Inappropriate or autonomous activation of intracellular signal transduction pathways
is a major cause for increased proliferation of tumour cells. In several major human
cancers, the WNT/β-catenin signal transduction pathway is constitutively activated,
regularly in colon carcinoma and hepatoma, and with considerable frequency in melanoma,
mammary carcinoma, medulloblastoma and in some cases of renal, ovarian, thyroid and
prostate carcinomas (Polakis, 2000). In normal tissues, this pathway is crucial for
regulation of cell growth and differentiation, from embryonic tissue patterning to
adult tissue homeostasis. In particular, WNT signalling may be important for the maintenance
of epithelial stem cell compartments (Taipale and Beachy, 2001). The WNT/β-catenin
pathway elicits activation of specific target genes including the proto-oncogene c-MYC,
the cell cycle activator CCND1 (CyclinD1) and the metalloprotease gene MMP7 (Crawford
et al, 1999) (see http://www.stanford.edu/~rnusse/wntwindow.html for a list of target
genes). In the absence of activating WNT signals, β-catenin is phosphorylated by a
multiprotein complex containing GSK3β, the tumour suppressors APC and axin/conductin.
Phosphorylated β-catenin is recognised by a ubiquitin–ligase complex with β-TrCP as
the crucial component, ubiquitinylated and degraded by the proteasome. Binding of
an activating WNT factor to the Frizzled receptor leads to the inactivation of GSK3β
via activation of Dishevelled and GBP/FRAT and thus to stabilisation of β-catenin,
which accumulates in the cytoplasm, translocates into the nucleus and heterodimerises
with TCF/LEF transcription factors to activate target genes containing specific TCF
binding sites in their promoters. Constitutive activation of WNT/β-catenin signalling
in cancer is caused by a variety of mutations in various components of the pathway,
leading either to inactivation of APC, axin/conductin or β-TrCP or to oncogenic activation
of β-catenin. In addition, recent evidence suggests that the activity of the pathway
may also be influenced by further factors, notably by the status of the E-cadherin
cell adhesion protein (Stockinger et al, 2001). Whichever mechanism causes constitutive
activation of the WNT/β-catenin pathway, its activity is reflected in increased transcription
of genes containing TCF binding sites.
While the importance of WNT/β-catenin signalling is well established in many human
carcinomas, its role in transitional cell bladder carcinoma (TCC) is unclear. WNT/β-catenin
signalling is implicated in urothelial development. Expression of Wnt2b in mesenchymal
renal cells induces ureter branching in the murine foetus (Lin et al, 2001). In the
human foetus, WNT11 is expressed in the tips of the ureter buds and other urogenital
tissues (Lako et al, 1998). Thus, a function of WNT/β-catenin signalling in adult
urothelial tissue homeostasis is conceivable. Indeed, differences in WNT7B expression
have been reported between normal urothelium, superficial and invasive bladder carcinomas
(Bui et al, 1998). However, mutations typically responsible for deregulated β-catenin/TCF
activity in other tumours, that is, in APC, AXIN, BTRC and CTNNB1, have not been found
in TCC to date (Stoehr et al, 2002). This might mean that mutations are rare or absent
in this cancer type, that they are difficult to detect, that they occur at unusual
sites in the above genes or that they affect different components of the pathway than
in other cancers. This is, of course, difficult to ascertain. Two established changes
potentially affecting the activity of this pathway in TCC are hypermethylation of
the APC promoter and diminished expression of E-cadherin, caused by CDH1 hypermethylation
or mutation. These changes have been reported to occur with moderate (APC) or high
frequency (CDH1) in advanced TCC and could conceivably lead to increased β-catenin/TCF
activity and proliferation in TCC (Maruyama et al, 2001; Ribeiro-Filho et al, 2002).
