Aso 2005 04 010
Aso 2005 04 010
Aso 2005 04 010
DOI: 10.1245/ASO.2005.04.010
Tracey A. Martin, PhD, Amit Goyal, MB ChB, Gareth Watkins, BSc, and Wen G. Jiang, MD
Metastasis & Angiogenesis Research Group, University Department of Surgery, Wales College of Medicine, Cardiff University,
Cardiff, CF14 4XN, Heath Park, United Kingdom
Background: Slug, Snail, and Twist are transcription factors that regulate the expression of
tumor suppressors such as E-cadherin. We examined the distribution and expression of these
three molecules together with the methylation of the Twist gene promoter in human breast
cancer to elucidate their clinical significance.
Methods: Frozen sections from breast cancer primary tumors (tumor, n = 114; back-
ground, n = 30) were immunostained with Slug, Snail, and Twist antibodies. RNA was
reverse-transcribed, quantified, and analyzed by quantitative polymerase chain reaction (Q-
PCR). Results were expressed as copy number of transcript per 50 ng of RNA (standardized
against b-actin).
Results: Immunohistochemistry revealed that all three molecules were stained in mammary
tissues, with an increase in Twist within tumor tissues; this was supported by Q-PCR analysis.
Q-PCR analysis showed that Slug was elevated with increasing tumor grade and prognostic
indices. Twist was elevated with increasing nodal involvement (tumor-node-metastasis status).
Conversely, Snail was reduced in expression corresponding with prognostic indices and tumor
grade. Increased levels of Slug were associated with tumors from patients with metastatic
disease or disease recurrence, and increased expression of Twist was associated with tumors
from patients who had died from breast cancer. It is interesting to note that Snail expression
was significantly reduced in patients with a poor outcome and those who had node-positive
tumors. In addition, tumors exhibited methylation of the Twist promoter.
Conclusions: These data demonstrate that all three transcription factors have inappropriate
expression in breast cancer and that this may play a part in the progression of human breast
tumors.
Key Words: Breast cancer—Twist—Snail—Slug.
Snail and Slug belong to the Snail family of genes, such as cell differentiation, cell motility, cell-cycle
which is conserved among species during evolution. regulation, and apoptosis.1,3,4 They share highly
They encode transcription factors that are expressed conserved C2H2-type zinc finger domains, which
at different stages of development in different tis- bind to the E-box and repress the transcription of
sues.1 These transcription factors encode a zinc fin- target genes.3 More recently, they have been impli-
ger–type transcription factor that is necessary for cated in the progression of human tumors via their
gastrulation and mesoderm formation2 and are in- regulatory action on the epithelial cell adhesion
volved in a broad spectrum of biological functions, molecule E-cadherin.5–7 Such transcriptional repres-
sion mechanisms have emerged as one of the crucial
Received April 8, 2004; accepted January 19, 2005; published processes for the downregulation of E-cadherin
online. expression during development and tumor progres-
Address correspondence and reprint requests to: Tracey A. sion with both Snail and Slug, acting through an
Martin, PhD; E-mail: martinta1@cf.ac.uk
interaction with proximal E-boxes of the E-cadherin
Published by Springer Science+Business Media, Inc. 2005 The Society of
Surgical Oncology, Inc. promoter.7
1
2 T. A. MARTIN ET AL.
Loss of expression of the E-cadherin cell/cell potential regulators of cell adhesion and migration,
adhesion molecule is important in carcinoma this study aimed to determine the levels of expression
development and progression.8 It has been shown of Snail, Slug, and Twist in human breast cancer
that Slug and Snail are potential repressors of E- tissues and to elucidate whether these levels are clin-
cadherin transcription in carcinomas that lack E- ically significant.
cadherin expression,6 and analysis of the expression
patterns of Slug, Snail, and E-cadherin in breast
cancer cell lines demonstrated that the expression of METHODS
Slug was strongly correlated with a loss of E-cadherin
transcripts. Slug is the more likely in vivo repressor of
Cell and Tissue Collection and Preparation
E-cadherin expression in breast cancer.
