Carcinogenesis vol.21 no.7 pp.

14531456, 2000
SHORT COMMUNICATION
APC truncation and increased -catenin levels in a human breast
cancer cell line
Peter W.Schlosshauer
1,3
, Stephen A.Brown
2
, 4, Wnt-7b) has been seen in human proliferative breast lesions
Katarina Eisinger
2
, Qingyou Yan
1
, (12). Overexpression of Wnt-1 and Wnt-3 in the murine
Enrica R.Guglielminetti
2
, Ramon Parsons
1
, mammary gland induces mammary hyperplasia and increases
Lora Hedrick Ellenson
3
and Jan Kitajewski
1,2,4
the incidence of mammary gland tumors (1). In addition,
APC
min
mice, which represent a model for the human FAP
1
Department of Pathology and
2
Department of Obstetrics and Gynecology,
syndrome, have an increased incidence not only for intestinal
Columbia University, College of Physicians and Surgeons, 630 West 168th
neoplasias but also for mammary tumors (13). Despite the link
Street, New York, NY 10032 and
3
Department of Pathology, Weill Medical
College of Cornell University, 1300 York Avenue, New York, NY 10021, between Wnt signal activation and mammary gland tumors in
USA
the mouse, comparable signal activation due to mutations in
genes encoding Wnt signaling components has not been
4
To whom correspondence should be addressed
Email: jkk9@columbia.edu documented in human breast cancer. In the present study, we
screened a panel of 27 cell lines, either derived from human
Mutations in the Adenomatous Polyposis Coli (APC) tumor
breast carcinomas or established by immortalization of normal
suppressor gene or the -catenin gene are present in most
breast epithelial cells, for alterations in the -catenin, -catenin
colon cancers and less frequently in other tumor types. In
and APC proteins and for mutations in the APC, - and -
this study, we screened 24 human breast cancer cell lines
catenin genes.
and three immortalized human breast epithelial cell lines
Cell lines BT 20, BT 474, BT 483, BT 549, CAMA 1, DU
for alterations in - and -catenin and APC by western
4475, HBL 100, HS 578 T, MCF7, MCF10A, MDA-MB 157,
blotting, protein truncation assay and DNA sequence
MDA-MB 175 VII, MDA-MB 231, MDA-MB 330, MDA-
analysis. In one cell line (DU 4475), an APC mutation
MB 361, MDA-MB 415, MDA-MB 435S, MDA-MB 436,
was identied (E1577stop) that resulted in expression of
MDA-MB 453, MDA-MB 468, SKBR 3, T 47 D, UACC 812,
truncated APC. This mutation was associated with elevated
UACC 893, ZR 75-1 and ZR 75-30 were purchased from the
cytosolic -catenin levels, probably due to loss of APC
American Type Culture Collection (ATCC; Rockville, MD).
function, as in colon cancers. No mutations were found in
MCF10A is a spontaneously immortalized line from normal
exon 3 of the - or -catenin genes. We conclude that APC
human breast epithelial cells, and HBL 100 is an SV40
mutations and -catenin upregulation may occur with low
immortalized human breast epithelial cell line. HB2, another
frequency in human breast cancer cells.
SV40 immortalized human breast epithelial cell line, was
obtained from Dr Joyce Taylor-Papadimitriou (London, UK).
Cells were grown under recommended conditions and harvested
-Catenin, a key cytosolic component of the Wnt signal
at 90100% conuency. For total cell protein extraction, cells
transduction pathway, is implicated in embryonic development
were lysed in TENT buffer (50 mM Tris pH 8.0, 1 mM EDTA,
and carcinogenesis. Normally, cytosolic -catenin is maintained
150 mM NaCl, 1% Triton X-100), centrifuged, the supernatant
at low levels by the APC gene product, which promotes -
admixed with protein sample buffer and boiled. Cytosolic
catenin degradation, and by cadherins, which integrate -
and membranous fractionation was carried out as described
catenin into adherens junctions (1). Wnt signal activation
previously (3). Total protein (30 g) was separated by electro-
results in stabilization of -catenin (24) and subsequent
phoresis on 8% SDSpolyacrylamide gels, transferred onto
translocation of -catenin to the nucleus, where it binds to
nitrocellulose (Micron Separations Inc., Westboro, MA) and
TCF-class transcriptional factors and induces expression of
identied using primary antibodies against - and -catenin
TCF-responsive genes (5,6). -Catenin has structural and
(Transduction Laboratories, Lexington, KY), and chemilumin-
functional similarities to -catenin (1) but its role in Wnt
escence detection (ECL; Amersham, Arlington Heights, IL).
signaling has not been elucidated. Several reports have docu-
To screen for mutations in exon 3 of the - and -catenin
mented that deregulation of the Wnt pathway is associated
genes, a portion of that exon was amplied from genomic
with human tumorigenesis. Truncations of the APC protein
DNA from all cell lines using the following primer pairs: -
are linked to the familial adenomatous polyposis (FAP) coli
catenin, Forward(F) 5-ATTTGATGGAGTTGGACATGGC-
syndrome and are found in the majority of sporadic colon
3 and Reverse(R) 5-GAGGAAGAGGATGTGGATACCT-
carcinomas (7). Mutations in either APC or -catenin, resulting
CC-3; -catenin, F 5-AGCCACGATGGAGGTGATGAAC-
in deregulation of -catenin turnover, increase -catenin/Tcf
3 and R 5-CCTGGGTGTAAGTGGTGGTTTTC-3. signaling in colon cancer (8) and melanoma cell lines (9).
To analyze the entire -catenin gene from DU4475, cDNA The APC encoding locus on chromosome 5q21 shows loss
was synthesized in 12 separate RTPCR reactions (primer of heterozygosity (LOH) in ~25% of breast cancers (10).
sequences available upon request) using total RNA as template. Frequent alterations in expression of E-cadherin and - and
cDNA was then PCR-amplied, submitted to automated -catenins have been reported in a survey of 18 human breast
cancer cell lines (11). High Wnt gene expression (Wnt-2, Wnt- sequencing and compared to the published -catenin sequence
Oxford University Press 1453
P.W.Schlosshauer et al.
Fig. 2. - and -catenin levels in the cytosolic fraction of selected breast
cancer cell lines, analyzed by western blot. HB2 serves as control. Note
increased content of cytosolic -catenin in DU 4475.
Fig. 1. Total cell lysates from breast cancer cell lines analyzed by western
blot for -catenin (A) and -catenin (B). HB2 serves as control. (A)
-Catenin blot. Note signicant overexpression of -catenin in DU 4475 as
compared to control and other cell lines. (B) -Catenin blot. -catenin
expression in DU 4475 is strong, but not exceeding levels in other cell
lines.
(14). To screen breast cancer cell lines for truncation mutations
in APC, the TNT