To circumvent the vagaries associated with mutation detection in multiple candidate
genes and to determine the significance of reported changes in CDH1 and APC in TCC,
we have determined WNT/β-catenin activity and inducibility in TCC cell lines and in
cultured normal uroepithelial cells (NUEC). Transitional cell bladder carcinoma lines
harbour genetic changes and gene expression patterns typical of advanced TCC and provide
a well-characterised and established experimental system to study properties of this
tumour. Normal uroepithelial cells can be maintained in primary cultures, where they
proliferate spontaneously or stimulated by external growth factors such as EGF. Furthermore,
we analysed the expression of β-catenin, APC and E-cadherin at the protein level and
expression of TCFs at the mRNA level. The effect of E-cadherin was investigated in
more detail. Overall, our data suggest that proliferation of TCC does not depend on
activity of the WNT/β-catenin pathway, which instead appears to be generally repressed.
E-cadherin may act as a buffer for β-catenin activity in urothelial cells and its
loss in TCC may not only affect cell adhesion, but also sensitivity towards WNT factors.
MATERIALS AND METHODS
Cell lines and culture
The bladder carcinoma cell lines, VMCub1, VMCub2, SW1710, SD, HT1376, 5637 and BFTC905,
obtained from the DSMZ, Braunschweig, Germany, were cultured in Dulbecco's minimal
essential medium (Gibco Life Technologies, Karlsruhe, Germany), supplemented with
15% fetal calf serum and 100 μg/ml penicillin/streptomycin as described (Grimm et
al, 1995). The hepatoma cell line HepG2, used as positive control because of its oncogenic
β-catenin mutation, was cultured as described (Schulz et al, 1988). Ureters from nephrectomy
patients were used to prepare NUEC as described by Southgate et al (1994). These were
routinely maintained in keratinocyte serum-free medium (KSFM, Gibco Life Technologies)
supplemented with 50 μg/ml bovine pituitary extract (BPE), 5 ng/ml epidermal growth
factor (EGF) and 30 ng/ml cholera toxin. After the first passage, they were used for
experiments as described (Swiatkowski et al, 2002).
Plasmids
The reporter plasmids pTopFlash and pFopFlash, containing wild-type or mutant TCF
binding sites, respectively, in front of the HSV thymidine kinase minimal promoter
driving the Photinus pyralis luciferase gene, were purchased from Upstate Biotechnology
(c/o Biomol, Hamburg, Germany). The Renilla reniformis luciferase reporter plasmid,
pTKRL, used as internal control for transfection efficiencies, and the negative control
vector pGL3 were purchased from Promega, Mannheim, Germany. pLINEluc, constructed
by insertion of bps −193 to +661 from the active LINE-1 element L1.2B into pGL3 (Steinhoff
et al, 2002) was used as positive control. The following expression plasmids were
used: pbcat, kindly donated by Dr H Clevers, Utrecht, NL, contains human β-catenin
cDNA with an activating S33Y mutation driven by the strong CMV promoter. pEGFP-UM
permits expression of murine E-cadherin cDNA, kindly provided by Dr R Kemler, Freiburg,
Germany, from the CMV promoter.
Transfections and reporter gene assays
Cells were grown in six-well plates to 30% confluence. Transient transfection was
carried out using FuGene/DMEM (Roche, Mannheim, Germany) at a 1 : 25 dilution. Per
well, 1 μg of reporter plasmid, 0.5 μg of each expression plasmid and 0.15 μg pTKRL
were transfected. At 80% confluence, cells were lysed and luciferase activity was
measured using the Dual Luciferase Reporter Assay System (Promega, Heidelberg, Germany)
as recommended by the manufacturer. Each experiment was repeated with at least five
independent passages or cultures.
Western blotting
Cells were grown in 75 cm2 tissue culture flasks to 80% confluence and lysed for 1 h
on ice in modified RIPA-buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 40 mM NaF, 5 mM EGTA,
5 mM EDTA, 1 mM sodium orthovanadate, 1% nonidet P-40, 0.1% sodium dodecylsulphate,
0.1% sodium deoxycholate, 10 μg/ml phenylmethylsulphonylfluoride). Protein amounts
were quantified by the Bradford method. For SDS–polyacrylamide gel electrophoresis
(10% polyacrylamide for β-catenin and E-cadherin, 5% for APC), 10 μg of protein were
used and subsequently transferred to an Immobilon-P membrane (Millipore Corp., Bedford,
MA, USA). After blocking in 10% nonfat milk powder in PBS overnight at 4°C, membranes
were incubated for 1 h at room temperature with primary antibodies at the following
dilutions: anti-β-catenin (BD Transduction Laboratories, Heidelberg, Germany) at 1 : 1500,
anti-E-cadherin (Santa Cruz Biotechnology, CA, USA) at 1 : 1000, anti-APC (Upstate
Biotechnology, c/o Biomol, Hamburg, Germany) at 1 : 500 and anti-α-tubulin (Sigma,
St Louis, MO, USA) at 1 : 5000. Incubation with HRP-conjugated rabbit anti-mouse secondary
antibody (Santa Cruz Biotechnology) at 1 : 5000 was carried out at room temperature
for 1 h, followed by luminescence detection with the ECL-Kit (Amersham-Pharmacia,
Freiburg, Germany).
