa, or rga with mutated  tcf <dig> binding sites .
when we re-analyzed the microarray data focusing our analysis only on the list of these  <dig> previously published β-catenin target genes, we identified  <dig> ,  <dig> , and  <dig>  of these genes as differentially regulated in the three cell lines, respectively .
the genes ascl <dig>  axin <dig>  myc, notch <dig>  s100a <dig>  and sp <dig> were regulated in dld <dig>  sw <dig>  and ls174t cells.
moreover, we found that the genes abcb <dig>  cd <dig>  fgf <dig>  hef <dig> , and itf <dig>  were also differentially regulated in dld <dig> and sw <dig> cells .
thus, our second analysis using a limited number of genes confirmed our first microarray analysis using all differentially expressed genes and identified the genes itf <dig> , notch <dig> and sp <dig> as β-catenin target genes in addition to abcb <dig>  ascl <dig>  axin <dig>  cd <dig>  fgf <dig>  myc, hef <dig> , and s100a <dig> found in the first analysis.
due to the large number of genes encoding members of the basic helix-loop-helix  family of proteins among the list of β-catenin target genes , we were interested in identifying more members of this family of genes in our microarray data set.
when we re-analyzed the microarray data limiting our analysis to the  <dig> bhlh proteins listed in the pfam database, we identified the genes ascl <dig> and myc as differentially regulated in dld <dig>  sw <dig>  and ls174t cells .
in dld <dig> and sw <dig> cells, the genes id <dig>  and itf <dig>  were also differentially regulated .
taken together, out of the six β-catenin target genes belonging to the bhlh family of proteins , three genes , myc) are commonly regulated in dld <dig> and sw <dig> cells.
using the software package “cytoscape” in combination with the “michigan molecular interactions”  plugin, we searched the list of  <dig> genes that are differentially regulated in dld <dig>  sw <dig>  and ls174t cells for known interactions.
we identified three networks that contained three or more nodes  .
the largest network centered on β-catenin comprised  <dig> genes, while the second largest network with  <dig> genes contained the gene ywhaz encoding the 14-3- <dig> protein isoforms ξ/δ at its center.
the smallest network contained the three nodes net <dig>  arhgap <dig>  and depdc <dig> .
when we focused our analysis on the list of  <dig> genes that are differentially regulated in dld <dig> and sw <dig> cells, we identified the same three networks that we found in the intersection of dld <dig>  sw <dig>  and ls174t cells.
however, the network centered on β-catenin was increased to  <dig> genes, while the network containing the gene ywhaz comprised two additional nodes .
the smallest network contained the four nodes net <dig>  arhgap <dig>  abr and depdc <dig> .
interestingly, both panels highlight the β-catenin target gene myc as a major player with  <dig> and  <dig> directly regulated genes, respectively .
apart from identifying potential β-catenin target genes, we were interested to find out if particular signaling pathways or cellular functions are regulated by β-catenin.
we used the gene set enrichment software  software  <cit>  to identify pathways associated with β-catenin sirna treatment.
in dld <dig> and sw <dig> cells treated with control sirna  <dig> kegg pathways were enriched.
these pathways comprised the glycine, serine and threonine metabolism as well as the renin angiotensin system.
after down regulation of β-catenin expression levels, the following  <dig> kegg pathways were enriched in dld <dig> and sw <dig> cells: endocytosis, insulin signaling pathway, lysosome fc gamma r mediated phagocytosis, apoptosis, regulation of actin cytoskeleton, adipocytokine signaling, glycerolipid metabolism, lysosome and aldosterone regulated sodium reabsorption .
in addition, the steroid hormone biosynthesis pathway was enriched in dld <dig>  sw <dig>  and ls174t cells after treatment with β-catenin sirna .
apart from the kegg pathway database, we also used the biocarta pathway database in the gene set enrichment analysis.
down regulation of β-catenin resulted in the enrichment of the m-calpain pathway, the creb pathway, and the igf1r pathway in dld <dig> and sw <dig> cells .
gene set enrichment analyses were performed after treatment of dld <dig>  sw <dig>  and ls174t cells with β-catenin rnai or β-galactosidase rnai using the kegg and the biocarta pathway database, respectively.
pathways that were enriched in more than one cell line were identified using a biovenn software package.
discussion
β-catenin plays a crucial role in embryogenesis, tissue homeostasis and carcinogenesis.
because of its role in regulating homeostasis of the intestinal tract as well as being a key player in sporadic forms of colorectal cancer, inhibition of β-catenin is not a suitable strategy to treat patients with colorectal cancer.
for this reason, identifying and characterizing β-catenin target genes could result in better understanding of colorectal carcinogenesis and development of new therapies.
here, we used two non-isogenic colorectal cancer cell lines, dld <dig>  and sw <dig>  and identified  <dig> commonly regulated β-catenin target genes.
