[CANCER RESEARCH 55, 5195-5199,
November 15, 19951
Advances in Brief
E-Cadherin Expression Is Silenced by DNA Hypermethylation in Human Breast
and Prostate Carcinomas1
Jeremy R. GrafT, James G. Herman, Rena G. Lapidus, Hemi Chopra, Rosa Xu, David F. Jarrard, William B Isaacs,
Paula M. Pitha, Nancy K Davidson, and Stephen B Baylin2
The Johns Hopkins Oncology Center fJ. R. G., J. G. H.. R. G. L, H. C.. R. X., P. M. P., N. E. D., S. B. B.J and The Brady Urologicai Institute (D. F. J.. W. B. 1.], Johns
Hopkins University Medical Center, Baltimore, Maryland 21231
Abstract
Expression
of the Ca2@-dependent,
homotypic
cell:ceH
adhesion
mole
cule, E-cadhenn (E-cad), suppresses tumor cell invasion and metastasis in
experimental tumor models. Decreased E-cad expression is common in
poorly differentiated,
advanced-stage
carcinomas. These data implicate
E-cad as an “invasionsuppressor― gene. The mechanism by which E-cad
is silenced
in advanced
stage
carcinomas
Is unclear.
In this
report,
we
show that: (a) the 5' CpG island of E-cad is densely methylated in
E-cad-negative breast and prostate carcinoma cell lines and primary
breast carcinoma
tissue but is unmethylated
in normal
breast tissue; (b)
treatment with the demethylating agent, 5-aza-2'-deoxycytidine, partially
restores
E-cad
RNA
and
protein
levels
in
E-cad-negative
breast
and
prostate carcinoma cell lines; and (c) an E-cad promoter/CAT construct is
expressed
in both E-cad-positive
noma
lines,
cell
indicating
that
and -negative breast and prostate
these
cells
have
the
active
carci
transcriptional
machinery necessary for E-cad gene expression. Our data demonstrate
that frequent loss of E-cad expression in human breast and prostate
carcinomas
results
from
hypermethylation
of
the
E-cad
promoter
c-erb-B2 proto-oncogene cDNA (20). Alternatively, aberrant hyper
methylation of 5' CpG islands within proximal promoter regions has
been implicated as a mechanism by which tumor suppressor genes can
be inactivated. Examples of this mechanism have been best demon
strated for the VHL and p16 tumor suppressor genes (21, 22). Indeed,
a CpG island has been identified within the 5' proximal promoter
region of the E-cad gene (23), and one recent study of colon, liver,
bladder, and gastric carcinoma cell lines has correlated aberrant hy
permethylation of HpaII sites in this CpG island with decreased E-cad
expression (24). In this report, we have investigated, in detail, whether
E-cad expression in breast and prostate carcinomas might be silenced
by aberrant hypermethylation of the 5' CpG island within the proxi
mal promoter region. We now show that hypermethylation of the
E-cad 5' CpG island accounts for loss of this “invasion
suppressor―
molecule in cells from breast and prostate carcinomas.
Materials
and Methods
region.
Cell Culture and Treatment All cell lines were culturedin 80-cm2tissue
Introduction
culture
flasks (Nunc)
cell lines (T47D,
E-cad3 is a Mr 120,000 glycoprotein that, when complexed with a-,
13-,and -y-catenins,
mediatesCa2tdependent,homotypiccell:cell
adhesion (1). Loss of E-cad expression and the subsequent loss of
homotypic cellular adhesiveness may be a critical step in the ability of
epithelial tumor cells to invade and metastasize. E-cad expression is
decreased or absent in poorly differentiated carcinomas of the breast,
prostate, colon/rectum, stomach, esophagus, kidney, pancreas, liver,
thyroid, and ovary and in squamous cell carcinomas of the head and
neck (2—1
1). In breast and prostate carcinoma, loss of E-cad is related
to tumor aggressiveness (2, 3). In experimental tumor models, re
stored expression of E-cad promotes epithelial differentiation charac
teristics and inhibits invasion in vitro and in vivo, implicating E-cad
as an “invasionsuppressor gene―(12—14).