RNA isolation and RT–PCR
Total mRNA was isolated from cultures grown to 80% confluence, using the RNeasy® Midi
Kit (Qiagen, Hilden, Germany). After quantification, mRNA was transcribed into first-strand
cDNA using AMV-RT (Promega, Mannheim, Germany). Polymerase chain reactions were carried
out in a total 20 μl volume containing 1 × PCR-buffer (Biometra, Göttingen, Germany),
150 μ
M of each nucleotide, 10 pmol of each primer, 1 U of Taq polymerase (Biometra) with
2% (CTNNB1), 3% (TCF1), 2% (TCF4) and 2.5% (hAES) formamide added. Each PCR cycle
consisted of 30 s denaturing at 95°C, 30 s at the annealing temperature and 1 min
at 72°C. Each initial denaturation was performed for 5 min at 95°C and each final
extension for 7 min. A total of 25 cycles were performed for CTNNB1 at 57°C annealing
temperature, 34 cycles at 59°C for TCF1, 35 cycles at 58°C for LEF1, 31 cycles at
59°C for TCF3, 29 cycles at 56°C for TCF4, and for hAES, 36 cycles at 59°C. Primer
pairs were selected according to Iwao et al (1998) for CTNNB1, Duval et al (2000)
for TCF4 and Brantjes et al (2001) for TCF1, LEF1, TCF3 and hAES. GAPDH, with primers
added to the mixture for the last 19 cycles, served as an internal control. Polymerase
chain reaction products were separated by agarose gel electrophoresis (2%) and visualised
by ethidium bromide staining.
DNA extraction and methylation analysis
High molecular weight genomic DNA from cell lines was isolated using the blood and
cell culture DNA kit (Qiagen, Hilden, Germany). MS-PCR was performed essentially as
described by Herman et al (1996). In short, 1 μg DNA from each cell line was bisulphite-treated
with the CpGenome DNA modification kit (Oncor, Heidelberg, Germany). Aliquots from
the reaction mixture were then used in two separate PCR amplifications with primer
pairs from the CDH1 and the APC gene promoter regions specific for converted, that
is unmethylated, or unconverted, that is methylated, DNA using the conditions specified
in Herman et al (1996) and Virmani et al (2001). Polymerase chain reaction conditions
were as follows: template DNA (100 ng) was amplified in a total volume of 50 μl containing
150 μ
M of each dNTP, 1.5 mM MgCl2, 15 pmol of each primer, and 1.25 U of HotStar Taq polymerase
(Qiagen, Hilden, Germany). Following initial denaturation at 94°C for 15 min, 35 cycles
of 30 s at 95°C, 30 s at T
m, and 45 s at 72°C were performed. All reactions included a final elongation step
at 72°C for 10 min.
RESULTS
Properties of cell lines used
Some important properties of the TCC lines used in this study are compiled in Table
1
Table 1
Properties of TCC lines
TCC line
TP53mutation
RB Western blot
CDKN2A
APCmethylation
APC
Western blot
CDH1methylation
E-Cadherin Western blot
VmCub1
Mutant
+
Mutant
−
+
−
+
Exon 5
VmCub2
Mutant
+
Deleted
−
+
−
−
Exon 5
SW1710
Mutant
+
Deleted
−
+
±
−
Exon 8
SD
Mutant
+
Deleted
−
+
−
+
Exon 4
HT1376
Mutant
−
+
+
+
−
+
Exon 7
5637
Mutant
−
+
−
+
−
±
Exon 8
BFTC905
Wild-type
+
Deleted
−
+
−
+
(MDM2↑↑)
For each cell line, TP53, RB and CDKN2A status are given as previously described (Steinhoff
et al, 2002; Swiatkowski et al, 2002). APC and CDH1 hypermethylation and expression
were determined in the present study as described in the Materials and methods section.