<dig> of these genes were also differentially regulated in ls174t cells.
compared to the number of differentially regulated genes in ls174t and dld <dig> cells  as well as in ls174t and sw <dig> cells , the number of commonly regulated genes in dld <dig> and sw <dig>  was low, resulting in a low number of commonly regulated genes in dld <dig>  sw <dig>  and ls174t cells .
while dld <dig> and sw <dig> cells both express a mutated form of apc, they differ with respect to the underlying genetic disturbance: dld <dig> cells show microsatellite instability , whereas sw <dig> cells are derived from a tumor with chromosomal instability .
however, sw <dig> cells  share a lot of commonly regulated genes with ls174t cells , suggesting that other factors apart from genetic instability contribute to the observed differences in gene expression between dld <dig> and sw <dig> cells.
looking at the mutational status, dld <dig>  sw <dig>  and ls174t cells differ with respect to the status of the genes apc, pi3k, and tp <dig>  but they are identical with respect to status of the genes kras and pten: dld <dig> , sw <dig> , and ls174t cells   <cit> .
deregulation of pi3k/akt signaling  has been implicated in the phosphorylation of β-catenin at ser <dig>  <cit> , thereby promoting nuclear accumulation and enhancing β-catenin dependent transcriptional activity even in the absence of aberrant wnt signaling  <cit> .
this observation suggests that in particular ls174t cells that express wild-type apc could benefit from a mutation in the pi3k gene.
but even dld <dig> cells  could profit from pi3k mutations: deming et al.
demonstrated that expression of a dominant active form of pi3k in apcmin/+ mice resulted in an increased tumor number and size and implicated the ccnd <dig> gene as one transcriptional target that contributes to the observed phenotype of these mice  <cit> .
in our experiments, ccnd <dig> mrna expression levels were up regulated in ls174t cells, but not in dld <dig> cells.
the tumor suppressor p <dig> has been implicated in down-regulating β-catenin expression and/or activity by siah- <dig> dependent  degradation  <cit> , reduction of tcf <dig> mrna and protein levels  <cit>  as well as reduction of β-catenin mrna by p <dig> dependent regulation of mirna mir- <dig>  <cit> .
accordingly, inactivation of the transcriptional activity of p <dig> by mutations –as observed in sw <dig> cells– should result in increased β-catenin mrna and protein levels and transcriptional activity as well as increased mrna and protein levels of tcf <dig> .
interestingly, ls174t cells showed a strong increase in the fold change value for ctnnb <dig>   and  <dig>  , respectively).
all three cell lines showed slightly reduced fold change values for tcf <dig> .
the effect of p <dig> inactivation on the development of intestinal adenomas was also analyzed in apcmin/+ mice: despite the fact that p <dig> protein expression is deregulated as a consequence of apc loss  and p <dig> is known to regulate β-catenin expression via a negative feedback loop, loss of p <dig> did not promote the initial steps of intestinal neoplasia in apcmin/+ mice  <cit> , suggesting that p <dig> has only a limited role in this mouse model.
apart from regulating β-catenin protein expression levels via proteasomal degradation,  apc has been implicated in affecting β-catenin activity by recruiting co-repressors of β-catenin to promoter regions, as well as sequestering and enhancing the nuclear export of β-catenin  <cit> .
mutated forms of apc still show a nuclear localization, but they are more frequently observed in the cytoplasm when compared to wild-type apc, suggesting that mutation of apc is not only contributing to higher β-catenin protein expression levels, but also contribute to enhanced β-catenin activity  <cit> .
when zeineldin et al.
generated mice expressing apc with mutated nuclear localization signals  and compared these apcmnls/mnls mice with apc+/+ mice, they found up regulation of the mrna for the genes axin <dig>  myc, and ccdn <dig> as well as down regulation of hath <dig> mrna in response to stimulation with wnt  <cit> .
accordingly, we expected an increase in the fold change values for axin <dig>  myc, and ccdn <dig> transcripts as well as a reduction of hath <dig> transcripts in dld <dig> and sw <dig> cells  compared to ls174t .
in our experiments, dld <dig> and sw <dig> showed a modest increase in the fold change values for myc  compared to ls174t cells.
however, ls174t  cells responded with a higher fold change for axin <dig> compared to dld <dig> cells.
ccnd <dig> transcript levels were increased only in ls174t cells, whereas hath <dig> transcript levels did not changed in any of three cell lines.
the differential expression of the genes axin <dig> and ccnd <dig> in the three colorectal cancer cell lines highlights the fact that the genetic background of a given cell has a major impact on the expression of a specific gene.
