From the Institute of Pathology, Medical School
Rheinisch-Westfälische Technische Hochschule (RWTH)
Aachen, D-52074 Aachen, Germany and § Unitat de
Biologia Cellular i Molecular, Institut Municipal d'Investigacio
Medica, Universitat Pompeu Fabra, 08003 Barcelona, Spain
Received for publication, December 13, 2001, and in revised form, April 24, 2001
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ABSTRACT |
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Malignant transformation of melanocytes
frequently coincides with loss of E-cadherin expression. Here we show
that loss of E-cadherin in melanoma cell lines does not involve
mutations in the E-cadherin gene, promoter methylation, or alterations
in expression of AP-2 transcription factors as suggested
previously. In a panel of different melanoma cell lines, E-cadherin
expression was negatively regulated by up-regulation of the
transcription factor Snail. In comparison with primary human
melanocytes, where Snail expression was not detected by reverse
transcription-polymerase chain reaction, significant expression was
found in all eight melanoma cell lines. In parallel, Western blot and
reverse transcription-polymerase chain reaction analysis revealed
strong reduction of E-cadherin expression in the melanoma cells.
Consistently, transient transfection of a Snail expression plasmid into
human primary melanocytes led to significant down-regulation of
E-cadherin, whereas transient and stable transfection of an antisense
Snail construct induced reexpression of E-cadherin in Mel Ju and Mel Im
melanomas. In summary, we conclude that activation of Snail expression
plays an important role in down-regulation of E-cadherin and
tumorigenesis of malignant melanomas.
The cell-cell adhesion molecule E-cadherin has been shown to
execute important functions in embryogenesis and tissue architecture by
forming intercellular junction complexes and establishing cell polarization (1). The extracellular domain of E-cadherin is involved in
a molecular zipper mediating cell-cell adhesion, whereas the
cytoplasmic tail is linked to the actin cytoskeleton via catenins (2,
3). Because of its critical function in intercellular adhesion,
E-cadherin has also been assumed to act as a tumor suppressor negatively regulating several critical steps of invasion and
metastasis. Loss of E-cadherin expression during tumor development was
recently observed in a variety of different tumor types, including
malignant melanomas (4-10). Transfection of E-cadherin cDNA into
invasive carcinoma cells led to significant reduction of their invasive capacity in vitro (11, 12), and activation of E-cadherin
resulted in growth retardation of tumor cell lines (13, 14).
Subsequently, results obtained from in vivo tumor models
provided consistent evidence for a role of E-cadherin as a potent tumor
suppressor (12, 15-17). Further, the tumor suppressor gene
fat in Drosophila melanogaster was revealed as a
member of the cadherin gene family (18).
Immunohistochemical studies of E-cadherin expression in malignant
melanomas demonstrated significant down-regulation of E-cadherin in the
tumor tissue compared with benign melanocytes and melanocytic nevi (4,
5). Evidence for the functional relevance of this phenomenon was
obtained by Herlyn and co-workers (19-21), who showed that
down-regulation of E-cadherin leads to deregulated control of
melanocyte proliferation by keratinocytes and in parallel to an
invasive growth behavior. E-cadherin is involved in a signaling pathway
mediated by The mechanism of down-regulation of E-cadherin in malignant melanoma is
still unknown. One possibility involves promoter inactivation attributable to hypermethylation, which has been observed in human breast, gastric, and prostate cancers and in leukemias (8, 26, 27).
Furthermore, loss of activating protein-2 transcription factor
expression as a potential activator of E-cadherin has been suggested
(28-31). Also, mutations in the E-cadherin gene resulting in a
functionally inactive protein have been detected in some tumors,
including colorectal, gastric, and breast carcinomas (10, 32-34). Only
very recently, up-regulation of the transcription factor Snail was
reported to mediate significant negative regulation of E-cadherin
expression in bladder, colorectal, and pancreatic carcinomas (35,
36).