Methylation status: + hypermethylated, − no hypermethylation; ± both hypermethylated
and unmethylated alleles detectable; protein expression: + present; − not detectable;
± weakly detectable.
, including APC and CDH1 hypermethylation status, which were newly determined. By
MS-PCR, all APC alleles were hypermethylated in HT1376, and one allele of CDH1 was
hypermethylated in SW1710.
Reporter gene analysis of endogenous and inducible β-catenin/TCF signalling activity
First, endogenous activity of the WNT/β-catenin pathway was determined in proliferating
NUEC and seven TCC lines under optimal growth conditions. The hepatoma cell line HepG2
harbouring an activating β-catenin mutation was used as a positive control. The cells
were transfected with either pTopFlash or pFopFlash plasmids. These plasmids are identical
but for mutations in the TCF/β-catenin binding sites in the promoter driving luciferase
expression. The ratio of luciferase expression from pTopFlash to pFopFlash is an established
indicator of Wnt/β-catenin pathway activity. Differences in transfection efficiencies
were corrected by cotransfection of a Renilla luciferase plasmid. Plasmids without
a promoter (pGL3) or with a cell-type-independent retrotransposon promoter (pLINEluc)
were included in each experiment as quality controls.
Figure 1A
Figure 1
Basal and inducible activities of a TCF/β-catenin-dependent promoter in TCC lines
and NUEC. The indicated TCC lines, NUEC and the hepatoma cell line HepG2 (hatched
bar) were transfected with reporter plasmids and luciferase activity was measured
2 days later. All data are derived from at least five independent triplicate experiments.
(A) Basal activity of a TCF/β-catenin-dependent promoter (contained in pTopFlash)
in TCC lines. Mean±s.d. of the pTopFlash/pFopFlash activity ratio are shown. The dotted
line indicates a ratio of 1 corresponding to lack of activity. The ratio is significantly
different in the HepG2-positive control cell line (*
t-test; P<0.05). (B) Induction of pTopFlash reporter activity by oncogenic β-catenin.
Data are mean±s.d. of the ratio pTopFlash+pbcat/pTopFlash. The dotted line indicates
a ratio of 1 indicating lack of inducibility. Statistically significant inducibility
(*
t-test: P<0.05) was observed in the TCC lines SW1710 and 5637. (C) Effect of oncogenic
β-catenin on activity of the pFopFlash reporter that contains mutated TCF sites. Data
are mean±s.d. of the ratio pFopFlash+pbcat/pFopFlash. The dotted line indicates a
ratio of 1 indicating lack of inducibility. No statistically significant inducibility
was observed.
shows the pTopFlash to pFopFlash ratio in seven TCC-lines, normal urothelial cells
and the positive control HepG2. As expected for a cell line with an activating β-catenin
mutation, HepG2 displayed significant endogenous signalling activity (12.3±3.3). In
contrast, the ratios found in normal urothelial cells (0.96±0.09) and in the TCC lines
(range: 0.64±0.20–1.33±1.3) indicate lack of β-catenin/TCF signalling.
Next, we tested whether mutationally activated β-catenin was able to induce promoter
activity from pTopFlash in TCC and NUEC in cotransfection experiments. Figure 1B displays
the ratios of luciferase activity with vs without β-catenin. Surprisingly, in normal
urothelial cells and four of seven TCC-lines, the ratio did not differ significantly
from 1 with values ranging from 0.2±0.1 in normal urothelial cells to 2.4±2.1 in VMCub1,
indicating that expression of activated β-catenin was not sufficient to increase transcription
from the β-catenin/TCF-dependent promoter. In contrast, significant induction was
observed in the TCC lines SW1710 (12.7±2.3) and 5637 (58.7±23.8). A slight, but not
statistically significant induction was observed in BFTC905 (4.9±2.0). The high basal
activity in HepG2 was not further increased by transfected β-catenin, as expected.