The mechanism by which E-cad expression is lost is presently
unclear. Allelic loss of the E-cad gene locus (16q22) has been re
ported in 30—50%of breast, prostate, and hepatocellular carcinomas
(1 1, 15, 16). Extensive analyses reveal that mutations within the E-cad
coding sequence in breast, gastric, and gynecological cancers are rare
(17—19).Additionally, in human mammary epithelial cells, expression
of the E-cad gene can be down-regulated by overexpression of the
I This
work
was
supported
by
National
Cancer
Institute
Grants
CA43318
(to
J.
R.
0.,
J. G. H., H. C., and S. B. B.) and DK49043 (to R. X. and P. M. P.) and a grant from the
Susan G. Komen Foundation (to R. G. L.).
2 To
whom
requests
for
reprints
should
be
addressed,
at The
Johns
Hopkins
University
Oncology Center, 424 North Bond Street, Baltimore, MD 21231. Phone: (410) 955-8506;
Fax: (410) 614-9884.
3 The abbreviations
chloramphenicol
used are: E-cad, E-cadherin;
acetyltransferase;
AzaC, 5-aza-2'-deoxycytidine;
f3Gal, @3-gaIactosidase.
CAT,
plates (Sarstedt).
MDA-MB-231,
MCF-7,
Breast
cancer
MCF-7/ADR,
ZR
75—1,Hs578t, and HBL100) were routinely cultured in DMEM (GIBCO-BRL,
Bethesda, MD) supplemented with 5% FCS. Prostate cancer cell lines (DuPro,
Du145, PC-3, LNCaP, TSUPr1, and FNC) were cultured in RPMI (GIBCO
BRL) supplementedwith 10% [email protected] cell lines were treatedwith a
final concentration of0.5 @tM
AzaC for 3 days (Sigma Chemical Co., St. Louis,
MO).
Restriction
ILg)
was
Digests
isolated,
and
Southern
electrophoresed,
Blot
Analysis.
transferred
to
Genomic
Zetaprobe
nylon
DNA
(5-8
membrane
(Bio-Rad, Richmond, CA), and hybridized as described previously (21, 22).
Fifteen units of each enzyme were used/p@g genomic DNA. All restriction
enzymes were purchased from New England Biolabs (Cambridge, MA).
To generate a DNA probe for Southern blotting, a 769-bp PCR fragment
corresponding
to nucleotides
403—i 172 of the E-cad proximal
promoter
region
(25, 26) was amplified from normal fibroblast DNA using the following
primers and conditions: 5'-GGAGGCCAAGGCAGGAGGATCGC-3' (up
stream); 5'-CGAGAGGCFGCGGCFCCAAGGG-3'
(downstream);
95°Cfor
35 s, 68°Cfor 45 5, and 72°C1 mm for 40 cycles. The 769-bp amplified
product was then subcloned into the TA PCR cloning vector as per the
manufacturer's instructions (Stratagene, La Jolla, CA). A 377-bp PstI fragment
prepared from the amplified product, which encompasses the active 5' prox
imal promoter (Fig. 1; Ref. 25), was used as the probe for all Southern blots
(Fig.
1).
Western
Received 8/15/95; accepted 10/2/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
or 10-cm2 tissue culture
MDA-MB-468,
Blot Analysis.
Western
blots were performed
as described
pre
viously (27) using 50 p@gprotein/lane. The mouse monoclonal anti-E-cad
antibody, HECD, was purchased from Zymed (San Francisco, CA). The goat
antimouse IgG conjugated to horseradish peroxidase was purchased from
Santa Crux Biotechnology
(Santa Cruz, CA). Western blots were visualized
with the ECL chemiluminescent detection kit (Amersham, IL).
