From the Division of Hematology/Oncology,
Cedars-Sinai Medical Center, and the Departments of
Pathology and ** Biomathematics, School of Medicine, University
of California, Los Angeles, California 90048 and the
¶ Saitama Cancer Center, 818 Komuro, Ina, Saitama 362, Japan
Received for publication, October 25, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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To identify genes involved in breast cancer,
polymerase chain reaction-selected cDNA subtraction was utilized to
construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early gene
Cyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and
neovascularization. Northern analysis revealed that Cyr61
was expressed highly in the invasive breast cancer cell lines
MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the
less tumorigenic MCF-7 and BT-20 breast cancer cells and barely
detectable amounts were expressed in the normal breast cells, MCF-12A.
Univariate analysis showed a significant or borderline significant
association between Cyr61 expression and stage, tumor size,
lymph node positivity, age, and estrogen receptor levels.
Interestingly, expression of Cyr61 mRNA increased 8- to
12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h
exposure to estrogen, respectively. Induction of Cyr61
mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the
estrogen receptor. Stable expression of Cyr61 cDNA
under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude
mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in
nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development.
Breast cancer is the most common form of malignancy and the
second-leading cause of cancer-related death among women in the United
States. The nature of the cellular and molecular changes that lead to
breast cancer remains poorly defined. Several of the aberrant tumor
suppressor genes that have been identified, include BRCA1,
BRCA2 (1, 2), and p53 (3); however, they are silenced or
mutated in only a fraction of breast cancers. Oncogenes associated with
breast cancers include myc, CCND1, and Her2 (4-8), but only 15-30% of invasive breast cancers
show increased expression of these genes.
We set out to isolate differentially expressed genes in human breast
cancer. Suppression subtractive hybridization
(SSH)1 and differential
screening (9) identified genes highly expressed in the carcinoma cell
line MDA-MB-231 and either absent or minimally expressed in the normal
breast cell line MCF-12A. Cyr61 was one of the genes
isolated from the screening of the subtracted cDNA library. This
gene codes for a growth factor-inducible, immediate-early gene first
identified in murine fibroblasts (10). Cyr61 is a secreted,
cysteine-rich, heparin-binding protein that associates with the
extracellular matrix. Purified Cyr61 protein has been reported to
mediate cell adhesion, stimulate chemotaxis, augment growth
factor-induced DNA synthesis, enhance cell survival, and induce
angiogenesis in vivo (10-12). Because these characteristics may foster the progression of breast cancer, we studied this gene in
detail and found it to be highly expressed in some invasive breast
cancer cell lines and 36% of primary breast tumors. Furthermore, characterization of the oncogenic activity of Cyr61
demonstrated that forced expression of Cyr61 enhanced MCF-7
cell growth in soft agar and promoted tumor growth in both normal
breast and breast cancer cells in nude mice.
SSH and Differential Screening
SSH was performed by using the PCR-Select cDNA subtraction
kit (CLONTECH). Tester double-stranded cDNA was
synthesized from 2 µg of poly(A)+ RNA isolated from the
breast cancer cell line, MDA-MB-231; and driver cDNA was made from
2 µg of RNA from the normal breast cell line, MCF-12A. The subtracted
library was differentially screened with 32P-labeled probes
synthesized as first-strand cDNA from tester and driver. The
differential clones were picked and confirmed by Northern analysis.
Cell Culture
The cell lines MCF-12A, MCF-10A, MDA-MB-231, MCF-7,
MDA-MB-157, MDA-MB-436, BT-474, BT-20, ZR-75-1, and T47D were obtained from the American Type Culture Collection (Rockville, MD). MCF-12A and
MCF-10A normal breast lines were maintained in a 1:1 mixture of
Dulbecco's modified Eagle's medium and Ham's F12 medium (Life Technologies, Inc.), 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5%
horse serum; MCF-7 was cultured in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.); MDA-MB-231, MDA-MB-436, MDA-MB-157, BT-474,
BT-20, and T47D were grown in RPMI 1640 (Life Technologies, Inc.).