coupled reticulocyte lysate system(Promega,

Madison, WI) was used, following the manufacturers instruc-
tions. Two overlapping segments of the APC gene, spanning
the region from codon 686 to 1693, were amplied from
cDNA using the following T7-modied primers (T7-trans
GGATCCTAATACGACTCACTATAGGGAGACCACCAT-
GG): segment A (codons 686-1217 ), F 5-T7-transATGCAT-
GTGGAACTTTGTGG-3 and R 5-GAGGATCCATT-
AGATGAAGGTGTGGACG-3; segment B (codons 1099
1693), F 5-T7-transTTTCTCCATACAGGTCACGG-3 and
Fig. 3. (A) Protein truncation assay of segment B (codons 1099 to 1693) of
the APC gene. The control was generated with a human genomic DNA R 5-GGAGGATCCTGTAGGAATGGTATCTCG-3.
sample known to have the wild type APC gene. A truncated protein is
Aliquots of 5 l of the in vitro transcription/translation
expressed in DU 4475. Representative for all other breast cancer cell lines,
reaction were separated on 420% TrisHCl SDSpolyacryl-
the protein segment in ZR 75-1 is of normal size. (B) Partial sequence of
amide gels (Ready Gel

; Bio-Rad, Hercules, CA) and the

the APC gene from genomic DNA of cell line DU4475 and a normal
product visualized by autoradiography (BioMax Kodak, Roch- control. As denoted by arrows, a GT transversion creates a stop codon at
amino acid 1577.
ester, NY). Segment B of the APC gene in cell line DU 4475
was sequenced using the Thermo Sequenase

kit (Amersham,
Arlington Heights, IL). Additional primers (sequences available MDA-MB 453 and UACC 812; -catenin was detected at very
low levels in MDA-MB 330, SKBR 3 and ZR75-30. In DU upon request) were obtained from Operon Technologies Inc.
(Alameda, CA). An aliquot of 2 l of the chain-termination 4475, -catenin expression was extremely strong, exceeding
the positive control (Figure 1A). In MDA-MB 468, a double reaction products were submitted to electrophoresis on a
CastAway