Immunofluorescence. Cells were plated onto glass coverslips and grown
to confluency in the presence or absence of 0.5 gtMAzaC for 3 days. Routine
immunofluorescence was performed on cells that were precleared with 0.1%
Triton X-100 and stained with HECD and a goat antimouse antibody conju
gated to FITC (Southern Biotechnology, Birmingham, Alabama; Ref. 3).
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METHYLATION SILENCES E-CADHERJN EXPRESSION
P
A
@
I
Hh.1
@‘[email protected]
IS
I•
I
ISJDE
Thall000B
aJOD
Fig. 1. Methylation-sensitive restriction map of
3llbp
the E-cad 5' CpG island. A. the methylation-sen
sitive site restriction map of the E-cad 5' CpG
island, based upon published sequences (23, 25).
P. the PstI sites
encompassing
the 377-bp
Pati flank
237bp PstllThal
292bpPall/Thai
active
promoter. H, S. B, and E. HpaII, SaclI, BssHII and
PstilHhal
EagI sites, respectively. H/wI and ThaI sites are
indicated as such. PstIJThaI and PstIJHhaI restric
tion fragment sizes are indicated below the restric
tion map. A —16-kb EcoRl restriction also flanks
the depicted region. B depicts the CpG density of
the island, and each vertical mark represents an
individual CpG site.
B
CpG Density
II III II I IIII I III I III III III 111111
11111111
11111
III 11111
I11111111
liii
I
@oD
Transient Transfection Analysis. The PstI fragment representing the pre
viously characterized active promoter of the mouse E-cad gene (26) was
subcloned
in front of a promoterless
CAT reporter
construct
and designated
E-cad/CAT. One pg pRSV-f3Gal and 2 @gpRSV-CAT (positive control),
pBLCAT3 (promoterless CAT as a negative control) or E-cad/CAT was
transfected
into cells with lipofectamine
reagent as per the manufacturer's
recommendations (GIBCO-BRL). Cells were harvested 48 h after removal of
lipofectamine.
f3Gal activity was measured using a @3Galactivity kit (Promega,
Madison, WI). CAT activity was measured as described previously (28). All
CAT activity values were normalized to /3Gal activity for each plate.
Results
Fig. 1 provides a methylation-sensitive
-.-
1500-bp
CpG
island
extending
from
the
restriction
promoter
map of the
region
through
intron 1 of the E-cad gene. To assess whether methylation of the
E-cad CpG island may be related to expression, we mapped the
methylation-sensitive restriction sites in breast and prostate carcinoma
cell lines, normal breast tissue, and primary breast carcinoma (Fig. 2
and Table 1). We first matched methylation patterns of the E-cad
promoter with well-documented expression profiles of E-cad in es
tablished breast and prostate cancer cell lines (29, 30). The E-cad
positive breast and prostate cancer cell lines are unmethylated at each
site tested within the 5' CpG island (breast: MCF-7, T47D, and
ZR-75—1; prostate: PC-3, LNCaP, and Du145). In contrast, all but one
of the E-cad-negative breast and prostate cancer cell lines are meth
ylated at each site examined (breast: MCF-7/ADR, MDA-MB-23i,
MDA-MB-435, Hs578t, and HBL 100; prostate: DuPro, TSUPr1, and
FNC;
Fig. 2, A and B; summarized
in Table
1). MDA-MB-468,
a
breast cancer cell line with reduced E-cad expression despite having
an unmethylated promoter, is discussed below.
To ensure that the E-cad CpG island is not methylated in DNA
from normal tissue and that the above findings are not limited to
cultured cells, the methylation status of the E-cad CpG island was
determined for DNA isolated from normal breast tissue and primary
breast cancer tissue. DNAS isolated from five normal breast tissue
samples were unmethylated at all sites examined within the CpG
island (summarized in Table 1). Changes in methylation, however,
could be detected by Southern analysis of DNA isolated from primary
tumor tissue samples, although these samples were not enriched for
tumor versus normal cells. A total of 12 breast carcinoma tissue
samples were analyzed, including two samples from breast carcinoma
I
1@D
2JOD
metastases. The majority of these 12 samples were analyzed at the
ThaI, HhaI, SacII, and Eagl sites depicted in Fig. 1 (summarized in
Table 1). Each sample had prominent bands, resulting from com
pletely unmethylated sites which may be attributed to associated
normal tissue within the samples. However, many samples revealed
distinct methylation at the ThaI sites (55%; Fig. 2C), at the HhaI site
(45%), SacII site (70%; Fig. 2D), and the EagI site (67%).