Therefore, we aimed to analyze regulation of E-cadherin in melanoma
cells and sequenced the E-cadherin gene in a panel of different
melanoma cell lines and studied the effect of AP-2 transcription factors, promoter methylation, and transient transfection of
Snail sense and antisense expression constructs.
Cell Lines and Cell Culture Conditions--
The melanoma cell
lines Mel Im, Mel Ei, Mel Wei, Mel Ho, Mel Juso, Mel Ju, SK-Mel-28, and
HTZ19d have been described in detail previously (37, 38). The cell
lines Mel Ei, Mel Wei, Mel Ho, and Mel Juso were derived from a primary
cutaneous melanoma, and Mel Im, Mel Ju, SK-Mel-28, and HTZ19d were
derived from metastases of malignant melanomas. For tissue culture the
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with penicillin (400 units/ml), streptomycin (50 µg/ml),
L-glutamine (300 µg/ml), and 10% fetal calf serum serum
(Sigma) and split 1:5 every 3 days.
Human primary melanocytes derived from normal skin were cultivated in
melanocyte medium melanocyte growth medium-3 (Life Technologies, Inc.) under a humidified atmosphere of 5% CO2 at 37 °C.
Cells were used in passages 6-10 and not later than 3 days after
trypsinization. Cells were detached for subcultivation or assay with
0.05% trypsin and 0.04% EDTA in phosphate-buffered saline
(PBS).1
Transfection Experiments--
For transient transfections,
2 × 105 cells per well were seeded into six-well
plates and transiently transfected with 0.5 µg of AP-2
A panel of Mel Im cell clones was established by stable transfection
with an antisense Snail expression plasmid (35) under the control of a
cytomegalovirus promoter and cotransfected with the neo-selectable
pcDNA3 plasmid (Invitrogen, Groningen, Holland). Transfection was
also performed using LipofectAMINE Plus (Life Technologies) according
to the manufacturer's instructions. One day after transfection, cells
were placed into the selection medium containing 50 µg/ml G418
(Sigma). Twenty-five days after selection, individual G418-resistant
colonies were subcloned.
Western Blots--
Cells (3 × 106)
were lysed in 200 µl of radioimmunoprecipitation assay buffer (Roche
Molecular Biochemicals) and incubated for 15 min at 4 °C. Insoluble
fragments were removed by centrifugation at 13,000 rpm for 10 min, and
the supernatant lysate was immediately shock frozen and stored at
RT-PCR--
Expression of Snail and E-cadherin mRNA was
measured by RT-PCR. First-strand cDNA was synthesized using 2 µg
of total cellular RNA as template, 1 µg of a random primer (Amersham
Pharmacia Biotech), 4 µl of 5× first-strand buffer (Life
Technologies), 2 µl of 10 mM dithiothreitol, 1 µl of 10 mM dNTPs, and 1 µl of Superscript II (Life Technologies)
in a volume of 20 µl. For Snail amplification, a nested PCR was
performed using 2 µl of the cDNA preparation and the following
program: 15 cycles of 45 s at 94 °C, 30 s at 60 °C, and
1 min at 72 °C and a final extension of 5 min at 68 °C. Two
microliters of the PCR product were applied to the same reaction
profile but with 25 cycles using nested primers (hsnail forward, 5'-TGC
GCG AAT CGG CGA CCC; hsnail reverse, 5'-CCT AGA GAA GGC CTT CCC GCA G;
hsnail forward nested, 5'-ACT ACA GCG AGC TGC AGG; and hsnail reverse
nested, 5'-GTG TGG CTT CGG ATG TGC). PCR products were separated on a
1.8% agarose gel and stained with ethidium bromide. For E-cadherin,
both conventional RT-PCR and quantitative real time PCR were performed
on a LightCycler (Roche Molecular Biochemicals). Two microliters of
cDNA template, 2 µl of 25 mM MgCl2, 0.5 µM forward and reverse primers
(CLONTECH), and 2 µl of SybrGreen LightCycler mix
in a total of 20 µl were applied to the following PCR program:
30 s at 95 °C (initial denaturation); 20 °C/s temperature
transition rate up to 95 °C for 15 s, 10 s at 58 °C,
22 s at 72 °C, and 10 s at 82 °C acquisition mode
single, repeated for 40 times (amplification). The PCR reaction was
evaluated by melting curve analysis following the manufacturer's
instructions and checking the PCR products on 1.8% agarose gels.