Cotransfection of β-catenin did not significantly alter the activity of pFopFlash
(Figure 1C) in any cell type.
Expression of β-catenin
To identify the cause for the different inducibility of the TCC lines by oncogenic
β-catenin, we first compared the expression of endogenous β-catenin. Qualitative RT-PCR
analysis revealed β-catenin mRNA to be present in all TCC lines and in normal urothelial
cells with only slight differences in expression levels (Figure 2A
Figure 2
Expression of β-catenin in TCC lines and normal uroepithelial cells. Expression of
β-catenin (CTNNB1) was determined in the indicated TCC lines, NUEC and HepG2 hepatoma
cells at the mRNA level (A) by RT–PCR using GAPDH for comparison and at the protein
level (B) using α-tubulin for comparison. Control in (A) refers to PCR without cDNA,
the size marker is shown on the right-hand lane. Control in (B) was supplied by the
antibody manufacturer.
). Western blot analysis confirmed that overall β-catenin expression was also similar
in all TCC lines and normal urothelial cells at the protein level (Figure 2B). Thus,
the differences in inducibility were not due to major differences in β-catenin expression
between inducible and noninducible TCC-lines.
Expression of TCF and hAES mRNA
β-catenin-mediated transcription depends on its interaction in the nucleus with TCF/LEF
transcription factors, which in the absence of β-catenin act as transcriptional repressors
by interacting with transcriptional corepressors of the Grg/TLE-family. Thus, expression
patterns of TCF/LEF factors and perhaps of TLE factors could account for the different
inducibilities between the TCC lines and were therefore investigated by RT–PCR analysis.
GAPDH served as internal control. First, we analysed mRNA expression of hTCF1, hLEF1,
hTCF3 and hTCF4. Among these, hTCF1 and hTCF4 mRNAs were found in all TCC lines, normal
urothelial cells and HepG2 (Figure 3A and D
Figure 3
Expression of TCF mRNAs in TCC lines and normal uroepithelial cells. Expression of
the indicated TCF mRNAs was determined in the indicated TCC lines, NUEC and HepG2
hepatoma cells by RT–PCR using GAPDH for comparison (except for hLEF1 and hTCF3 yielding
similar size PCR products as GAPDH). Control refers to PCR without cDNA, the size
marker is shown on the right-hand lane. Note the double band for hLEF1 corresponding
to known splice variants and the additional band for hAES in NUEC, which may be a
novel splice variant.
). Interestingly, in the inducible TCC lines SW1710 and 5637 hTCF1 expression was
slightly, but reproducibly increased relative to GAPDH (Figure 3A). Expression of
hLEF1 was more heterogeneous (Figure 3B). The TCC lines SW1710, HT1376 and BFT905,
normal urothelial cells and HepG2 displayed only faint bands or lacked hLEF1 expression,
which was robustly detected in VMCub1, VMCub2, SD and 5637. Two bands were found which
correspond to known hLEF1 splice variants (Carlsson et al, 1993). While four TCC lines,
as well as normal urothelial cells and HepG2 showed hTCF3 expression, only weak expression
could be detected in SD and HT1376, and also a very faint band in the inducible cell
line SW1710 (Figure 3C). Neither hLEF1 nor hTCF3 expression patterns correlated with
inducibility among the TCC lines. Since other Grg/TLE factors have been described
as ubiquitous (Brantjes et al, 2001), we investigated only hAES mRNA by RT–PCR, which
is the only member in the Grg/TLE-family that is not a repressor. Figure 3E demonstrates
that hAES was expressed in all TCC lines, normal urothelial cells and HepG2, except
for BFTC905 showing only weak expression. In normal urothelial cells, a second faint
band could be observed, which may correspond to a splice variant. As for hTCF1, both
inducible TCC lines displayed slightly increased hAES expression relative to GAPDH.
Role of E-cadherin
Since E-cadherin has been reported to act as an inhibitor of β-catenin/TCF-mediated
transcription by sequestering β-catenin at the plasma membrane (Stockinger et al,
2001) and loss of E-cadherin to be frequent in TCC (Rieger et al, 1995), the differences
in WNT/β-catenin signalling between the TCC lines could be related to E-cadherin expression.