Methyl
ation at the ThaI site was also evident in a primary prostate tumor
sample (PT-i; Fig. 2c; Table 1) previously shown not to express
E-cad (3). These data demonstrate that hypermethylation of the E-cad
CpG island is tumor specific and is not limited to cultured cells.
If methylation of the E-cad CpG island is responsible for sup
pressing E-cad expression, treatment of the breast and prostate
cancer cell lines with the demethylating agent, AzaC, might reac
tivate expression. Treatment of cell lines with AzaC reactivates the
expression of the VHL and pi6 tumor suppressor genes for cell
lines in which the 5' CpG islands are methylated (21, 22). Slight
reactivation of E-cad expression was evident in the methylated
breast cancer cell lines, Hs578t and MDA-MB-23i,
as evidenced
by low levels of E-cad protein detected by Western blots only after
AzaC
treatment
of cells
(Fig.
3). Greater
activation
was detected
for the methylated prostate cancer cell line, TSUPr1 (Fig. 3).
Prostate cancer cell line DuPro, which is also methylated, ex
pressed low basal levels of E-cad protein but much higher levels
after AzaC treatment (Fig. 3). Partial reactivation in the same cell
lines was also confirmed by reverse transcription-PCR
and by
FACS analysis for one prostate and one breast cancer cell line (for
DuPRO, less than 1% of cells were positive prior to AzaC treat
ment, whereas 14% of cells were positive after AzaC treatment; for
Hs578t, 1.5% of cells were positive prior to AzaC treatment,
whereas 15% were positive after AzaC treatment). E-cad gene
reactivation could also be clearly detected by indirect immunoflu
orescence. Surface localization of E-cad was evident in numerous
clusters of cells from MDA-MB-231, Hs578t, TSUPr1 (data not
shown), and DuPro (Fig. 3) after a 3-day treatment with AzaC. The
level of demethylation at the ThaI site, as quantitated by phospho
rimaging scanning of blots, ranged from 11% in Hs578t, where
only minimal protein was detected (Fig. 3), to 45% in DuPro, in
which E-cad expression was sharply increased after AzaC treat
ment (Fig. 3). AzaC treatment of the E-cad-positive
cell lines
5196
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m
m. .@
;@
=
m
,@
A.
r,@
237bp
METHYLATION SILENCES E-CADHERIN ExPRESSION
@
A
B!
2
.@
I,
;;
(‘I
@
@
@:
tL
0
th
@
@
@
0
p@tLiL
@
@
—
‘0
I,
—
377bpS
__
.—
—@
—
.
292bp
t•
237bp
@
LLLL
C.)
C)
0
I-.
0
<
N
0
I
w
@.
..
.
Br.ast Cancsr Call Uns
Prostat.
I
.
294bp
Cancer Call Lln.s
C
D
a
A.
0
E
@
@
;a
@- r',i
C')
In
(0
r.-.
• a,
0@-
,.:.
@
.@
@
317bps
.
@
-.16kb
@.