In vertebrates, the basic helix-loop-helix protein Human breast cancer cell lines (BT-549,
Twist may be involved in the negative regulation of BT-474KC, MDA-MB-231, MDA-MB-453, MDA-
cellular determination and in the differentiation of MB-231, MDA-MB-468, MDA-MB-436, MCF-7,
several lineages, including myogenesis, osteogenesis, MCF-10a, and ZR-751), putative melanoma cell line
and neurogenesis.9–11 Twist is able to inhibit onco- MDA-MB-435S, prostate cancer cell line PC-3, and
gene- and p53-dependent cell death and is thus HeLa and human fibroblast cell line MRC-5 (both
known as an antiapoptotic factor. Twist is also from ECACC, Wiltshire, UK) were maintained in
known to trigger epithelial-mesenchymal transition DulbeccoÕs modified EagleÕs medium with 10% fetal
(EMT) mechanisms, possibly regulating the E- to calf serum. Breast tissue samples23 (tumor and mat-
N-cadherin switch during EMT.12 Twist has been ched background; Table 1) were collected and
found to be inappropriately suppressed in 50% of immediately frozen in liquid nitrogen before pro-
rhabdomyosarcomas.10 Twist is also an activator of cessing—a portion of each sample for quantitative
GLI1 reporter expression, where defects in the sig- PCR (Q-PCR) analysis, a portion for immunohisto-
naling pathway lead to severe birth defects and can- chemical analysis, and a portion for routine histo-
cer formation in humans.13 Methylation-specific logical examination.
polymerase chain reaction (PCR) has been used to RNA was isolated from tissue samples and cells by
detect cancer cells from ductal lavage fluid for cyclin using standard RNAzol procedures. For reverse
D2, RAR-b, and Twist,14 which was frequently found transcription-PCR, complementary DNA (cDNA)
to be methylated in patients with carcinomas (17 of was synthesized in a 20-lL reaction mixture with 1 lg
20). Upregulation of Twist is associated with cellular of RNA, as described in the protocol (AB Gene Re-
resistance to paclitaxel but not with other drugs that verse Transcription System; ABGene, Surrey, UK).
have different mechanisms of action.15 This indicates
a novel mechanism that leads to resistance to
Quantitative PCR
microtubule-disrupting anticancer drugs through
upregulation of Twist. The Q-PCR system used the Amplofluor Unipri-
EMT results in the loss of polarity of epithelial cell mer system (Intergen Company, Oxford, UK) and
layers and cell/cell contacts, with a corresponding Thermo-Start (ABgene, Epsom, Surrey, UK), as we
remodeling of the cell cytoskeleton.16 Evidence has recently reported.16 Specific primer pairs for Snail,
accumulated showing that EMT is an important in Slug, and Twist (Table 2) were designed by the au-
vitro correlation of late-stage tumor progression.17–20 thors by using Beacon Designer software and were
In mouse mammary cells, it has been demonstrated manufactured by Invitrogen (Invitrogen Life Tech-
that EMT occurs before the acquirement of an nologies, Paisley, Scotland, UK), each amplifying a
invasive, metastatic tumor phenotype.21 Twist has region that spans at least one intron (primer details
been shown to play a role in metastasis in murine are given in supplement 1), thus generating an
models.22 Ectopic expression of Twist resulted in the approximately 100–base pair product from both the
loss of E-cadherin–mediated cell/cell adhesion, acti- control plasmid and cDNA.
vation of mesenchymal markers, and induction of cell By using the iCycler IQ system (Bio-Rad), which
motility; this suggests that Twist may contribute to incorporates a gradient thermocycler and a 96-chan-
metastasis by promoting EMT. Increased Twist nel optical unit, the plasmid standards and breast
expression has also been correlated with invasive cancer cDNA were simultaneously assayed in dupli-
lobular cancers associated with E-cadherin loss in cate reactions with a standard hot-start Q-PCR
human breast cancer.22 Because Snail and Slug are master mix. Q-PCR conditions were as follows:
TABLE 1. Clinical information of patient samples analyzed in the breast cancer samples were calculated. Q-PCR
Variable n for b-actin was also performed on the same samples
to correct for any residual differences in the initial
Tissue type
Background 33
level of RNA in the specimens (in addition to spec-
Tumor 124 trophotometry). The products of Q-PCR were veri-
Tumor grade fied on agarose gels (not shown).