Media were supplemented with 10% fetal calf serum (Gemini Bio-Products, Calabasas, CA), 10 units/ml penicillin-G, 10 mg/ml streptomycin (Gemini Bio-Products). All cells were incubated at 37 °C in 5% CO2. In experiments in which the effects of
estrogen were studied, MCF-12A and MCF-7 cells were first cultured in
phenol red free medium with charcoal-treated newborn calf serum. Cells were then treated with estradiol (1 × 10 Proteins and Antibodies
Recombinant human Cyr61 protein was purified from an
Escherichia coli host strain (BL21) programmed for synthesis
of the Cyr61 protein via pGEX-5X-2 expression vector (Amersham
Pharmacia Biotech). Anti-Cyr61 antibodies were prepared from polyclonal
rabbit antisera raised against a GST-Cyr61 fusion protein. Monoclonal
antibodies to CD31 were obtained from DADO Corp. (Carpinteria, CA).
RNA Preparation and Northern Analysis
Total RNA was isolated from cell lines and patient tissue by
using TRIzol reagent (Life Technologies, Inc.) according to the standard protocol. Cyr61 cDNA probe was labeled with
[32P]dCTP by using a random primer (Life Technologies,
Inc.). Total cellular RNA was separated on 1.2% formaldehyde-agarose
gels and was immobilized on a Hybond-N+ membrane by
standard capillary transfer and UV cross-linking. The membrane was
hybridized with the Cyr61 probe by standard protocol and was
rehybridized with a 32P-labeled glyceraldehyde-3-phosphate
dehydrogenase cDNA to confirm equal loading of the samples.
Cell Transfection and Soft Agar Assays
The expression vector pcDNA61 was constructed by placing
full-length human Cyr61 cDNA into the pcDNA3.1
eukaryotic expression vector containing the neomycin gene under the
control of the same promoter (Invitrogen). The constructs were
transfected into MCF-12A and MCF-7 cells by using LipofectAMINE, and
transfectants were selected for G418 resistance (400 and 450 µg/ml,
respectively). The selected clones were confirmed by Northern analysis.
For clonogenic assay, cells were plated into 24-well flat-bottomed
plates using a two-layer soft agar system with a total of 1 × 103 cells/well in a volume of 400 µl/well, as described
previously (13). After 14 days of incubation, the colonies were counted and measured. All experiments were done at least three times using triplicate plates per experimental point.
Cell Migration Assays
Cell migration assays were performed according to the protocol
from Chemicon (Temecula, CA). 5 × 104 cells were
added to the top of each modified Boyden chamber (10-µm thickness and
8-µm pores) containing polycarbonate membranes (6.5-mm diameter)
coated on the underside of the membrane with 10 µg/ml vitronectin and
with the lower chamber containing 500 µl of migration buffer (medium
with 0.5% bovine serum albumin). Cells were allowed to migrate to the
underside of the top chamber for 4-8 h. The migratory cells attached
to the bottom surface of the membrane were stained with 0.1% crystal
violet in 0.1 M borate, pH 9.0, and 2% ethanol for 20 min
at room temperature. The stained cells were extracted by using
extraction buffer (Chemicon). The number of migratory cells per
membrane was determined by absorbance at 550 nm.
Tumorigenicity Assay
Stably transfected MCF-12A/61 and MCF-12A/V cells (1.0 × 106 cells/flank) and MCF-7/61 and MCF-7/V cells (5 × 104 cell/flank) were injected subcutaneously into
8-week-old female nude mice. Each animal was injected at two sites in
the flanks. The resulting tumors were measured once a week, and tumor
volume (mm3) was calculated by using the standard formula:
length × width × height × 0.5236. Tumors were
harvested 6 weeks after injection and individually weighed before
fixation. Data were presented as both tumor volume (mean ± S.D.)
and tumor weight (mean ± S.D.). Statistical analysis was
performed with software (GraphPad, San Diego, CA) using the Student's
t test.
Real-time Quantitative PCR
Quantitative PCR analysis was performed using the TaqMan PCR
Core Reagent kit (PE Biosystems). cDNA of breast cancer samples were diluted, and real-time PCR was performed following the
protocol. Her2/neu-specific primers were 5'-ACAGTGGCATCTGTGAGCTG and
5'-CCCACGTCCGTAGAAAGGTA. The TaqMan probe for Her2/neu was
5'-CCAGCCCTGGTCACCTACAACACAG. Statistical Analysis
Univariate--
Chi square methods were used to compare stage,
tumor size category, and node status category in Cyr61-positive
versus Cyr61-negative individuals. Age, ER levels, and PgR
levels were compared using t tests and Wilcoxon rank sum
tests. The kappa statistic was used to assess concordance among stage,
tumor size category, and node status and is reported with its standard error.