292bp
-.4kb
Fig. 2. Southern blot analyses of methylation-sensitive restriction digests. All restrictions were performed with 15 units enzyme/p@gDNA. In each case, DNA is restricted, both with
a nonmethylation-sensitive enzyme (PstI or EcoRI) that detects sites flanking the methylation sensitive sites and the indicated methylation-sensitive enzyme (see Fig. 1). in each panel,
the size of the band(s), indicating a hypermethylated site(s). The 377-bp PstI fragment (Fig. 1) is used as the probe. A, the PstI/ThaI restriction of breast and prostate carcinoma cell
lines. The 377-bp fragment produced by cutting with Pstl alone is represented in the first lane. Note hypermethylation for MDA-MB-231, MDA-MB-435, Hs578t, HBL100,
MCF-7/Adr, FNC, DuPro, and TSUPr1. B, the PstI/HhaI restriction of breast carcinoma cell lines. The 377-bp flanking cut is represented in thefirst lane. Note hypermethylation in
MCF-7/ADR, HBL100, MDA-MB-231, MDA-MB-435 and Hs578t. C, the PstI/Thal restriction of primary breast and prostate carcinoma tissue. Independent breast tumor tissue
samples arc numbered BT1—BT1O.Breast tumors BT4, BT6, BT8, BT9, and prostate tumor PT-i show distinct evidence of ThaI site methylation. The PstI flanking cut of breast tumor
DNA is represented in thefirst lane. The prominent 230-bp band reflects fully unmethylated sites, which may be attributed to normal tissue in these tumor samples. D, the EcoRI/SacIl
restriction digests of primary breast tumor tissue. MBT-1 and MBT-2 are independent metastatic breast tumors. B'F-l, -4, -6, -7, -8, and -10 and MBT-2 display a —16-kb band,
indicating for some alleles, methylation of all Sacll sites within the EcoRI-flanked region. The fully restricted band at —4kb, evident in all lanes, may represent heavy contribution
from unmethylated normal tissue DNA that was not removed from these noncultured breast and prostate tumor tissue samples.
MCF-7 and PC-3, which are unmethylated, did not affect E-cad
MDA-MB-468 is the only cell line studied that is not methylated in
expression as evidenced by FACS analysis, Western blot analysis,
the E-cad proximal promoter and yet does not highly express the
reverse transcription-PCR,
or indirect immunofluorescence
(data
E-cad gene. In a previous report, the expression of E-cad in MDA
not shown).
MB-468 was reduced at the RNA level relative to either MCF-7 or
Aberrant hypermethylation of CpG islands is thought to silence
T47D and could not be detected by immunofluorescence (29). By
transcription by altering local chromatin structure and preventing
Western blot analysis, we find that MDA-MB-468 expresses less than
the binding of transcription factors to their respective promoter
10% of the E-cad protein levels expressed by MCF-7 (data not
elements. In this manner, aberrant hypermethylation
may block
shown). The MDA-MB-468 cell line overexpresses the c-erb-B2
transcription without affecting the expression or activity of tran
proto-oncogene (R. X. and P. P.; data not shown), which has been
scription factors (31, 32). If aberrant hypermethylation is respon
shown to down-regulate transcription of the E-cad gene via decreased
sible for silencing E-cad expression in this manner, then the cell
transcription factor activity (20). Hence, the mechanism associated
lines with methylated E-cad CpG islands should retain the active
with reduced expression of E-cad in this line should be different from
transcriptional machinery necessary for E-cad gene expression. To
that in the others.
test this hypothesis, a CAT reporter gene construct driven by the
As shown in Fig. 4, MCF-7, PC-3, DuPro, and Hs578t express
well-characterized murine E-cad proximal promoter region, known
substantial CAT activity, whereas MDA-MB-468 does not. Interest
to contain maximal activity in both murine and human cells (26),
ingly, the methylated cell lines, DuPro and Hs578t, express slightly
was transfected into two prostate cell lines, DuPro and PC-3, and
less (2-fold) CAT activity than the unmethylated lines, MCF-7 and
three breast cancer cell lines, Hs578t, MCF-7, and MDA-MB-468.
PC3. However, this reduction cannot solely account for the vast
MCF-7 and PC-3 are unmethylated at the E-cad 5 â€C̃pG island and
differences in expression of the endogenous E-cad gene between these
express high levels of E-cad endogenously, whereas Hs578t and
cell lines. That the methylated
cell lines do not express E-cad endo
DuPro have an extensively methylated E-cad 5'CpG island and
genously, but are able to express CAT activity from the exogenous
have little or no endogenous E-cad expression.