however, linking a specific somatic mutation in a tumor suppressor gene or proto oncogene, e.g.
apc, pi3k, or tp <dig>  to these differences in expression is difficult because the expression of a specific gene is affected by multiple transcription factors .
therefore, we are not able to attribute the observed differences in the gene expression profiles for dld <dig> and sw <dig> to a single mutation.
compared to ls174t cells, the total number of differentially regulated β-catenin target genes was much lower in dld <dig> and sw <dig> cells.
this could be either specific to the genetic background of the cell lines used or due to different experimental approaches.
while we transiently transfected dld <dig> and sw <dig> cells with β-catenin sirna and analyzed the gene expression  <dig> hours after transfection, mokry et al.
<cit>  stably transfected ls174t cells with a doxycycline inducible expression vector encoding β-catenin shrna and stimulated the cells for  <dig> hours with doxycycline before analyzing the gene expression profile.
both research groups used affymetrix human genome u <dig> plus  <dig>  dna microarrays for their experiments.
the longer incubation period used to identify potential β-catenin target genes in ls174t cells might contribute to secondary effects like the differential expression of indirect β-catenin target genes, thus explaining why the absolute number of differentially regulated genes was higher in ls174t cells when compared to dld <dig> and sw <dig> cells.
for this reason, we were particularly interested in genes that are up and down regulated in the non-isogenic colorectal cancer cell lines dld <dig> and sw <dig> or dld <dig>  sw <dig>  and ls174t cells, assuming that these genes are commonly and directly regulated by β-catenin.
remarkably, the number of β-catenin regulated genes was very similar in the three cell lines when focusing on the list of  <dig> previously described β-catenin target genes.
here, dld <dig>  sw <dig> and ls174t cells differentially regulated  <dig>   <dig> and  <dig> of these genes, respectively.
there are at least two reasons why the number of detected β-catenin target genes did not include  all  <dig> β-catenin target genes presented in table  <dig>  first, the detection of β-catenin target genes is dependent on the cellular context.
dld <dig>  sw <dig>  and ls174t represent colorectal cancer cell lines.
therefore, the expression pattern of β-catenin target genes in these cells is likely to differ from β-catenin target genes found in healthy colon cells, colonic adenomas, or metastatic colonic cells that are also listed in table  <dig>  second, the gene expression profile was analyzed  <dig> and  <dig> hours after beginning with the rnai treatment, respectively.
different periods of time influence the identification of β-catenin target genes as well, since some of these genes might be regulated only for a short period of time immediately after induction of β-catenin activity.
this phenomenon has been described by van de wetering et al.
: they transfected ls174t cells with doxycycline  inducible expression vectors encoding dntcfs and analyzed the changes in gene expression using dna microarrays.
<dig> h after treatment with dox,  <dig> genes where differentially regulated.
after an incubation period of  <dig> h, they identified  <dig> differentially regulated genes.
comparison of the two sets of genes revealed that the expression of  <dig> genes was abrogated between  <dig> and  <dig> h after dox treatment, while  <dig> additional genes appeared in this time frame  <cit> .
similarly, another group analyzed the gene expression profiles of several known β-catenin target genes in a time-course experiment and classified these genes according to their expression profiles  <cit> .
based on this criterion, they identified genes that were “immediately up  regulated”, “early up ”, or “late up ” in response to the activation of the wnt/β-catenin signaling cascade.
when comparing their results to the regulation of the same genes in different cellular contexts, this group found that the regulation of these genes was similar, but not necessarily identical in different cell lines.
this observation suggests that the expression levels and activities of a given transcription factor and its cofactors  influence the starting point and duration of the gene expression of a specific target gene.
therefore, it is recommendable to identify target genes of a given transcription factor in different cell lines at least at the same time points, better yet in a time-course experiment.
using the kegg pathway database, we identified  <dig> signaling pathways or cellular functions in dld <dig> and sw <dig> cells that are differentially regulated in response to treatment of the colorectal cancer cell lines with β-catenin sirna.
in addition, the steroid hormone biosynthesis pathway was similarly regulated in all three cell lines analyzed.
whereas the steroid hormone biosynthesis pathway  <cit>  as well as the endocytosis pathway  <cit> , the insulin signaling pathway  <cit> , apoptosis  <cit> , regulation of the actin cytoskeleton  <cit> , and focal adhesion  <cit> , have been associated with wnt/β-catenin signaling, there are no publications in the pubmed database linking β-catenin to the remaining six pathways in colorectal cancer cells.
interestingly, the list of kegg pathways/functions did not include the canonical wnt signaling pathway.