Multivariate--
The simultaneous relationship between the six
predictors and Cyr61 was modeled using classification tree methods.
Comparison of stage versus tumor size showed that these two
variables are proxies for each other (kappa = 0.94 ± 0.05, observed agreement = 42/44 = 95%). Therefore, tumor size was
not included as a candidate in the multivariate analysis. The
concordance between stage and lymph node status was only moderate
(kappa = 0.54 ± 0.12). A logistic regression analysis was
also carried out but gave poor results and is therefore not reported.
Cyr61 Is Highly Expressed in Some Breast Cancer Cell Lines and
Primary Breast Tumors--
Using a differential screening technique
(see "Materials and Methods"), we identified 36 genes highly
expressed in breast cancer cell line MDA-MB-231 as compared with normal
breast cell line MCF-12A (data not shown). The genes each displayed
6-fold or greater expression in MDA-MB-231 than in MCF-12A, as
determined using Northern blots and densitometric analysis (Fig.
1). Cyr61, a growth
factor-inducible immediate-early gene, is one of these differentially
expressed genes. Cyr61 expression was examined in a panel of
normal breast and breast cancer cell lines. Northern analysis showed
that Cyr61 mRNA was prominently expressed in the highly
invasive and tumorigenic breast cancer cell lines MDA-MB-231, MDA-MB-436, MDA-MB-157, BT-474, and T47D; it was expressed at a low
level in the less tumorigenic tumor cell lines MCF-7, BT-20, and
ZR-57-1 and was barely detectable in the normal breast cell lines,
MCF-10A and MCF-12A (Fig.
2A).
To determine the pattern of Cyr61 expression in primary
breast tumors, RNAs were isolated from quick-frozen breast samples obtained at initial surgery from 44 individuals with breast cancer (Table I). Each breast carcinoma and
matching normal breast tissue was confirmed histologically. Expression
of Cyr61 was easily detectable in sixteen of 44 (36%)
primary breast cancer samples (Table I), but levels were
negligible in normal breast tissues as shown by Northern analysis (Fig.
2B, representative autoradiogram of Northern blot).
We analyzed the Her2/neu status for the clinical samples by
performing real-time PCR using Her2/neu-specific primers.
Breast cancer cell lines BT-474 and MCF-7 were used as high expressor and low expressor controls, respectively. Her2/neu was
highly expressed in 7 of 16 (44%) Cyr61-positive samples compared with 6 of 28 (21%) Cyr61-negative samples, suggesting Cyr61
expression is positively correlated with Her2/neu
expression (Table I).
Univariate analysis (Tables II and III) showed either
significant or borderline significant
association between breast cancer stage, tumor size, lymph node status,
ER levels in the primary tumor as well as age at onset of disease
compared with whether the primary tumor expressed Cyr61 on Northern
blot. For analysis of stage, only 6 of 28 (21%) of women having stages
I and II were positive for Cyr61; in contrast, 10 of 16 (63%) of women
having stages IIIA, IIIB, or IV expressed Cyr61 (p < 0.006). Similarly, only 6 of 28 (21%) individuals with primary breast
cancer size of either 1A (
Our classification tree multivariate model predicted that, if the
patient at initial diagnosis was less than 57 years of age and had a
breast cancer with an ER of less than 24 fmol/g, Cyr61 would be
negative (%Cyr61-positive, 0/17 = 0%). If the individual was
less than 57 years old and had an ER greater than 24 fmol/g, the model
predicted that Cyr61 would be positive (%Cyr61-positive, 4/4 = 100%). If age was greater than 57 and the stage was I or II, the model
predicted that Cyr61 would be negative (3/10 = 30% Cyr61-positive); if the age of the patient was greater than 57 and had
either stage IIIA or IIIB, Cyr61 was predicted to be positive (9/9
(100%) were Cyr61-positive). This model has an observed sensitivity of
81% and observed specificity of 86% and gave an overall observed correct classification of 84% correctly classified. According to the
tree model, when predicting who is Cyr61-positive, ER is only important
in younger women under the age of 58, whereas stage is only important
in older women over the age of 57. In summary, the model posits that
the proportion who have Cyr61-positive breast cancer increases with
age, stage, and ER level.