E-cad/CAT construct, implies that methylation acts in cis to silence
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@
@
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METHYLATION SILENCES E-CADHERIN EXPRESSION
Table I Mapping of the methylation-sensitive
E-cad 5' CpG island
restriction sites in the
mci―HhnlaH,@€ri@Eagl―Breast
carcinoma
MCF-7―
ZR-75-l―U
UMDA-MB-468
UU
UU
UU
UU
ery required for E-cad expression. This latter finding further impli
cates methylation of the endogenous promoter as a functional, cis
acting block to expression. However, our data cannot rule out the
additional possibility that trans-acting mechanisms work in concert
with methylation to suppress E-cad gene expression. Indeed, the
T47D―U
UU
UU
UU
UMDA-MB-435PMMNDNDHBL100MMMNDNDMDA-MB-231MMMMMMCF-7/ADRMMMMMHs578tMMMPMBreast
UU
A
tissueNIUUUUN2UUUUN3UUUUN4UUUUN5UUUUBT1UUMMBT2UMUMBT3UUUUBT4MMMMBTSMUUUBT6MMMMBT7UUMMBThMUMMBT9MUMMBT1ONDMMUMBT-1UUNDUMBT-2MMNDMProstate
@
,@
120
B
E
carcinoma
PC-3―
,‘
LNCaP@'
U
U
U
UDuProMMMTSUPrIMMMFNCPNDNDPT-lMNDND
DuI4S―U
PU
UU
@
@
..-@
@
.
“@
. ‘54•@@A@\
_r@@_S:
t@_S
@
@
@
@L@:;!@o
.
;:t@@:.
.
@.
. S,
a ND, not determined, M, methylated sites; P, partially methylated sites; U, the
presence of unmethylated sites only.
b
@lllines
that
express
. ,
1-@-@
:@-;@
@‘-r'@@
.@
@:,@,
-S
\@@SS..:-—
E-cad.
The flanking restriction for ThaI, H/wI, and HpaII was PstI. The flanking restriction
for Sacll and EagI was EcoRI. All blots were probed with the 377-bp PstI fragment
representing the active promoter region of E-cadherin (26). Ni through N5 are normal
breast tissue samples. BT1 through 10 are independent primary breast carcinoma tumor
samples. MBTI and 2 are independent metastatic breast tumor samples. MBT1 and T2 are
unrelated to BT-l and 2. FT-i, E-cad-negative prostate tumor tissue sample (3).
@
@
transcription. Likewise, the failure of the MDA-MB-468 line to use
the E-cadiCAT construct is consistent with a different, trans-acting
mechanism for silencing E-cad gene transcription in these cells. The
possibility that, in the methylated cell lines, additional trans-acting
deficiencies may account for the slightly lower CAT activities will be
of importance for subsequent studies.
@
Discussion
@
@
@
@
S.
;@
Data in this report demonstrate that loss of expression of the E-cad
gene, which encodes an invasion suppressor protein for which loss is
integral to the progression of carcinomas, is accompanied by aberrant
methylation of a 5' promoter region CpG island in human breast and
prostate carcinoma cells. Methylation of the E-cad CpG island cor
relates not only with a lack of expression in breast and prostate
carcinoma cell lines (Table 1) but also is evident in more than 50% of
primary
breast carcinomas
. ,@,
I'!1'*@@@
cij'.,
.@
:
‘ )
4@A
S
throughout
the entire E-cad
5' CpG island
.tS:.@
j@';,•p@@@
::,@. t.ç.@..
;@.
,, .
@)
;@..
. “l..@;'“,@f ,.. ‘:@.â€ẫ€˜
,‘
...
