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Women’s Health Research Institute, Division of Endocrinology, Wyeth-Ayerst Research, Inc., Radnor, Pennsylvania 19087
Address all correspondence and requests for reprints to: Deepak Sampath, Ph.D., or Zhiming Zhang, Ph.D., Wyeth-Ayerst Research, Inc., 145 King of Prussia Road, Radnor, Pennsylvania 19087. E-mail: sampatd@war.wyeth.com or zhangz{at}war.wyeth.com
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Abstract |
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(ER) in a dose-dependent and
immediate early fashion. Cyr61 gene induction by E2 is
transcriptionally regulated by ER as the antiestrogen, ICI 182,780,
and actinomycin D blocked induction completely. In addition, Cyr61 is
up-regulated in MCF-7 cells by epidermal growth factor (EGF) in an
immediate early fashion as well. The functional relevance of steroid
induction of Cyr61 in breast cancer cell growth is demonstrated by
anti-Cyr61 neutralizing antibodies, which diminished E2 and
EGF-dependent DNA synthesis and dramatically reduced
E2-driven cell proliferation by more than 70%. Most
importantly, Cyr61 is overexpressed in 70% (28 of 40) of breast cancer
patients with infiltrating ductal carcinoma and is localized
exclusively to hyperplastic ductal epithelial cells. Moreover, the
levels of Cyr61 protein are higher in breast tumors that are
ER+/EGF receptor+ than those that are
ER-/EGF receptor+, suggesting that estrogens
may mediate Cyr61 expression in vivo. Collectively, our
data suggest that Cyr61 may play a critical role in estrogen- as well
as growth factor-dependent breast tumor growth. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An emerging group of growth factor-regulated immediate-early genes that
mediate diverse roles in development, cell proliferation, and
tumorigenesis collectively belong to the CCN
(CTGF/Cyr61/Cef10/NOVH)
family. Cyr61, a prototypical member of this family, was originally
identified by differential hybridization screening of a complementary
DNA (cDNA) library of serum-stimulated BALB/c 3T3 fibroblasts
(4). Cyr61 is not expressed in quiescent fibroblasts, but
is transcriptionally activated within minutes after stimulation by
serum, epidermal growth factor (EGF), basic fibroblast growth
factor (bFGF), platelet-derived growth factor, transforming growth
factor-ß, and 12-O-tetradecanoylphorbol 13-acetate
(4, 5, 6, 7). Rapid and transient induction of Cyr61 by vitamin
D3, tumor necrosis factor-, as well as
interleukin-1 has also been reported in human fetal osteoblasts
(8). Cyr61 is a secreted, cysteine-rich heparin-binding
protein that associates with the cell surface and the extracellular
matrix, biochemical features that resemble the Wnt-1 protooncogene and
a number of known growth factors (9). The human Cyr61 cDNA
encodes a protein 379 amino acids (aa) in length with a molecular mass
of 42 kDa, and the gene is located on the short arm of chromosome 1
(1p22–31) (10, 11). All CCN proteins 1) display a high
degree of conservation among family members and across species, 2) are
cysteine-rich and structurally similar to extracellular
matrix-associated molecules, 3) are composed of multifunctional modular
domains, and 4) have been shown to mediate functions as diverse as cell
adhesion, cell migration, mitogenesis, cell survival, and
differentiation (reviewed in Ref. 12). More specifically,
Cyr61 has been shown to be directly involved in a number of
developmentally regulated processes such as angiogenesis and
chondrogenesis (13, 14).
In addition to growth factors, Cyr61 is regulated by 17ß-estradiol (E2) in ovariectomized rat uteri in vivo (15). Given that estrogen induction of growth factors appears to be critical to breast tumorigenesis, we sought to determine whether Cyr61 is induced by E2 in hormone-responsive MCF-7 mammary adenocarcinoma cells. In addition, the role of Cyr61 in mammary tumor epithelial cell growth was addressed by determining the effects of anti-Cyr61 neutralizing antibodies on hormone-dependent DNA synthesis and cell proliferation. Finally, estrogen receptor (ER)-positive and -negative tumor lysates from a cohort of patients with invasive ductal carcinomas, the most common form of breast cancer, were screened for Cyr61 protein expression and compared with autologous healthy mammary tissue controls to ascertain the relevance of hormonal regulation of Cyr61 in breast carcinogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
MCF-7 and MDA-MB-231 adenocarcinoma cell lines were obtained
from American Type Culture Collection (Manassas, VA) and
propagated in DMEM/Ham’s F-10 medium containing 10% FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 2 mM Glutamax
(Life Technologies, Inc., Gaithersburg, MD). For steroid
and/or growth factor treatments, cells were cultured in phenol-red free
DMEM/F-12 medium containing 2% charcoal-stripped FBS (HyClone Laboratories, Inc., Logan, UT).
Cyr61 antibodies
Two anti-Cyr61 polyclonal antisera were generated at the
Louisiana State University Medical Center Core Facilities (Baton Rouge,
LA) to peptides corresponding to aa 163–229 or 371–381 of the human
Cyr61 as antigens. Peptides were coupled to keyhole limpet hemocyanin
and injected into female New Zealand White rabbits. After preliminary
screening of crude antisera by Western analysis of human uterine smooth
muscle cell lysates, which contain high basal levels of Cyr61 protein,
polyclonal antibodies were purified by affinity chromatography using
their respective peptides as the immunoabsorbent.
Study subjects and tissue procurement
Breast tumor biopsies and matched normal mammary tissue
specimens were obtained from Clinomics, Inc. (Pittsfield, MA), after
obtaining informed patient consent and internal review board approval.