Expression of Cyr61 Is Modulated through the Estrogen Receptor
Pathway in MCF-12A and MCF-7 Cells--
Previous studies have shown
that Cyr61 is inducible in the uterus by estrogen treatment
in ovariectomized rats (14). Furthermore, the correlation of
Cyr61 expression in ER+ breast cancers observed
in our experiments suggested a potential interaction between the
estrogen receptor pathway and expression of Cyr61. To
determine whether expression of Cyr61 was regulated by
estrogen and estrogen blockade, the estrogen-responsive normal breast
cells, MCF-12A, and breast cancer cells, MCF-7, were harvested at
different times after estrogen and antiestrogen treatment. Expression
of Cyr61 mRNA was induced at 16 h after estradiol
(10 Cyr61 Promotes Cell Proliferation in Soft Agar and Stimulates Cell
Migration of Breast Cell Lines--
Previous studies indicated that
Cyr61 promoted DNA synthesis and cell proliferation of mesenchymal
cells from the limb (15) and stimulated cell migration in fibroblasts
(12, 16). To study if similar activities occurred in breast cells, both
normal breast cells (MCF-12A) and breast tumor cells (MCF-7) were
stably transfected with pcDNA61 containing either full-length
cDNA of Cyr61 (MCF-12A/61 and MCF-7/61) or empty vector
pcDNA3.1 (MCF-12A/V and MCF-7/V) as control. As expected,
Cyr61 was highly expressed in the MCF-12A/61 and MCF-7/61
but not in the MCF-12/V and MCF-7/V transfected cells as shown by
Northern studies (data not shown) and Western analysis (Fig.
4A). The MCF-7/61 cells
expressing the Cyr61 vector formed significantly more
colonies in soft agar (mean, 2.2- ± 0.6-fold more colonies;
p < 0.05) than MCF-7/V cells harboring the empty
vector or MCF-7 control cells (Fig. 4B). The MCF-7/61
colonies also were substantially larger than the MCF-7/V and MCF-7
colonies (data not shown). The results indicated that forced expression
of Cyr61 promoted anchorage-independent clonogenic growth of
MCF-7 cells. Neither MCF-12A/V nor MCF-12A/61 formed colonies after 4 weeks.
To assess the effects of estrogen and tamoxifen on
anchorage-independent growth of MCF-7 cells, clonogenic proliferation
of MCF-7/V and MCF-7/61 in soft agar containing either estradiol (10
To determine whether enhanced expression of Cyr61 influenced
cell migration of MCF-12A and MCF-7 cells, migration assays of MCF-12A/V, MCF-12A/61, MCF-7/V, and MCF-7/61 were performed in vitronectin-coated Boyden chambers. As shown in Fig.
5, both MCF-12A/61 and MCF-7/61 cells
(Cyr61 stably transfected cell lines) had significantly (p < 0.05) increased migration compared with the
empty-vector-transfected MCF-12A/V and MCF-7/V cells in
vitronectin-coated Boyden chambers.
Cyr61 Promotes Tumor Growth and Vascularization in Nude
Mice--
On the basis of our in vitro studies indicating
that overexpression of Cyr61 promotes anchorage-independent
clonogenic proliferation in soft agar and cell migration, we
investigated the effect of expression of Cyr61 on tumor
development and neovascularization by comparing tumor formation of
MCF-12/V and MCF-12A/6 cells, as well as MCF-7/V and MCF-7/61 cells, in
nude mice. These cells were injected subcutaneously into 8-week-old
nude mice, and tumor growth was measured once a week. Tumors from the
normal breast cells expressing Cyr61 (MCF-12A/61) first became apparent
3 weeks after injection, and all of the mice developed tumors ranging from 0.6 to 1.4 g at 6 weeks after injection (Figs.