.- •aS
Fig. 3. Treatment of breast and prostate carcinoma cell lines with the demethylating
agent AzaC partially reactivates E-cad expression. Top, Western blot using the antibody
HECD-l. The E-cad-specific band at M, 120,000 is indicated by an arrow to the right and
by asterisks. Note faint bands in both MDA-MB-23l
and Hs578t and stronger bands in
TSUPr1, and especially DuPro, following AzaC treatment of cells. Protein from culture
(Table 1). The direct involvement of hypermethylation in the suppres
line T47D is included in the first lane as an E-cad-expressing positive control. Bottom,
indirect immunofluorescence for E-cad expression. A, background fluorescence in the
sion of E-cad gene expression is supported by the observation that
E-cad-expressing prostate cell line, PC-3, stained only with secondary FITC-conjugated
expression of the E-cad gene can be reactivated by treatment with the
goat antimouse IgG. E. the typical “honeycomb―
fluorescence of the E-cad-positive
demethylating agent, AzaC, in four carcinoma cell lines. Finally, these
prostate cancer cell line, PC-3. C, a typical field of the E-cad-negative prostate cancer cell
line, DuPro, without AzaC treatment. G, a typical field of DuPro cells following a 3-day
cell lines, which do not express an endogenously methylated E-cad
treatment of the cell line with 0.5 @M
AzaC. Note the “honeycomb―
pattern of fluores
gene, can substantially utilize an E-cad promoter/CAT construct,
cence similar to that of PC-3 cells (E). B, D, F, and H, the phase contrast photographs
indicating that they retain much of the active transcriptional machin
corresponding to A, C, E, and G. respectively.
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METHYLATION SILENCES E.CADHERIN ExPRESSION
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correlation with tumor dedifferentiation and lymph node metastasis. Cancer Rca., 51:
__
DuPro PC-3
Prostate
Hs578t
6328—6337,1991.
MCF/7
Breast
468
Fig. 4. Both methylated and unmethylated breast and prostate carcinoma cell lines can
transactivate an exogenous E-cad promoter. All data are normalized to expression of a
RSV-@3Gal construct, which was always cotransfected to control for transfection effi
ciency. Data are expressed as the fold increase of E-cad/CAT expression relative to
PBLCAT3 expression (the promoterless CAT construct). Bars. SEM for 6 to 12 mdc
pendent transfection dishes. Breast cancer cell line MDA-MB-468 is represented as 468.
12. Vleminckx, K., Vakaet, L., Marcel, M., Fiers, W., and Van Roy, F. Genetic manip
ulation of E-cadherin expression by epithelial tumor cells reveals an invasion sup
pressor role. Cell, 66: 107—119,1991.
13. Frixen, U. H., Behrens, J., Sachs, M., Eberle, G., Voss, B., Warda, A., Lochner, D.,
and Birchmeier, W. E-Cadherin-mediated cell-cell adhesion prevents invasiveness of
human carcinoma cells. J. Cell Biol., 113: 173—185,
1991.
14. Navarro, P., Gomez, M., Pizarro, A., Gamallo, C., Quintanilla, M., and Cano, A. A
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15. Sato, T., Tanigami, A.. Yamakawa, K., Akiyama, F., Kasumi, F., Sakamoto, 0., and
Nakamura, Y. Allelotype of breast cancer: cumulative allele losses promote tumor
prostate cancer cell line TSUPr1, which has a fully methylated E-cad
CpG island, is unable to express a similar E-cad promoter CAT
construct (25) but can express E-cad following AzaC treatment
(Fig. 3).
As we were completing our current study, Yoshiura em'a!. (24)
reported similar data, indicating that hypermethylation of HpaII sites
in the E-cad promoter is associated with loss of E-cad expression in
colon, bladder, liver, and gastric carcinoma cell lines. Taken together,
these two reports demonstrate that hypermethylation of the CpG
island within the 5 ‘
proximal promoter is a prevalent mechanism by
which E-cad expression is inactivated in multiple types of carcinoma.
This report provides further evidence that aberrant DNA hypermethy
lation critically alters gene expression patterns in cancers, thereby
promoting tumor progression. Finally, these data indicate that loss of
E-cad expression from tumor cells, a critical step in the acquisition of
invasiveness and the subsequent metastatic spread of carcinomas,
results from the potentially reversible process of aberrant DNA meth
ylation. These findings suggest a potential therapeutic strategy for
preventing or reducing the metastatic dissemination of epithelial
tumors.