this was probably due to the definition of the “kegg wnt signaling pathway” that summarized the canonical, the planar cell polarity , and the wnt/ca2+ signaling pathway under one database entry, despite different functions of these signaling pathways.
while the canonical wnt pathway has been implicated in regulating the activity of the β-catenin protein, pcp signaling results in remodeling of the cytoskeleton that is a prerequisite for migration and cell polarization via the gtpases rhoa and rac <dig>  wnt/ca2+ signaling, however, plays a role in the regulation of cell adhesion and cell movements during gastrulation and results in the activation of protein kinase , calcium calmodulin mediated kinase ii  and calcineurin  <cit> .
furthermore, all three biocarta pathways that were enriched in dld <dig> and sw <dig> cells have been associated with wnt/β-catenin signaling based on pubmed publications: the m-calpain pathway  <cit> , the creb pathway  <cit> , and the igf1r pathway  <cit> .
since  <dig> out of the  <dig> previously described β-catenin target genes encode proteins that belong to the bhlh protein family , we focused our analysis on this group of genes.
our analysis revealed that the genes ascl <dig>  id <dig>  itf <dig>  and myc were regulated in a β-catenin dependent manner in dld <dig> and sw <dig> cells.
interestingly, the proteins ascl <dig> and itf-2b as well as id <dig> and itf-2b are known to interact with each other  <cit> , suggesting that these bhlh proteins form a functional network in colorectal carcinoma cells.
CONCLUSIONS
in conclusion, by comparing potential β-catenin target genes in dld <dig> and sw <dig> cells with 1) corresponding data from ls174t cells and 2) a list of previously identified β-catenin target genes, we found and confirmed a large number of β-catenin target genes, suggesting that dld <dig> and sw <dig>  are suitable for identifying β-catenin target genes as well as β-catenin dependent signaling pathways and functions.
therefore, the presented list of commonly regulated genes in dld <dig> and sw <dig> cells as well as the annotated list of previously published β-catenin target genes are useful resources for further studies.

 the majority of sporadic forms of colorectal cancer are characterized by deregulation of wnt/β-catenin signaling resulting in increased transcriptional activity of the protein β-catenin. in colon cells, a protein complex consisting of the protein adenomatous polyposis coli, the scaffolding protein axin, casein kinase and glycogen synthase kinase 3β tightly controls the transcriptional activity of the β-catenin protein by targeting it for proteasomal degradation. stimulation of colon cells with wnt proteins results in the disruption of the apc protein complex and stabilization of the β-catenin protein. β-catenin is then transported into the nucleus where it recruits cofactors of the tcf/lef family to activate the transcription of its target genes. in colorectal tumor cells, the tumor suppressor gene apc is frequently affected by loss of heterozygosity as well as mutations, resulting in inactivation of the apc protein complex targeting β-catenin for degradation. consequently, β-catenin activity is deregulated and promotes aberrant transcription of its target genes. β-catenin target genes have been implicated in regulating different cellular processes including proliferation, stem cell fate, survival, differentiation, migration, and angiogenesis. while these β-catenin functions play an important role in embryonic development and tissue homeostasis, they can also contribute to the initiation and progression of colon cancer. in particular, deregulation of genes involved in proliferation and migration is frequently observed in colorectal carcinomas. genes encoding members of the basic helix-loop-helix family of proteins, like ascl hath and itf represent one of the largest groups of genes on the list of previously published β-catenin target genes. members of the bhlh protein family are characterized by the presence of a basic dna binding domain and a hlh protein-protein interaction domain. based on their primary sequence, bhlh proteins are grouped into seven classes and are known to form homo- and heterodimers to regulate their functions. for example, the class i bhlh protein itf-2b encoded by the gene itf is known to interact with class ii and class v bhlh proteins. the colon carcinoma cell lines, dld and sw are frequently used as model systems to study colorectal carcinogenesis in general and β-catenin function in particular. to identify target genes that are commonly regulated by β-catenin, we chose these non-isogenic colorectal cancer cell lines for our experiments. while dld cells are derived from a microsatellite instable tumor, sw tumor cells are characterized by chromosomal instability. in both cell lines the apc gene is affected by loh on chromosome and contains inactivating mutations in the remaining apc allele. in contrast, the msi colon carcinoma cell line ls174t –that was used for validation purposes– expresses wild-type apc and a mutated β-catenin protein. here, we analyzed the gene expression profiles of dld and sw cells after down regulation of β-catenin expression and compared the potential β-catenin target genes that are similarly regulated in dld and sw cells with a comparable data set published for ls174t cells in the gene expression omnibus database as well as confirmed β-catenin target genes found in the pubmed database. we also identified kyoto encyclopedia of genes and genomes pathways that are regulated similarly in dld and sw cells. in addition, one kegg pathway was regulated in all three cell lines. based on the large number of genes that are regulated in a similar way in dld sw and ls174t cells as well as the large overlap with previously published β-catenin target genes, we conclude that dld and sw are good model systems to study β-catenin target genes and signaling pathways. furthermore, we were able to confirm a large number of previously published β-catenin target genes and identified many new potential β-catenin target genes. therefore, this data is a useful resource for further studies to elucidate the role of β-catenin signaling in colorectal cancer.  