6A, 6B, and 7A). In contrast, the control mice that received MCF-12A/V
cells containing empty vector remained tumor-free even at 12 weeks
after injection (Fig. 7A). In
the other experimental group, the MCF-7/61 cells expressing Cyr61 at a
high level developed tumors with a significantly shorter latency
(p < 0.05) and marked increased tumor growth compared
with the tumors from the control MCF-7/V cells (Fig. 6, A
and B). All 16 tumors that developed from MCF-7/61 cells
appeared within 3 weeks of subcutaneous injection of the cells, whereas
only eight tumors from 16 separate injections of the MCF-7/V developed
during the entire 6 weeks of observation.
To examine whether the tumorigenic ability of Cyr61
in vivo was associated with angiogenic activity, tumors that
developed from both MCF-7/61 and MCF-7/V cells were analyzed
histochemically using antibody against CD31. Immunohistochemical
analysis demonstrated robustly increased blood vessel density in
MCF-7/61 tumors compared with those from the MCF-7/V controls (Fig.
7B). The tumors that developed from both MCF-7/61 and
MCF-12A/61 had a similar histologic appearance. Both were classified as
high grade infiltrating ductal carcinomas (Bloom-Richardson Grade III).
The tumors had marked nuclear pleomorphism, absence of tubule
formation, and a high mitotic count (12-15 per high power field). In
contrast, the control MCF-7 tumors were moderately well differentiated
infiltrating ductal carcinomas (Bloom-Richardson grade I) with tubule
formation in greater than 75% of the tumor and less than one mitosis
was observed per high power field.
Cyr61 is a member of a growing family termed the CCN
(Connective tissue growth factor/Cysteine-rich
61/Nephroblastoma-overexpressed) gene family that is characterized by a
high degree of amino acid sequence homology ranging from 50 to 90% and
includes Cyr61 (cysteine-rich protein); CTGF
(connective tissue growth factor) (17, 18), nov
(nephroblastoma-overexpressed gene) (19, 20); elm1, also termed WISP-1 (expressed low in metastasis 1 gene) (21, 22); rCop-1, also termed WISP-2 (heparin-inducible
CCN-like protein) (23); and WISP-3 (354-residue
protein containing 36 cysteine residues) (24). All members of the CCN
gene family possess a secretory signal peptide at the N terminus,
indicating that they are secreted proteins.
Several lines of evidence support a role for CCN molecules in
tumorigenesis. Although elevated expression of chicken nov mRNA was
consistently found in all MAV1 (myeloblastosis-associated virus 1)- and
MAV2-induced avian nephroblastomas (19), the human homolog of chicken
nov (novH) is mainly overexpressed in tumors of
predominantly stromal origin such as Wilms' tumors (25). Consistent
with its profibrotic properties, CTFG is overexpressed in
pancreatic cancers (26), mammary tumors (27), and melanomas (28).
WISP-1 is strongly expressed in the fibrovascular stroma of
breast tumors developing in Wnt-1 transgenic mice (22).
Recent studies showed that the forced overexpression of
WISP-1 in normal rat kidney fibroblasts (NRK-49F) was
sufficient to induce their transformation (29).
In the present study, Cyr61 was found to be prominently
expressed in the highly tumorigenic breast cancer cell lines such as
MDA-MB-231, MDA-MB-157, MDA-MB-436, and T47D, but the levels were low
in the less tumorigenic cells, MCF-7 and BT-20. It was barely
detectable in normal breast cell lines MCF-10A and MCF-12A (Fig.
2A). Furthermore, Cyr61 was expressed in 36%
primary breast tumor samples (16/44) and was undetectable in normal
breast tissues (samples N1 and N15) as shown by Northern analysis (Fig.
2B). After submission of our study, Tsai et al.
(30) also noted that Cyr61 was expressed in about 30% of
breast cancers. We found that expression of Cyr61 in breast
tumor samples correlated with ER positivity and lymph node involvement.
The latter is routinely used as a prognostic and predictive marker in
the clinical management of breast cancer (Table I). These results
suggest that Cyr61 is closely associated with the malignant
phenotype in breast cancer and may serve as a marker of potential
progression of the cancer. Statistical analysis of the 44 fresh tumors
showed that tumors that were at a more advanced stage at diagnosis,
with large primary tumors, that expressed Her-2/neu, and that had lymph
node involvement, were more likely to express Cyr61.