sequencing device (Stratagene, La Jolla, CA) using band was seen with a second signal at ~80 kDa (Figure 1A).
-Catenin expression was strong in BT 20, BT 474, BT 483, precast 6% polyacrylamide7 M urea gels and visualized
by autoradiography (BioMax; Kodak, Rochester, NY). All BT 549, CAMA1, DU4475, HB2, HBL 100, MCF7, MCF10A,
MDA-MB 157, MDA-MB 175 VII, MDA-MB 361, MDA- experiments were performed at least twice.
Total cell lysate western blot analysis (Figure 1) showed MB 415, MDA-MB 468, SKBR 3, T47D, UACC 812, UACC
893 and ZR 75-1; weak in HS 578 T. MDA-MB 231, MDA- strong expression of -catenin in BT 20, BT 474, BT 483, BT
549, DU 4475, HB2, HBL 100, HS 578 T, MCF7, MCF10A, MB 435 S, MDA-MB 436, MDA-MB 453, ZR 7530 and C
57-Wnt-1; and not detectable in MDA-MB 330 (Figure 1B). MDA-MB 157, MDA-MB 175 VII, MDA-MB 231, MDA-
MB 361, MDA-MB 415, MDA-MB 468, T 47 D, UACC 893, All detectable - and -catenin signals migrated at the expected
position for an apparent molecular weight of 92 and 82 kDa, ZR 75-1 and C57-Wnt-1 (positive control); weak expression
was seen in CAMA 1, MDA-MB 435 S, MDA-MB 436, respectively.
1454
APC truncation in human breast cancer
On the basis of these results, the following cell lines were been reported, both are point mutations leading to amino acid
selected for analysis of cytosolic (Figure 2) and membranous substitutions (18). In addition, an APC mutation in a primary
protein fractions (data not shown): CAMA 1 (weak expression breast cancer has been reported (20), but no documentation of
of -catenin), DU 4475 (high expression of -catenin), HS alterations in -catenin levels was presented.
578 T (low levels of -catenin), MDA-MB 468 (-catenin The DU 4475 cell line was derived from a recurrent thoracic
double band), SKBR 3 (low levels of -catenin) and HB2 wall tumor following mastectomy for a poorly differentiated
(control). As expected, in most lines - and -catenin levels invasive ductal breast carcinoma in a postmenopausal patient
were very low in the cytoplasmic fraction (Figure 2), whereas (21). DU4475 cells are highly transformed and do not adhere
both proteins were prominent in the membranous fraction to the culture dish, but rather grow in suspension. It is not
(data not shown). Of the cell lines analyzed, -catenin was clear whether upregulation of -catenin contributes to these
signicantly elevated in the cytosolic fraction of DU 4475 growth characteristics. Since APC mutations are rare in breast
(Figure 2). -Catenin levels were slightly stronger in the cancer, we considered the possibility that this mutation was
cytosolic fraction of DU 4475, as compared with the other acquired in culture. In our analysis, there was no evidence of
cell lines analyzed, but not to the extent exhibited for - a second normal allele. This is interpreted as a hemizygous
catenin. Low levels of -catenin in CAMA 1 (Figure 1) were loss of the other allele since homozygosity for a point mutation
conrmed in the fractionated analysis (Figure 2). would be extremely unlikely. Presumably, the point mutation
We analyzed the serine/threonine region encoded by exon in one allele (Figure 3B) was complemented by loss of
3 of the - and -catenin genes from all cell lines and the the other allele (Figure 3A). The acquisition of these two
entire -catenin gene from DU 4475. However, no mutations independent mutations is unlikely to occur during propagation
were identied in our study. in vitro; although it cannot be formally excluded. There is no
The protein truncation assay was performed on two APC evidence from the original description (21) that the patient