E-cadherin Sequencing--
Genomic DNA was isolated from
melanoma cell lines as described previously (40). Exons and intron-exon
boundaries of E-cadherin were amplified using the primers described by
Berx et al. (10). The PCR products were purified, and direct
cycle sequencing reactions were performed using a PRISM dye primer
cycle sequencing kit with AmpliTaq FS (Perkin-Elmer) and analyzed by
capillary electrophoresis (Applied Biosystems ABI 310).
Analysis of E-cadherin Promoter Methylation
Status--
This method is based on the selective conversion of
unmethylated cytosine to uracil by the effect of bisulfite (41).
Genomic DNA (1 µg) was denaturalized with NaOH and treated with 2.5 M sodium bisulfite in the presence of 0.5 mM
hidroquinone for 16 h at 50 °C. DNA was purified from the
excess of salts, and 100 ng of modified DNA were used as a template in
the PCR reaction. This reaction was performed under the following
conditions: 67 mM Tris HCl, pH 8.8, 16.6 mM
ammonium sulfate, 6.7 mM MgCl2, 10 mM Fluorescence-activated Cell Sorting Analyses--
Cultured cells
were detached with 10 mM EDTA in PBS, washed once with 2%
bovine serum albumin in PBS, and stained for 40 min with a 1:200
dilution (0.2 µg/ml) of anti E-cadherin antibody at 4 °C (clone
M106, Takara). After final incubation with Cy2-conjugated goat
anti-mouse IgG (1:200; Dianova, Hamburg, Germany), cells were fixed for
5 min at room temperature with 0.5% formaldehyde and analyzed by flow
cytometry using a Facsalibur (Becton Dickinson). As a negative control,
unrelated mouse IgG was used instead of E-cadherin antibody.
Previously, down-regulation of E-cadherin has been described as an
important molecular event in tumorigenesis of melanomas. Therefore, we
first aimed to verify the deregulated E-cadherin expression pattern in
a panel of different melanoma cell lines and then addressed potential
molecular mechanisms involved in down-regulation of the gene. Western
blotting with radioimmunoprecipitation assay lysates derived from eight
different melanoma cell lines and from primary human melanocytes
revealed significant E-cadherin down-regulation in all melanoma cell
lines. As depicted in Fig. 1A,
an ~120-kDa E-cadherin signal was present in the skin melanocyte culture (primary human melanocytes) and in the colon carcinoma cell
line LoVo, which was not detected in any of the melanoma cell lines. In
parallel to the results obtained by Western blots, we observed
significantly reduced mRNA expression levels by RT-PCR analyses.
After 26 PCR cycles, E-cadherin amplification products were clearly
detected in primary human melanocytes and LoVo cells but not in the
melanoma cell lines. Consistently, PCR reactions prepared with RNA
template from human skin fibroblasts failed to amplify the E-cadherin
cDNA fragment (Fig. 1B). Because down-regulation of
E-cadherin protein was closely paralleled by reduction of mRNA in
all eight different melanoma cell lines, we hypothesized that defective
E-cadherin expression in these cell lines involved transcriptional but
not translational mechanisms.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin and lymphocyte enhancer factor and T cell
factor transcription factors. Deregulation of this pathway results in constant activation of
-catenin, lymphocyte enhancer factor, and T cell factor target genes, including c-myc and
cyclin D1, and occurs in many kinds of malignant tumors (22). In
melanoma both loss of E-cadherin and mutations of
-catenin, leading
to a more stable, nondegradable protein, have been reported (5, 19,
23-25).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or AP-2
cytomegalovirus promoter expression plasmids (39), E-cadherin
expression plasmid, or sense- and antisense Snail cDNA in
pcDNA3 (35) using the LipofectAMINE Plus method (Life Technologies)
according to the manufacturer's instructions. The cells were harvested
48 h after transfection and E-cadherin expression was evaluated
using fluorescence-activated cell sorting, Western blotting, and
reverse transcription-polymerase chain reaction (RT-PCR). All
transfections experiments were repeated three times.