The duration of follow-up is too short to know if the prognosis of
these individuals parallels their Cyr61 expression. This
becomes more complex, because Cyr61 expression also
correlated with ER expression in the primary tumors. The ER-positive
breast tumors are responsive to estrogen blockade, and ER expression in
the breast cancer is a good prognostic indicator. Thus, we find a
paradox that cyr61 expression is frequently associated with advance
disease at diagnosis, but these tumors are often ER-positive.
Another interesting finding of this study is that expression of
Cyr61 in breast cell lines can be modulated by estrogen and antiestrogen (Fig. 3). Estradiol markedly increased the expression of
Cyr61 in both normal and breast cancer cells in a
time-dependent manner. Thus, induction of Cyr61
is not specific for transformation of breast cells. Nevertheless,
Cyr61 may play a role in tumor progression, because
overexpression of Cyr61 in the normal breast cells (MCF-12A)
allowed them to form tumors in nude mice. Fresh breast tumors that
expressed Cyr61 had a greater level of expression of ER
(mean, 102 fmol/g) than those tumors that did not express Cyr61 (mean,
40 fmol/g). Because we have not found an estrogen response element in
the promoter region of the human Cyr61 (data not shown), we
speculate that estrogen acts through one or more primary
estrogen-responsive genes whose product regulates the expression of
Cyr61. Consistent with this hypothesis is the finding that
estradiol required a lag of 8-16 h to stimulate the visible accumulation of Cyr61 mRNA in the breast cells.
The role of Cyr61 in breast tumor growth was evaluated in
several experimental tumor models. The forced expression of
Cyr61 in MCF-7 cells (MCF-7/61) markedly stimulated their
anchorage-independent cell growth in soft agar and significantly
enhanced their tumorigenicity and vascularization in vivo
(Fig. 6). MCF-7/61 cells that highly expressed Cyr61 developed larger
and more vascularized tumors in nude mice (Fig. 7B) and had
a much shorter latency in their development of tumors than did the
MCF-7/V cells containing the empty vector. This is consistent with an
earlier observation showing that the gastric adenocarcinoma cell line
RF-1 became tumorigenic when induced to express Cyr61 (31).
Furthermore, we found that the overexpression of stably transfected
Cyr61 in the normal breast cell line MCF-12A, which does not
normally express Cyr61, resulted in tumor formation in nude mice (Fig.
6, A and B). Taken together, these results
suggest that prominent expression of Cyr61 may facilitate transformation of breast tissue.
Recently, WISP-1, another CNN family member closely related to
Cyr61, was found to be a Wnt-1- and Previous studies have suggested that Cyr61 is involved in
angiogenesis through its interaction with the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
9
M; Sigma Chemical Co.) for different durations. In the
experiments in which antiestrogens were examined, cells were pretreated
with tamoxifen for 6 h (1 × 10
7 M;
Sigma) or ICI182,780 (1.5 × 10
7 M;
Tocris Cooksson Inc., Ballwin, MO) before estradiol treatment.
-Actin was used for normalization, and
-actin-specific primers were 5'-GATCATTGCTCCTCCTGAGC and
5'-ACTCCTGCTTGCTGATCCAC. The TaqMan probe for
-actin was
5'-CTCGCTGTCCACCTTCCAGCAGAT.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Northern blots of differentially expressed
genes isolated from breast cancer cells by PCR-selected subtractive
hybridization. Northern blots demonstrate representative clones
isolated using suppression subtractive hybridization and differential
screening which are differentially expressed. The cDNAs used as
probes on Northern blots were isolated from the subtracted cDNA
library. The same blots were hybridized with glyceraldehyde-3-phosphate
dehydrogenase cDNA to confirm similar loading. MCF-12A is a normal
breast cell line and MDA-MB-231 is a breast cancer cell line.
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Fig. 2.
Expression of Cyr61 in human
breast cancer cell lines and primary breast cancer samples.
Cellular RNA was extracted, subjected to electrophoresis (10 µg of
total RNA/lane for cell lines and 5 µg of total RNA/lane for primary
tissue), Northern blotted, and probed with 32P-labled Cyr61
cDNA. A, MCF-10A and MCF-12A are
normal breast cell lines; LNCaP is a prostate cancer cell
line; the others are breast cancer cell lines. B,
N1 and N15 are normal breast tissue; all other
samples are from primary breast tumors.