Indeed, the CDH1 gene encoding E-cadherin was found to be hypermethylated in SW1710
(Table 1). In Western blot analysis, the TCC lines VMCub1, SD, HT1376 and BFTC905
as well as normal urothelial cells were found to express E-cadherin (Figure 4A
Figure 4
Expression and function of E-cadherin. (A) Expression of E-cadherin was determined
in the indicated TCC lines and in NUEC by Western blotting (B) using α-tubulin for
comparison. Control refers to a lysate from cells transfected with an E-cadherin expression
construct. (B) Effect of E-cadherin cotransfection on induction of pTopFlash reporter
activity by oncogenic β-catenin in the TCC lines SW1710 and 5637. Values are mean±s.d.
of the relative activity of pTopFlash or pFopFlash (as indicated) without any cotransfection
(white bars), with transfected pbcat only (grey bars) or pbcat and pEGFP-UM expressing
E-cadherin (dark bars). Activity of LINEluc was set as 100%. The differences in pTOP-Flash
activation caused by E-cadherin cotransfection are statistically significant (t-test:
P<0.05) in both cell lines.
). Diminished expression or loss of E-cadherin protein was observed in both inducible
TCC lines, SW1710 and 5637, and in the noninducible VMCub2 line. We therefore tested
whether re-expression of E-cadherin in the inducible TCC lines SW1710 and 5637 might
restore repression of β-catenin/TCF signalling in reporter gene analysis. In both
cell lines, significant inhibition of promoter activation by β-catenin was elicited
by E-cadherin cotransfection (Figure 4B). In SW1710 and 5637, induced levels of pTOPluc
promoter activity were diminished by 60 and 90%, respectively. Smaller effects were
observed on pFOPluc.
APC expression
In addition to methylation analysis of the APC gene, its expression was investigated
at the protein level by Western blot analysis. APC protein was present in all TCC
lines and normal urothelial cells and even detectable in HepG2 cells, albeit at a
lower level. In particular, diminished levels were neither observed in the TCC lines
SW1710 and 5637, which were inducible by β-catenin, nor in HT1376 cells, which displayed
hypermethylation of all APC alleles (data not shown).
DISCUSSION
Constitutive activity of the WNT/β-catenin signalling pathway is an important step
in the development of many human cancers. However, none of the typical mutations in
components of this pathway have been reported in TCC. Few studies have appeared addressing
this issue explicitly, which is likely due to publication bias against negative results
(Stoehr et al, 2002). Still, a lack of mutations in known components of a pathway
does not permit the conclusion that it is intact unless its actual functional state
has been determined. This is difficult to perform in TCC tissues, but can be done
by reporter gene analysis in TCC lines that show the typical genetic aberrations of
advanced bladder cancers. The results of the present study are in accord with the
impression from the literature that activating mutations in the WNT/β-catenin signalling
pathway are rare or absent in TCC. Moreover, the data suggest that this pathway is
inactive in and not required for proliferation of TCC cells. Since the same result
was obtained with proliferating NUEC, the lack of basal activity in TCC cells is likely
an extension of the state in the normal tissue. Moreover, even in normal cells, the
pathway could not be activated by oncogenically activated β-catenin. Thus, the WNT/β-catenin
signalling pathway is not only inactive, but turned off in urothelial cells, likely
because it is not required for proliferation and perhaps to avoid inappropriate target
gene activation. More speculatively, it is interesting to consider the downregulation
of the WNT/β-catenin pathway in urothelial cells and carcinomas in the light of current
hypotheses on the function of the pathway in maintaining stem cell properties (Taipale
and Beachy, 2001). According to our data, such a function in uroepithelial tissue
seems unlikely. This might be related to its particular organisation as a transitional
epithelium with its low turnover, in which stem cells may behave differently as in
colon.