identification of β-catenin target genes in dld and sw colon carcinoma cells to test the efficacy of the β-catenin sirna used for our dna microarray experiments, dld and sw cells were treated with this β-catenin sirna and β-catenin protein expression was analyzed using immuno detection. since we observed the strongest down regulation of β-catenin protein after sirna treatment for hrs, cells were treated for this period of time. compared to dld and sw cells treated with control sirna, treatment with β-catenin sirna attenuated β-catenin protein expression levels in both cell lines. similarly, treatment of dld and sw cells with β-catenin sirna strongly reduced the activity of the β-catenin dependent 8×topflash reporter gene construct, confirming the efficacy of the β-catenin sirna. to identify commonly regulated β-catenin target genes in colorectal cancer cell lines, we analyzed the expression of β-catenin target genes in dld and sw cells after sirna treatment of these cells using dna microarrays. treatment of dld and sw cells with sirna targeting β-catenin resulted in differential expression of and genes, respectively. the intersecting set comprised genes, which are regulated in the same direction in both cell lines. we compared the lists of potential β-catenin target genes obtained for dld and sw cells to a previously published data set containing differentially expressed genes after treatment of the colon carcinoma cell line ls174t with β-catenin shrna. out of the differentially expressed genes in ls174t cells, genes and genes were regulated similarly in dld and sw cells, respectively. furthermore, we identified genes that are regulated in the same direction in all three cell lines. out of the genes that are up or down regulated in dld sw and ls174t cells, genes have previously been described as β-catenin target genes as well as the fold change for each gene). in addition, we identified the genes abcb cd fgf and hef as potential β-catenin target genes in dld and sw cells as well as the fold change for each gene). based on a pubmed search, we identified β-catenin target genes. nusse et al. have previously reported of these genes on their web site.. the gene and protein names follow the naming convention of the swiss-prot database. a regulation of a given target gene by β-catenin had to be confirmed by chromatin immune precipitation, electrophoretic mobility shift assay, or reporter gene assays with mutated tcf binding sites to be classified as “direct”. based on a literature search in the pubmed database, we identified genes that are regulated in a β-catenin dependent manner in addition to the β-catenin target genes published by roel nusse. out of these genes are directly regulated by β-catenin as demonstrated by chip, emsa, or rga with mutated tcf binding sites. when we re-analyzed the microarray data focusing our analysis only on the list of these previously published β-catenin target genes, we identified,, and of these genes as differentially regulated in the three cell lines, respectively. the genes ascl axin myc, notch s100a and sp were regulated in dld sw and ls174t cells. moreover, we found that the genes abcb cd fgf hef, and itf were also differentially regulated in dld and sw cells. thus, our second analysis using a limited number of genes confirmed our first microarray analysis using all differentially expressed genes and identified the genes itf, notch and sp as β-catenin target genes in addition to abcb ascl axin cd fgf myc, hef, and s100a found in the first analysis. due to the large number of genes encoding members of the basic helix-loop-helix family of proteins among the list of β-catenin target genes, we were interested in identifying more members of this family of genes in our microarray data set. when we re-analyzed the microarray data limiting our analysis to the bhlh proteins listed in the pfam database, we identified the genes ascl and myc as differentially regulated in dld sw and ls174t cells. in dld and sw cells, the genes id and itf were also differentially regulated. taken together, out of the six β-catenin target genes belonging to the bhlh family of proteins, three genes, myc) are commonly regulated in dld and sw cells. using the software package “cytoscape” in combination with the “michigan molecular interactions” plugin, we searched the list of genes that are differentially regulated in dld sw and ls174t cells for known interactions. we identified three networks that contained three or more nodes. the largest network centered on β-catenin comprised genes, while the second largest network with genes contained the gene ywhaz encoding the 14-3- protein isoforms ξ/δ at its center. the smallest network contained the three nodes net arhgap and depdc. when we focused our analysis on the list of genes that are differentially regulated in dld and sw cells, we identified the same three networks that we found in the intersection of dld sw and ls174t cells. however, the network centered on β-catenin was increased to genes, while the network containing the gene ywhaz comprised two additional nodes. the smallest network contained the four nodes net arhgap abr and depdc. interestingly, both panels highlight the β-catenin target gene myc as a