Acknowledgments
We thank Tammy Hess for secretarial assistance, Christopher M. Kovac for
photoimaging
provision
assistance,
of normal
breast
and Drs. Helene
and breast
Smith
cancer
and Sigmund
Weitzman
for
progression in primary breast cancer. Cancer Rca., 50: 7184—7189, 1990.
16. Carter, B. S., Ewing, C. M., Ward, W. S., Treiger, B. F., Aalders, T. W., Schalken,
J. A., Epstein, 3. I., and Isaacs, W. B. Allelic loss of chromosomes 16q and lOq in
human prostate cancer. Proc. NatI. Acad. Sci. USA, 87: 8751—8755, 1990.
17. Kanai, Y., Oda, T., Tsuda, H., Ochiai, A., and Hirohashi, S. Point mutation of the
E-cadherin gene in invasive lobular carcinoma of the breast. Jpn. J. Cancer Res., 85:
1035—1039,1994.
18. Becker, F., Atkinson, M. J., Reich, U., Becker, I., Nekarda, J., Siewert, .1. R., and
Hofler, H. Cadherin gene mutations provide clues to diffuse type gastric carcinomas.
Cancer Res., 54: 3845—3852,
1994.
19. Risinger, J. I., Berchuck, A., Kohler, M. F., and Boyd, J. Mutations ofthe E-cadherin
gene in human gynecologic cancers. Nat. Genet., 7: 98—102, 1994.
20. D'souza, B., and Taylor-Papadimitriou,
J. Overexpression of ERBB2 in human
mammary epithelial cells signals inhibition of transcription of the E-cadherin gene.
Proc. Natl. Acad. Sci. USA, 91: 7202-7206, 1994.
21. Herman, J. 0., Latif, F., Weng, Y., Lerman, M. I., Zbar, B., Liu, S., Samid, D., Duan,
D-S., Onarra, J. R., Linehan, W. M., and Baylin, S. B. Silencing of the VHL
tumor-suppressor gene by DNA methylation in renal carcinoma. Proc. Natl. Acad.
Sci. USA, 91: 9700—9704,1994.
22. Merlo, A., Herman, J. G., Lee, D. J., Gabrielson, E., Burger, P. C., Baylin, S. B., and
Sidransky, D. 5' CpG island methylation is associated with transcriptional silencing
of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat. Med., 1:
686—692,1995.
23. Berx, 0., Staes, K, van Hengel, J., Molemans, F., Bussemakers, M. J. G., van
Bokhoven, A., and van Roy, F. Cloning and characterization of the human invasion
suppressor gene E-cadherin (CDHJ). Genomics, 26: 281—289,1995.
24. Yoshiura, K., Kanai, Y., OChiai, A., Shimoyama, Y., Sugimura, T., and Hirohashi, S.
Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human
carcinomas. Proc. Natl. Acad. Sci. USA, 92: 7416—7419,1995.
25. Bussemakers, M. 3. G., Giroldi, L A., vab Bokhoven, A., and Schalken, J. A.
Transcriptional regulation of the human E-cadherin gene in human prostate cancer
cell lines. Biochem. Biophys. Rca. Commun., 203: 1284—1290, 1994.
26. Behrens, J., Lowrick, 0., Klein-Hitpass, L, and Birchmeier, W. The E-cadherin
promoter: functional analysis of a GC-rich region and an epithelial cell-specific
tissue samples.
palindromic regulatory element. Proc. NatI. Acad. Sci. USA, 88: 11495—1
1499, 1991.
27. Graff, J. R., Boghaert, E. R., Dc Benedetti, A., Tudor, D. L, Zimmer, C. C., Chan,
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5199
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
E-Cadherin Expression Is Silenced by DNA Hypermethylation in
Human Breast and Prostate Carcinomas
Jeremy R. Graff, James G. Herman, Rena G. Lapidus, et al.
Cancer Res 1995;55:5195-5199.
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