segments encompassing codons 686 through 1693 for all cell
had FAP, suggesting a germline APC mutation.
lines listed except HB2, MCF10A, MDA-MB 330 and MDA-
The variability of expression levels of - and -catenin in
MB 435S. Of all protein products, one showed an aberrant
the other lines may represent the normal range of variation.
size: The segment B product from DU 4475 had an apparent
Alternatively, it may be due to translational and post-transla-
molecular weight of 70 kDa instead of the expected 100 kDa
tional inuences or mutations in other regions of the respective
(Figure 3A). No wild-type allele, or full length protein product,
genes. Our results of - and -catenin expression are largely
was detected in this cell line (Figure 3A). Sequencing of the
concordant with other reports (11,22). The signicance of
respective APC gene segment revealed a point mutation at
minor deviations [in contrast to Pierceall et al. (11) we found
position 4747 of the coding sequence (GT), converting
strong -catenin expression in BT 474, MCF7, MDA-MB 231,
codon 1577 into a stop codon (E1577stop) (Figure 3B).
MDA-MB 468 and ZR 75-1] is uncertain. Concurring with
We analyzed 24 human breast carcinoma cell lines and three
our results, a study of 11 breast carcinomas revealed no
immortalized human breast epithelial cell lines for alterations
mutations in exon 3 of the - or -catenin genes by RT
in -/-catenin and APC. Mutations in the APC or -catenin
PCR and single strand conformation polymorphism (SSCP)
genes that result in accumulation of -catenin have been
analysis (23).
reported in several carcinoma cell lines (8,9,15,16). We hypo-
In conclusion, we screened 27 human cell lines derived
thesized that similar aberrancies may play a role in human
from breast carcinomas or immortalized breast epithelial cells
mammary tumorigenesis. Our analysis focused on cancer cell
for alterations in the downstream elements of the Wnt signaling
lines to correlate mutations in APC or -catenin with changes
pathway; APC, - and -catenin. We report the rst APC
in -catenin levels. Our APC analysis comprised the region
mutation with associated -catenin upregulation in a human
from codon 686 to codon 1693 of the APC coding sequence.
breast carcinoma cell line (DU 4475). Unlike colon carcinomas
This includes the mutation cluster region (MCR; codons 1286
and melanomas, however, deregulation of the Wnt signaling
1513) which contains 65% of the APC mutations in colorectal
pathway does not seem to be a frequent event in human breast
tumors (17). We identied an APC mutation in one cell line
carcinomas.
(DU 4475) that is associated with upregulation of -catenin.
This is the rst report of an APC truncation resulting in
Acknowledgements
deregulation of -catenin in human breast cancer cells. The
increased -catenin levels in DU 4475 is in keeping with the
The authors thank Martin Julius, Nick Papadopoulos and Dan Smith for
current concept that the truncated APC protein fails to promote providing comments on the manuscript. This work was supported by grants
NIH RO1CA75353 and Marilyn Bokemeier Sperry Fund (to J.K.) and
-catenin degradation (8,9). The APC mutation in DU 4475
R21CA66224 (to L.H.E.)
leads to a profound excess of -catenin in total cellular extracts
(Figure 1A) and cytosolic pools (Figure 2), whereas -catenin
levels are less affected (Figures 1B and 2).
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Received January 11, 2000; revised March 22, 2000; accepted March 29, 2000 413424.
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