80 °C. Eight micrograms of radioimmunoprecipitation assay cell
lysate were loaded per lane and separated on SDS-polyacrylamide gel
electrophoresis gradient gels (Invitrogen) and subsequently blotted
onto a polyvinylidene difluoride membrane. After blocking for 1 h
with 3% bovine serum albumin and PBS, the membrane was incubated for
16 h with a 1:150 dilution of a mouse monoclonal E-cadherin
antibody (clone M106, 0.2 µg/µl; Takara, Shiga, Japan). Then the
membrane was washed three times in PBS, incubated for 1 h with a
1:300 dilution of an alkaline phosphate-coupled secondary anti-mouse
IgG antibody (AP303A; Chemicon, Hofheim, Germany), and then washed
again. Finally immunoreactions were visualized by nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma) staining.
-mercaptoethanol, and 1.25 mM dNTPs using
as primers oligonucleotides 5'-TTAGGTTAGAGGGTTATCGCGT-3'
(sense) and
5'-TAACTAAAAATTCACCTACCGAC-3' (antisense) for the methylated DNA and
5'-TAATTTTAGGTTAGAGGGTTATTGT-3' (sense) and
5'-CACAACCAATCAACAACACA-3' (antisense) for unmethylated DNA. The nucleotides in bold correspond to
the uracils generated from cytosine by effect of bisulfite; the
nucleotides underlined correspond to the cytosines protected by
methylation. These four oligonucleotides correspond to sequences
177/
156 (sense) and
84/
62 (antisense) (methylated) and
182/
158 (sense) and
104/
85 (antisense) (unmethylated) from the
human E-cadherin promoter. The conditions of amplification used were 37 cycles composed of 20 s at 96 °C, 30 s at 53 °C
(unmethylated reaction) or 57 °C (methylated reaction), and 30 s at 72 °C. The presence of amplification products (116 base pairs
bp for methylated and 97 base pairs for unmethylated) was visualized in
7.5% Tris-borate EDTA-polyacrylamide gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (45K):
[in a new window]
Fig. 1.
Western blot (A) and RT-PCR
(B) experiments showing reduced E-cadherin in
melanoma cell lines. Displayed from left to
right are melanoma cell lines (Mel Wei, SK-Mel 28, Mel
Ju, Mel Ei, Mel Juso, Mel Ho, Mel Im, and HTZ19d),
primary human skin melanocytes (PHM), and the LoVo colon
cancer cell line.
To exclude the possibility that structural gene mutations lead to failures in processing mature mRNA, we sequenced the entire 16 exons and flanking intron regions of the E-cadherin gene in all eight melanoma cell lines. No mutations were detected either in the coding region or in the flanking intron-exon boundaries (data not shown). Three new intronic polymorphisms were detected, none of which affected a region critical for exon-intron processing.
It has been speculated previously that loss of E-cadherin possibly
involves absence of AP-2 transcription factors, hypermethylation of the
promoter region, or up-regulation of the transcription factor Snail
(26, 28, 35). To analyze the effect of AP-2 on E-cadherin expression in
melanoma cells, we reintroduced the AP-2 protein by transfecting AP-2
cDNA expression vectors. Results shown in Fig.
2 revealed that transient transfections
of both AP-2 and AP-2
expression plasmids did not significantly
activate E-cadherin expression. Fluorescence-activated cell sorting
analyses of AP-2
-transfected (Fig. 2B) and
AP-2
-transfected (Fig. 2C) Mel Im provided only minimally
enhanced E-cadherin profiles compared with mock-transfected control
cells (Fig. 2A). Identical results were obtained from
AP-2-transfected Mel Ju cells, and RT-PCR analyses consistently failed
to result in amplification of an E-cadherin cDNA product (data not
shown). In contrast, transient transfection of an E-cadherin expression
plasmid led to significant E-cadherin immunostaining in a large portion
of transfected Mel Im cells (Fig. 2D).