major player with and directly regulated genes, respectively. apart from identifying potential β-catenin target genes, we were interested to find out if particular signaling pathways or cellular functions are regulated by β-catenin. we used the gene set enrichment software software to identify pathways associated with β-catenin sirna treatment. in dld and sw cells treated with control sirna kegg pathways were enriched. these pathways comprised the glycine, serine and threonine metabolism as well as the renin angiotensin system. after down regulation of β-catenin expression levels, the following kegg pathways were enriched in dld and sw cells: endocytosis, insulin signaling pathway, lysosome fc gamma r mediated phagocytosis, apoptosis, regulation of actin cytoskeleton, adipocytokine signaling, glycerolipid metabolism, lysosome and aldosterone regulated sodium reabsorption. in addition, the steroid hormone biosynthesis pathway was enriched in dld sw and ls174t cells after treatment with β-catenin sirna. apart from the kegg pathway database, we also used the biocarta pathway database in the gene set enrichment analysis. down regulation of β-catenin resulted in the enrichment of the m-calpain pathway, the creb pathway, and the igf1r pathway in dld and sw cells. gene set enrichment analyses were performed after treatment of dld sw and ls174t cells with β-catenin rnai or β-galactosidase rnai using the kegg and the biocarta pathway database, respectively. pathways that were enriched in more than one cell line were identified using a biovenn software package. discussion β-catenin plays a crucial role in embryogenesis, tissue homeostasis and carcinogenesis. because of its role in regulating homeostasis of the intestinal tract as well as being a key player in sporadic forms of colorectal cancer, inhibition of β-catenin is not a suitable strategy to treat patients with colorectal cancer. for this reason, identifying and characterizing β-catenin target genes could result in better understanding of colorectal carcinogenesis and development of new therapies. here, we used two non-isogenic colorectal cancer cell lines, dld and sw and identified commonly regulated β-catenin target genes. of these genes were also differentially regulated in ls174t cells. compared to the number of differentially regulated genes in ls174t and dld cells as well as in ls174t and sw cells, the number of commonly regulated genes in dld and sw was low, resulting in a low number of commonly regulated genes in dld sw and ls174t cells. while dld and sw cells both express a mutated form of apc, they differ with respect to the underlying genetic disturbance: dld cells show microsatellite instability, whereas sw cells are derived from a tumor with chromosomal instability. however, sw cells share a lot of commonly regulated genes with ls174t cells, suggesting that other factors apart from genetic instability contribute to the observed differences in gene expression between dld and sw cells. looking at the mutational status, dld sw and ls174t cells differ with respect to the status of the genes apc, pi3k, and tp but they are identical with respect to status of the genes kras and pten: dld, sw, and ls174t cells. deregulation of pi3k/akt signaling has been implicated in the phosphorylation of β-catenin at ser, thereby promoting nuclear accumulation and enhancing β-catenin dependent transcriptional activity even in the absence of aberrant wnt signaling. this observation suggests that in particular ls174t cells that express wild-type apc could benefit from a mutation in the pi3k gene. but even dld cells could profit from pi3k mutations: deming et al. demonstrated that expression of a dominant active form of pi3k in apcmin/+ mice resulted in an increased tumor number and size and implicated the ccnd gene as one transcriptional target that contributes to the observed phenotype of these mice. in our experiments, ccnd mrna expression levels were up regulated in ls174t cells, but not in dld cells. the tumor suppressor p has been implicated in down-regulating β-catenin expression and/or activity by siah- dependent degradation, reduction of tcf mrna and protein levels as well as reduction of β-catenin mrna by p dependent regulation of mirna mir-. accordingly, inactivation of the transcriptional activity of p by mutations –as observed in sw cells– should result in increased β-catenin mrna and protein levels and transcriptional activity as well as increased mrna and protein levels of tcf. interestingly, ls174t cells showed a strong increase in the fold change value for ctnnb and, respectively). all three cell lines showed slightly reduced fold change values for tcf. the effect of p inactivation on the development of intestinal adenomas was also analyzed in apcmin/+ mice: despite the fact that p protein expression is deregulated as a consequence of apc loss and p is known to regulate β-catenin expression via a negative feedback loop, loss of p did not promote the initial steps of intestinal neoplasia in apcmin/+ mice, suggesting that p has only a limited role in this mouse model. apart from regulating β-catenin protein expression levels via proteasomal degradation, apc has been implicated in affecting β-catenin activity by recruiting co-repressors of β-catenin to promoter regions, as well as sequestering and enhancing the nuclear export of β-catenin. mutated forms of apc still show a nuclear localization, but they are more frequently observed in the cytoplasm when compared to wild-type apc, suggesting that mutation of apc is not only contributing to higher β-catenin protein expression levels, but also contribute to enhanced β-catenin activity. when zeineldin et al. generated mice expressing apc with mutated