Clinic information of the breast cancer samples
Univariate associations with Cyr61-positive breast cancer discrete
variables
2 cm in diameter) or 2A (
5 cm) had tumors
that were Cyr61-positive; in comparison, 10 of 16 (63%) of individuals with a stage of either 3A or 4B (
5 cm) breast cancer had primary tumors that expressed Cyr61 (p = 0.006). Furthermore,
of the 17 patients who were lymph node-negative, only two were
Cyr61-positive (12%) compared with 14 Cyr61-positive tumors among the
27 individuals who were lymph node-positive (52%, p = 0.01). The median and mean ages of patients who were Cyr61-negative,
were 52 and 53 (±10 S.D.), respectively, compared with a median and
mean age of 66 and 64 (±11.7 S.D.) in those who were Cyr61-positive
(p = 0.003). Tumors that were Cyr61-negative had a mean
ER score of 40 fmol/g (±69 S.D.), whereas those that were
Cyr61-positive had a mean ER score of 102 fmol/g (±110 S.D.)
(p = 0.03). No statistical difference in the mean
progesterone (PgR) values was noted in those that were Cyr61-negative
(mean 94.56 fmol/g) versus those that were Cyr61-positive
(mean 129.5 fmol/g) (p = 0.55).
Univariate associations with Cyr61-positive breast cancer continuous
variables
9 M) treatment and reached maximum (8- to
12-fold) levels at 48 h and started to decrease slightly after
72 h in the MCF-12A cells (Fig.
3A). Induction of expression
of Cyr61 mRNA was inhibited when either tamoxifen or
ICI182,780 (estrogen receptor antagonists, 10
7
M) were added to these cultures. A parallel induction (3- to 5-fold) of Cyr61 mRNA occurred in MCF-7 cells after
estrogen treatment (10
9 M), which was
detectable at 8 h, reached a plateau at 24 h, and decreased
at 72 h. The induction was completely blocked by either tamoxifen
or ICI182,780 (Fig. 3B).
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Fig. 3.
Modulation of Cyr61
expression in MCF-12A and MCF-7 cells by estrogen and an estrogen
receptor antagonist. Normal breast cell line MCF-12A and breast
cancer cell line MCF-7 were treated with -estradiol (1 × 10
9 M) for different durations; cellular RNA
was extracted, subjected to electrophoresis (10 µg of total
RNA/lane), Northern blotted, and probed with 32P-labled
Cyr61 and glyceraldehyde-3-phosphate dehydrogenase
cDNAs. Maximum induction of Cyr61 mRNA expression
was observed at 48 h for MCF-12A (A) and 24 h for
MCF-7 (B). Induction of Cyr61 expression was
blocked by pretreatment of the cells with the estrogen receptor
antagonists, tamoxifen (1.0 × 10
7 M) or
ICI182,780 (1.5 × 10
7 M).
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Fig. 4.
Formation of colonies in soft agar by MCF-7,
MCF/V, and MCF-7/61 cells. Expression of Cyr61 protein
by stably transfected MCF-7 (MCF-7/61) cells as shown by
Western analysis. Equal loading was shown by the internal nonspecific
bands at 90 kDa (A). The MCF/V and MCF-7/61 cell lines were
stably transfected with either the empty pcDNA3.1 vector or the
Cyr61 expression vector, respectively. The MCF-7/61 clone
was selected for high expression of Cyr61. B,
cells (1.0 × 103/well) were seeded in soft agar for 2 weeks, and colonies were enumerated. Each experiment was performed in
triplicate, and the results represent the mean ± S.D. of three
experiments. C, MCF-7/V and MCF-7/61 cells (1.0 × 103/well) were seeded in soft agar with either estradiol
(E2, 10 9 M) or
E2 (10
9 M) and tamoxifen
(TAM, 10
7 M) for 2 weeks, and
colonies were counted. Each experiment was performed in triplicate, and
the results represent the mean ± S.D. of three experiments.
9 M) or estradiol and tamoxifen
(10
7 M) was evaluated. Estrogen treatment
significantly (p < 0.05) enhanced colony formation of
both MCF-7/V and MCF-7/61 cells, and tamoxifen blocked the
estrogen-stimulated colony formation in both of these cell types (Fig.
4C).
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[in a new window]
Fig. 5.