1 24
2 42
3 58
NPI
Immunohistochemistry
1 68
2 38
Cryostat sections of frozen tissue were cut at 6 lm,
3 16 placed on Super Frost Plus (Optech Scientific In-
Unknown 2 struments, Oxon, UK) slides, air-dried, and fixed in a
Tumor-node-metastasis
1 70
50:50 solution of alcohol and acetone. The sections
2 40 were then air-dried again and stored at )20C until
3 7 use. Immediately before immunostaining began, the
4 4
Unknown 3
sections were washed in buffer for 5 minutes and
Histology treated with horse serum/buffer solution for 20 min-
Ductal 94 utes as a blocking agent to nonspecific binding. Sec-
Lobular 14
Other 16
tions were stained with Snail, Slug, and Twist
Outcome antibodies (Insight Biotechnology, Wembley, Cam-
Disease free 85 bridgeshire, UK). Primary antibodies were used at a
Metastatic disease 7
Local recurrence 5
1/50 dilution for 60 minutes and then washed in
Death 15 buffer. The secondary biotinylated antibody at 1/100
Poor outcomesa 27 dilution (universal secondary, Vectastain Elite ABC;
NPI, Nottingham Prognostic Index. Vector Laboratories Inc., Burlingham, CA) was ad-
a
Metastatic disease, local recurrence, or death. ded (in horse serum/buffer solution) for 30 minutes,
followed by numerous washings in buffer. Avidin/
biotin complex was added for 30 minutes, again fol-
TABLE 2. Quantitative polymerase chain reaction primer
pairs for analysis of angiogenic marker molecules lowed with washes in buffer. Diaminobenzidine was
used as a chromogen to visualize the antibody/anti-
Molecule Primer sequence gen complex. Sections were counterstained in MayerÕs
Slug hematoxylin for 1 minute, dehydrated, cleared,
SLUG1F ctccaaaaagccaaactaca mounted in dextropropoxyphene, and screened with
SLUG1ZR actgaacctgaccgtacagaggatctctggttgtggta
Snail an ·25 objective.
SNAILF1 tctttcctcgtcaggaagc
SNAILZR actgaacctgaccgtacactgctggaaggtaaactctg
Twist Evaluation of Twist Promoter Hypermethylation
TWISTF aagctgagcaagattcagac
TWISTZR actgaacctgaccgtacagaggacctggtagaggaagt Genomic DNA from multiple sections from frozen
b-Actin tissue and human breast cancer cells were extracted
b-Actin F atgatatcgccgcgctcgt
b-Actin R cgctcggtgaggatcttca by using a standard DNA-extraction protocol.
Twist methylated Methylation-specific PCR was performed as previ-
TwistMF tttcggatggggttgttatc ously described.17 One microgram of DNA was
TwistMR aaacgacctaacccgaagg
Twist unmethylated denatured in NaOH (to a final concentration of .2
TwistUF tttggatggggttgttattgt mol/L) for 10 minutes at 37C, followed by 10 mmol/
TwistUR cctaacccaaacaaccaacc L of freshly prepared hydroquinone and 3 mol/L of
F, forward; R, reverse primer. sodium bisulfite (pH 5.0). After incubation at 50C
for 16 hours, the DNA was purified with the Wizard
enzyme activation at 95C for 12 minutes for 1 cycle, DNA purification kit (Promega UK Ltd., South-
followed by 60 cycles of denaturing at 95C for 15 ampton, UK) and standard ethanol precipitation.
seconds, annealing at 55C for 40 seconds, and Hypermethylation was analyzed by PCR with prim-
extension at 72C for 25 seconds. With use of purified ers listed in Table 2. Methylation-specific PCR con-
plasmids as internal standards, the levels of each tight ditions were as follows: enzyme activation at 95C for
junction molecule cDNA (copies per 50 ng of RNA) 5 minutes for 1 cycle, followed by 36 cycles of
Statistical Analysis
Statistical analysis was performed with Minitab
version 13.32 (Minitab Inc., State College, PA) by
using a two-sample StudentÕs t-test and the non-
parametric Mann-Whitney confidence interval and
test, where appropriate.
RESULTS
FIG. 2. Immunostaining of Slug (A), Snail (B), and Twist (C) in background (left panel) and tumor (right panel) sections. Representative cases
of increased positive staining in tumor sections are shown at ·4, ·10, and ·20.
fect over E-cadherin function in breast cancer cells.38 through upregulation of Twist expression in human
N-cadherin expression in normal epithelial cells cau- cancer.
ses a reduction in E-cadherin expression, and N- In conclusion, the EMT regulatory proteins Slug
cadherin is also able to enhance tumor cell motility and Twist are upregulated in human breast cancer,
and migration.39 Twist has been found to be inap- whereas Snail is downregulated. Such disparate
propriately suppressed in 50% of rhabdomyosarco- expression levels may contribute to the progression of
mas.10 Twist is also an activator of GLI1 reporter tumors in breast cancer, and this deserves further
expression through E-box interaction via the binding investigation.
of upstream stimulatory factors (USF) proteins.
GLI1 encodes a critical transcription activator in the
sonic hedgehog signaling pathway, which regulates ACKNOWLEDGMENTS
vertebrate patterning, where defects lead to severe We thank Cancer Research UK for supporting this
birth defects and cancer formation in humans.13 work and Dr. Anthony Douglas-Jones for his
Here we demonstrated that Twist was increased in invaluable advice on histology.
tumor tissues (both in intensity of staining with
immunohistochemistry and in Q-PCR analysis) and
showed a progressive increase with tumor-node-
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