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To determine the effect of promoter hypermethylation on E-cadherin
expression, the methylation status of all melanoma cell lines was
analyzed. In none of the cell lines was promoter methylation detectable
(Fig. 3).
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Encouraged by recent studies of colon cancers revealing repression of
E-cadherin expression by up-regulation of the transcription factor
Snail (35, 36), we further analyzed Snail expression patterns and
the effect of transfected Snail in melanoma cells. RT-PCR experiments
demonstrated significant Snail mRNA expression in all eight
different melanoma cell lines and human skin fibroblasts but not in
primary human melanocytes and the colon cancer cell line LoVo (Fig.
4). From these results we concluded that
significant expression of Snail closely correlates with down-regulation
of E-cadherin. On the basis of these data, we transfected both sense and antisense Snail expression plasmids into primary human melanocytes and Mel Im or Mel Ju melanoma cells. Transient transfection of Snail
led to 2.7-fold reduced expression of E-cadherin in primary human
melanocytes as measured by quantitative real-time RT-PCR (LightCycler).
In contrast, transfection of a Snail antisense plasmid caused 4.3-fold
E-cadherin mRNA up-regulation in Mel Im cells and 3.9-fold
up-regulation in Mel Ju cells.
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Because results from transient transfections are limited by
transfection efficiency (~5% in primary melanocytes and up to 30%
in the melanoma cell lines) and rapid degradation of plasmid DNA
molecules, we also aimed to examine E-cadherin expression levels in
stably antisense Snail-transfected Mel Im cell clones. Therefore, we
selected a panel of six different neo-resistant Mel Im cell clones
and compared E-cadherin expression in these clones with a
neo-transfected control clone. Results shown in Fig.
5A clearly demonstrated
significant E-cadherin up-regulation in all six antisense
Snail-transfected cell clones by Western blot. Two of the cell clones
with the strongest E-cadherin expression (Mia as4 and MI as5) were
further analyzed for E-cadherin expression by flow cytometry (Fig.
5B) and snail mRNA expression (Fig.
6). A marked increase in E-cadherin
expression and a down-regulation of snail could be shown.
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DISCUSSION |
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A variety of phenotypical alterations distinguish tumor cells from normal cells, the most prominent of which include changes in cellular morphology (referred to as "dedifferentiation"), differences in adhesion, and acquisition of an invasive and metastatic phenotype. In many tissues E-cadherin was identified as a critically important cell-cell adhesion molecule, being a potential molecular target involved in these alterations. Several groups provided clear evidence that E-cadherin is frequently down-regulated in a variety of different human tumors, correlating with dedifferentiation and increased invasiveness in vitro. Disturbance of E-cadherin function by inhibitory antibodies led to induction of invasiveness and vice versa, and enhanced expression of E-cadherin in tumor cells by cDNA transfection experiments inhibited invasion (11, 12, 42). Recent studies have offered even further evidence for a causal tumor suppressor function of E-cadherin in vivo. Germ line-inactivating mutations in the E-cadherin gene have been detected in families with inherited predisposition to gastric and breast carcinoma (43-45).
In malignant melanomas, loss or down-regulation of E-cadherin was first described in vitro (23, 46, 47) and later substantiated by immunohistochemical studies of tumor specimens (4, 5) in vivo. Functional consequences resulting from loss of E-cadherin expression were elicited by Herlyn and co-workers (19-21). Several studies revealed that down-regulation of E-cadherin leads to defects in control of melanocyte proliferation by keratinocytes and to acquisition of an invasive tumor cell phenotype (19-21).