Nevertheless, the activation of typical WNT/β-catenin target genes such as CCND1 and
MYC is required for the proliferation of uroepithelial cells, too. Proliferation of
NUEC is stimulated by growth factors such as EGF and, in an autocrine fashion, HB-EGF
(Freeman et al, 1997). Since these factors act via MAPK signalling (Swiatkowski et
al, 2002), they seem quite sufficient to stimulate the necessary transcription of
CCND1 and MYC in an alternative fashion to WNT/β-catenin signalling. Almost all advanced
TCC display defects in either RB1 or CDKN2A, which obliterate the requirement for
cyclin D1 (cf. Table 1). Furthermore, in some cases CCND1 itself is amplified (Proctor
et al, 1991). Increased expression of MYC is found in almost all TCC, and is correlated
with increased gene copy numbers (Christoph et al, 1999) and/or overexpression of
the EGF receptor (Lipponen, 1995). Thus, upregulation of MYC or CCND1, required for
the proliferation of TCC cells, is likely achieved by mechanisms other than activation
of WNT/β-catenin signalling.
This interpretation implies that the inducibility of TCF-dependent gene expression
found in the TCC lines 5637 and SW1710 is a deviation from the normal state in the
urothelium and that at least one of the mechanisms ensuring inactivity of the WNT/β-catenin
pathway has become defective. We did not observe significant differences between these
two cell lines and the others in expression of overall β-catenin, hLEF1, hTCF3 and
hTCF4. However, both cell lines displayed decreased expression of E-cadherin. Indeed,
restoration of E-cadherin expression caused repression of TCF/β-catenin-induced transcriptional
activity (Figure 4B). On a note of caution, our data indicate that E-cadherin expression
cannot be the only factor determining inducibility of WNT/β-catenin signalling, since
VMCub2 cells also lacked the protein, but did not respond to β-catenin transfection.
Interestingly, both inducible TCC lines, but not VMCub2, displayed slightly increased
mRNA expression of hTCF1 and hAES. If hTCF1 acts as a feedback repressor of β-catenin/TCF4
signalling (Roose et al, 1999), increased hTCF1 expression in SW1710 and 5637 obviously
cannot account for their inducibility by β-catenin. However, the hAES homologue Grg5
is known to act as a de-repressor of TCF-mediated transcription (Roose et al, 1998).
Thus, in addition to E-cadherin loss increased hAES levels could contribute to inducibility
of β-catenin/TCF signalling in SW1710 and 5637. The potential roles of hTCF1 and hAES
are more difficult to address experimentally than that of E-cadherin, since the differences
in hTCF1 and hAES expression between the cell lines were only quantitative.
The finding that E-cadherin modulates WNT/β-catenin signalling in urothelial cells
is in line with recent data suggesting this as a function of E-cadherin additional
to or coordinate with mediating cell adhesion (Stockinger et al, 2001). In this regard,
our findings suggest that while constitutive activation by ‘classical’ mutations does
not occur, the WNT/β-catenin pathway may play a certain role in a subset of TCC, likely
those with loss of E-cadherin expression. This subset of tumours may show increased
sensitivity towards WNT factors present in the tissue. Indeed, changes in WNT7B expression
have been reported in some TCC (Bui et al, 1998). Loss of E-cadherin expression in
TCC is associated with a worse clinical prognosis (Bornman et al, 2001). It might
be worthwhile to investigate the emerging connection between WNT expression, E-cadherin
loss and clinical prognosis in more detail.
Finally, hypermethylation of the CDH1 and APC genes has been reported in TCC tissues.
Hypermethylation of CDH1 was found to be associated with loss of protein expression,
although not all cases with loss of protein also displayed hypermethylation (Bornman
et al, 2001; Ribeiro-Filho et al, 2002). This was reflected in the TCC cell lines
investigated here. Downregulation of E-cadherin expression was found in 3/7 cell lines,
but hypermethylation in only one. While the significance of CDH1 hypermethylation
is thus likely, that of APC hypermethylation, reported at frequencies up to 30% (Maruyama
et al, 2001), is not obvious in TCC. The HT1376 cell line showed methylation of all
APC alleles, but neither basal activity nor inducibility of the WNT/β-catenin pathway
were observed. In fact, APC protein expression could be detected in HT1376. While
this suggests that APC hypermethylation does not implicate activation of WNT/β-catenin
signalling, more extensive studies are required on this issue.