Cyr61 stimulates cell migration in
MCF-12A and MCF-7 cells. Cells (5 × 104) were
placed into either bovine serum albumin-coated (control) or
vitronectin-coated Boyden chambers. Cells were allowed to migrate for
4-8 h and quantified as described in QCM-VN (Chemicon, Temecula, CA).
The number of cells that migrated through the membrane was determined
by absorbance at 550 nm. Each bar represents the mean ± S.D. of triplicate experiments.
View larger version (18K):
[in a new window]
Fig. 6.
Effect of forced expression of
Cyr61 on the ability of MCF-12A and MCF-7 cells to
form tumors in nude mice. MCF-12A/V (normal breast, control),
MCF-12A/61 (normal breast, Cyr61 expressor), MCF-7/V (breast
cancer, control), and MCF-7/61 (breast cancer, Cyr61
expressor) were mixed with Matrigel (1:1) and injected subcutaneously
into BNX nude mice (1 × 106 cells/flank for MCF-12A
and 5 × 104 for MCF-7 cells). A, time
course of tumor growth. Tumor volumes were measured every week. Each
point represents the mean volume ± S.D. of eight tumors.
B, tumor weights at autopsy. At 6 weeks after injection,
tumors were removed and weighed. Results are shown as means ± S.D. of tumor weights. Statistical significance was determined with a
Student's t test using the program GraphPad (San Diego,
CA), and p values of <0.05 are indicated by an
asterisk.
View larger version (89K):
[in a new window]
Fig. 7.
Effect of forced Cyr61
expression on tumor growth and tumor neovascularization in nude
mice. A, xenografts growing in nude mice for 6 weeks after injection with either MCF-12A/V (left, control)
or with MCF-12A/61 (right, Cyr61 expressor).
B, immunohistochemical analysis demonstrating robustly
increased blood vessel density in MCF-7/61 tumors (left) in
nude mice compared with those from the MCF-7/V controls
(right) when immunostained with anti-CD31 antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
b-catenin-responsive
oncogene (29). Transfected and overexpressed WISP-1 in normal rat
kidney fibroblast cells (NRK-49F) induced their morphological
transformation, accelerated cell growth, enhanced saturation density
in vitro, and permitted the formation of tumors in nude
mice. Considering that Cyr61 has four identical structural
domains and is closely related to WISP-1, it might also be involved in
the Wnt-1 and
b-catenin pathways, especially those that enhance
tumor development and progression.
v
3 integrin (31-33). This could provide
insight into how it might enhance tumorigenesis. Angiogenesis, the
formation of new capillaries from pre-existing blood vessels, is
required for growth of solid tumors. This process is complex,
encompassing migration and proliferation of endothelial cells and tube
formation. It is regulated by many factors (34), which may include
Cyr61 by promoting migration of human microvessel endothelial cells and inducing neovascularization via
v
3 integrin (31). Integrins comprise a
large family of cell surface receptors that enhance cellular adhesion
to the extracellular matrix, which can exert profound control over
cells. The effects of the matrix are primarily mediated by integrins.
Integrin signals are involved in different cellular activities,
including cell migration, proliferation, and survival, as well as
diverse biological processes, including embryogenesis, angiogenesis,
immune response, and tumor metastasis (35-37). Integrin
v
3 may have an important role in tumor
vascularization and progression (38). Antagonists of integrin
v
3 significantly decrease tumor
vascularization and size (39). The interaction of Cyr61 with
integrin
v
3 provides a molecular
mechanism to account for the angiogenic and tumorigenic activities that
occurred in our breast cancer model system.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health and Department of Defense grants, the Parker Hughes Trust, the C. and H. Koeffler Research Fund, and the Horn Foundation.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: Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048. Tel.: 310-423-7758; Fax: 310-423-0225; E-mail: xied@ucla.edu.
A member of the Jonsson Comprehensive Cancer Center and
holder of the endowed Mark Goodson Chair of Oncology Research at
Cedars-Sinai Medical Center/UCLA School of Medicine.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M009755200
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ABBREVIATIONS |
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The abbreviations used are: SSH, suppression subtractive hybridization; PCR, polymerase chain reaction; ER, estrogen receptor; PgR, progesterone; CCN, connective tissue growth factor/cysteine-rich 61/nephroblastoma-overexpressed; NRK, normal rat kidney.
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