Results from our present study confirm that significant down-regulation of E-cadherin is a frequent event in melanoma cell lines but does not involve mutations in the E-cadherin gene. These data agree with other reports indicating that loss of E-cadherin expression because of structurally inactivating gene mutations occurs very infrequently (48). Mutational inactivation of the gene resulting in uniform loss of E-cadherin expression has been detected in a few gynecological cancers, lobular carcinomas of the breast, and gastric carcinomas (10, 33, 43, 49). In all of these cases, uniform E-cadherin loss correlated with typical scattered, highly dissociated tumor cell growth (10, 27). However, in the vast majority of tumors, loss of E-cadherin is heterogeneous (50) and can be modulated by the tumor microenvironment (51, 52), suggesting that irreversible genetic alterations are not common.
Furthermore, loss of expression of the transcription factor AP-2 as a
potential positive regulator of E-cadherin expression was described in
other tumors (28). Because loss of AP-2 expression in melanoma was
shown by several groups (30, 31, 53), the same mechanism was speculated
to be involved in loss of E-cadherin in melanoma but until now was not
experimentally proven (29). In this study we could clearly show that
expression of AP-2, either or
, in melanoma cell lines by
transient transfection did not lead to expression of E-cadherin. This
is in contrast to c-kit (53), in which AP-2 plays an
important role in regulating gene expression. Also, melanoma cell
adhesion molecule/mucin (31) was first demonstrated to be
up-regulated because of loss of AP-2, but recent studies indicated that
loss of AP-2 in melanoma is not required for MCAM expression in
melanoma (54).
Enhanced methylation of CpG islands in the E-cadherin gene promoter has been proposed as a possible mechanism for E-cadherin inactivation in carcinomas by several reports (26, 27, 55, 56). Our results indicate clearly that hypermethylation of the promoter region is not molecularly linked to down-regulation of E-cadherin expression in melanoma cells. Also Tamura et al. (27) demonstrated that loss of E-cadherin expression in vivo was not uniformly associated with promoter hypermethylation and therefore concluded that alterations in promoter methylation patterns are unlikely the only mechanisms involved in switching off E-cadherin expression. Possibly, promoter hypermethylation may result from long-term promoter inactivation by other transcriptional mechanisms rather than representing the primary cause of gene inactivation. In addition, Graff et al. (56) recently showed that hypermethylation may be dynamic, unstable, and changing in relation to signals provided by the tumor microenvironment.
We then analyzed the expression of the transcription factor Snail, which has recently been identified as a potent inhibitor of E-cadherin expression in colon, gastric, and pancreas carcinomas (35, 36).
Significant levels of Snail mRNA expression were detected in all melanoma cell lines that we analyzed but not in primary human melanocytes, indicating that Snail up-regulation is a very frequent event in melanoma cells. Snail expression in these cells is clearly causally involved in regulating E-cadherin expression, because we observed up-regulation of E-cadherin mRNA and protein levels both after transient and stable antisense Snail expression plasmids and, in addition, down-regulation of E-cadherin after transient Snail expression in benign skin melanocytes. Interestingly, Snail was also shown to activate vimentin expression, a cytoskeletal protein frequently up-regulated in melanoma cells compared with benign melanocytes (36). These data suggest that Snail may play a general role in changing gene expression patterns during malignant transformation of melanocytic tumors and call for further investigations addressing the regulation of Snail expression.
In summary, our results provide further insights into the regulation of
E-cadherin expression in malignant melanoma cells and reveal that Snail
up-regulation plays significant roles in switching off E-cadherin
expression in malignant melanoma cells.
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ACKNOWLEDGEMENTS |
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We are indebted to Astrid Hamm, Claudia Abschlag, and Inge Losen for technical assistance. We also thank Michael Weidner for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Deutsche Krebshilfe (to A. K. B. and R. B.) and by Grant PM 99-0132 from the Ministerio de Ciencia y Tecnologia (to A. G. d. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Institute of Pathology, Medical School Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany. Tel.: 49-241-8088080; Fax: 49-241-8888439; E-mail: bosserhoff@pat.rwth- aachen.de.
Published, JBC Papers in Press, April 25, 2001, DOI 10.1074/jbc.M011224200
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ABBREVIATIONS |
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The abbreviations used are: PBS, phosphate-buffered saline; RT, reverse transcription; PCR, polymerase chain reaction.
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