nuclear localization signals and compared these apcmnls/mnls mice with apc+/+ mice, they found up regulation of the mrna for the genes axin myc, and ccdn as well as down regulation of hath mrna in response to stimulation with wnt. accordingly, we expected an increase in the fold change values for axin myc, and ccdn transcripts as well as a reduction of hath transcripts in dld and sw cells compared to ls174t. in our experiments, dld and sw showed a modest increase in the fold change values for myc compared to ls174t cells. however, ls174t cells responded with a higher fold change for axin compared to dld cells. ccnd transcript levels were increased only in ls174t cells, whereas hath transcript levels did not changed in any of three cell lines. the differential expression of the genes axin and ccnd in the three colorectal cancer cell lines highlights the fact that the genetic background of a given cell has a major impact on the expression of a specific gene. however, linking a specific somatic mutation in a tumor suppressor gene or proto oncogene, e.g. apc, pi3k, or tp to these differences in expression is difficult because the expression of a specific gene is affected by multiple transcription factors. therefore, we are not able to attribute the observed differences in the gene expression profiles for dld and sw to a single mutation. compared to ls174t cells, the total number of differentially regulated β-catenin target genes was much lower in dld and sw cells. this could be either specific to the genetic background of the cell lines used or due to different experimental approaches. while we transiently transfected dld and sw cells with β-catenin sirna and analyzed the gene expression hours after transfection, mokry et al. stably transfected ls174t cells with a doxycycline inducible expression vector encoding β-catenin shrna and stimulated the cells for hours with doxycycline before analyzing the gene expression profile. both research groups used affymetrix human genome u plus dna microarrays for their experiments. the longer incubation period used to identify potential β-catenin target genes in ls174t cells might contribute to secondary effects like the differential expression of indirect β-catenin target genes, thus explaining why the absolute number of differentially regulated genes was higher in ls174t cells when compared to dld and sw cells. for this reason, we were particularly interested in genes that are up and down regulated in the non-isogenic colorectal cancer cell lines dld and sw or dld sw and ls174t cells, assuming that these genes are commonly and directly regulated by β-catenin. remarkably, the number of β-catenin regulated genes was very similar in the three cell lines when focusing on the list of previously described β-catenin target genes. here, dld sw and ls174t cells differentially regulated and of these genes, respectively. there are at least two reasons why the number of detected β-catenin target genes did not include all β-catenin target genes presented in table  first, the detection of β-catenin target genes is dependent on the cellular context. dld sw and ls174t represent colorectal cancer cell lines. therefore, the expression pattern of β-catenin target genes in these cells is likely to differ from β-catenin target genes found in healthy colon cells, colonic adenomas, or metastatic colonic cells that are also listed in table  second, the gene expression profile was analyzed and hours after beginning with the rnai treatment, respectively. different periods of time influence the identification of β-catenin target genes as well, since some of these genes might be regulated only for a short period of time immediately after induction of β-catenin activity. this phenomenon has been described by van de wetering et al.: they transfected ls174t cells with doxycycline inducible expression vectors encoding dntcfs and analyzed the changes in gene expression using dna microarrays. h after treatment with dox, genes where differentially regulated. after an incubation period of h, they identified differentially regulated genes. comparison of the two sets of genes revealed that the expression of genes was abrogated between and h after dox treatment, while additional genes appeared in this time frame. similarly, another group analyzed the gene expression profiles of several known β-catenin target genes in a time-course experiment and classified these genes according to their expression profiles. based on this criterion, they identified genes that were “immediately up regulated”, “early up ”, or “late up ” in response to the activation of the wnt/β-catenin signaling cascade. when comparing their results to the regulation of the same genes in different cellular contexts, this group found that the regulation of these genes was similar, but not necessarily identical in different cell lines. this observation suggests that the expression levels and activities of a given transcription factor and its cofactors influence the starting point and duration of the gene expression of a specific target gene. therefore, it is recommendable to identify target genes of a given transcription factor in different cell lines at least at the same time points, better yet in a time-course experiment. using the kegg pathway database, we identified signaling pathways or cellular functions in dld and sw cells that are differentially regulated in response to treatment of the colorectal cancer cell lines with β-catenin sirna. in addition, the steroid hormone biosynthesis pathway was similarly regulated in all three cell lines analyzed. whereas the steroid hormone biosynthesis pathway as well as the endocytosis pathway, the insulin signaling pathway, apoptosis, regulation of the actin cytoskeleton, and focal adhesion, have been associated with wnt/β-catenin signaling, there are no publications in the pubmed database linking β-catenin to the remaining six pathways in colorectal cancer cells. interestingly, the list of kegg pathways/functions did not include the canonical wnt signaling pathway. this was probably due to the definition of the “kegg wnt signaling pathway” that summarized the canonical, the planar cell polarity, and the wnt/ca2+ signaling pathway under one database entry, despite different functions of these signaling pathways. while the canonical wnt pathway has been implicated in regulating the activity of the β-catenin protein, pcp signaling results in remodeling of the cytoskeleton that is a prerequisite for migration and cell polarization via the gtpases rhoa and rac wnt/ca2+ signaling, however, plays a role in the regulation of cell adhesion and cell movements during gastrulation and results in the activation of protein kinase, calcium calmodulin mediated kinase ii and calcineurin. furthermore, all three biocarta pathways that were enriched in dld and sw cells have been associated with wnt/β-catenin signaling based on pubmed publications: the m-calpain pathway, the creb pathway, and the igf1r pathway. since out of the previously described β-catenin target genes encode proteins that belong to the bhlh protein family, we focused our analysis on this group of genes. our analysis revealed that the genes ascl id itf and myc were regulated in a β-catenin dependent manner in dld and sw cells. interestingly, the proteins ascl and itf-2b as well as id and itf-2b are known to interact with each other, suggesting that these bhlh proteins form a functional network in colorectal carcinoma cells.  in conclusion, by comparing potential β-catenin target genes in dld and sw cells with 1) corresponding data from ls174t cells and 2) a list of previously identified β-catenin target genes, we found and confirmed a large number of β-catenin target genes, suggesting that dld and sw are suitable for identifying β-catenin target genes as well as β-catenin dependent signaling pathways and functions. therefore, the presented list of commonly regulated genes in dld and sw cells as well as the annotated list of previously published β-catenin target genes are useful resources for further studies. 
to identify genes commonly regulated by β-catenin in colorectal cancer cell lines, we analyzed β-catenin target gene expression in two non-isogenic cell lines, dld and sw using dna microarrays and compared these genes to β-catenin target genes published in the pubmed database and dna microarray data presented in the gene expression omnibus database. deregulation of wnt/β-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein β-catenin. based on the large number of potential β-catenin target genes found to be similarly regulated in dld sw and ls174t cells as well as the large overlap with confirmed β-catenin target genes, we conclude that dld and sw colon carcinoma cell lines are suitable model systems to study wnt/β-catenin signaling and associated colorectal carcinogenesis. treatment of dld and sw cells with β-catenin sirna resulted in differential expression of and genes, respectively. furthermore, the confirmed and the newly identified potential β-catenin target genes are useful starting points for further studies. comparison of these data with published β-catenin target genes for the colon carcinoma cell line ls174t revealed genes that are regulated similarly in all three cell lines. the overlapping gene set includes confirmed β-catenin target genes like axin myc, and ascl we also identified kyoto encyclopedia of genes and genomes pathways that are regulated similarly in dld and sw cells and one pathway – the steroid biosynthesis pathway – was regulated in all three cell lines. of these genes were regulated in the same direction in both cell lines.
 deregulation of wnt/β-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein β-catenin. β-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto-oncogenes myc and ccnd as well as the genes encoding the basic helix-loop-helix proteins ascl and itf-2b. to identify genes commonly regulated by β-catenin in colorectal cancer cell lines, we analyzed β-catenin target gene expression in two non-isogenic cell lines, dld and sw using dna microarrays and compared these genes to β-catenin target genes published in the pubmed database and dna microarray data presented in the gene expression omnibus database.  treatment of dld and sw cells with β-catenin sirna resulted in differential expression of and genes, respectively. of these genes were regulated in the same direction in both cell lines. comparison of these data with published β-catenin target genes for the colon carcinoma cell line ls174t revealed genes that are regulated similarly in all three cell lines. the overlapping gene set includes confirmed β-catenin target genes like axin myc, and ascl we also identified kyoto encyclopedia of genes and genomes pathways that are regulated similarly in dld and sw cells and one pathway – the steroid biosynthesis pathway – was regulated in all three cell lines.  based on the large number of potential β-catenin target genes found to be similarly regulated in dld sw and ls174t cells as well as the large overlap with confirmed β-catenin target genes, we conclude that dld and sw colon carcinoma cell lines are suitable model systems to study wnt/β-catenin signaling and associated colorectal carcinogenesis. furthermore, the confirmed and the newly identified potential β-catenin target genes are useful starting points for further studies. colorectal cancerβ-catenintarget genesdld1sw480ls174tdna microarray 