Patients (n = 40) were between the ages of 42–68 yr (average, 54
yr) and had been diagnosed with stage II invasive ductal carcinoma
after histological examination. In addition, tumors were classified as
ER+/EGF receptor+
(EGFR+; n = 20) or
ER-/EGFR+ (n = 20) by
immunohistochemical staining of formalin-fixed tumor biopsies using
monospecific anti-ER and anti-EGFR antibodies. Tissue specimens were
immediately frozen in liquid nitrogen after surgery for protein
extraction or were fixed in 10% neutral-buffered formalin for in
situ hybridization.
Northern blotting for Cyr61
Total cellular RNA was isolated from cultured adenocarcinoma
cells by guanidium isothiocyanate lysis followed by phenol/chloroform
extraction. Subsequently, total cellular RNA (20 µg) was subjected to
electrophoresis in an 1% agarose gel containing 1 M
formaldehyde and transferred onto nylon membranes by capillary
electrophoresis. A 0.41-kb human Cyr61 cDNA fragment was radiolabeled
with [-32P]deoxy-CTP (3000 Ci/mmol) using
the random primer technique (Rediprime II, Amersham Pharmacia Biotech, Arlington Heights, IL) and used as the hybridization
probe. Relative levels of Cyr61 were normalized to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after reprobing
membranes with a 32P-radiolabeled oligonucleotide
according to manufacturer’s protocol (end labeling kit, Life Technologies, Inc.).
Protein extraction and immunoblotting for Cyr61
Tissue protein extracts were prepared from breast tumors and
matched normal mammary tissue specimens by homogenization in 50
mM Tris (pH 8.0), 250 mM NaCl, 1.0% Nonidet
P-40, 1.0% Triton-X 100, 2.0% SDS, 0.5% deoxycholate, 1
mM EDTA, and protease inhibitor cocktail containing 10
µg/ml pepstatin, aprotinin, and leupeptin (Sigma-Aldrich Corp.). Protein extracts (20 µg) were subjected to SDS-PAGE
under reducing conditions in 10% bis-acrylamide and
electrophoretically transferred to a polyvinyl difluoride membrane
(Immobilon-P, Bio-Rad Laboratories, Inc., Redding, CA).
Membranes were blocked with 5% dry milk on TBS/0.1% Tween-20 and
incubated with anti-Cyr61 pAb (10 µg/ml). Primary antibody binding
was detected using a donkey antirabbit IgG antibody conjugated to
horseradish peroxidase and an enhanced chemiluminescence detection
system (Amersham Pharmacia Biotech). All immunoblots were
subsequently reprobed with 1 µg/ml anti-pan-cytokeratin monoclonal
antibodies (Sigma-Aldrich Corp.) to verify equivalent
protein loading.
In situ hybridization
For riboprobe synthesis, a 0.28-kb human Cyr61 cDNA fragment was
positionally cloned into the EcoRI and HindIII
sites of pGEM4Zf- plasmid (Promega Corp., Madison, WI) to
generate pGEM4Zf-/Cyr61. Radiolabeled [35S]UTP
sense and antisense complementary RNA transcripts were transcribed
in vitro with T3 and T7 RNA polymerases, respectively, using
the Gemini Riboprobe system (Promega Corp.). In
situ hybridization was performed as described previously
(16), using formalin-fixed breast tumor biopsies and
matched normal mammary specimens.
Cyr61 neutralization assays with anti-Cyr61 antibodies
Polyclonal antibodies were raised to a 65-aa peptide
corresponding to the central domain of Cyr61, which was selected based
on a lack of homology to other CCN family members and the published
effectiveness of the antibodies in neutralizing bFGF-mediated DNA
synthesis in human microvascular endothelial cells (17).
The antibodies were affinity purified and assayed for specificity by
Western blot analysis using E2-treated MCF-7
whole cell lysates. A major band was detected at 42 kDa, which is the
molecular mass of Cyr61, along with a minor band at 77 kDa. For DNA
synthesis, MCF-7 and MDA-MB-231 cells were treated with 0.3
nM E2, 20 ng/ml EGF, or 20 ng/ml
HB-EGF in the presence or absence of 10 µg/ml anti-Cyr61 antibodies
for 18 h, and bromodeoxyuridine (BrdU) incorporation was measured
using the BioTrak enzyme-linked immunosorbent assay kit (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the
manufacturer’s instructions. For cell proliferation assays, MCF-7
cells were cultured in 2% charcoal-stripped FBS medium containing 0.3
nM E2 or 20 ng/ml EGF for 5 days in
the presence or absence of 10 µg/ml of anti-Cyr61-neutralizing
antibodies or nonimmune IgG at 37 C in 5% CO2.
After steroid or growth factor treatment, monolayers were trypsinized,
combined with cells in the culture supernatant, and counted in a
Coulter Multisizer II counter (Coulter Corp., Miami, FL).
Densitometric and statistical analysis of Cyr61 levels
Densitometric analysis of Cyr61 messenger RNA (mRNA) levels was
accomplished with a PhosphorImager and image quantitation software
(Molecular Dynamics, Inc., Sunnyvale, CA). Numerical
values are based on the relative volume of the radioactive band, and
the level of Cyr61 was normalized to the level of GAPDH. Cyr61 protein
levels detected by Western analysis were quantitated by densitometric
analysis using a molecular imager (Bio-Rad Laboratories, Inc.). Numerical values are based on the relative OD of the
band, and the level of Cyr61 was normalized to the level of
cytokeratin. All experiments were repeated at least three times.
Arbitrary units derived from densitometric measurements of RNA and
protein bands were analyzed using SAS statistical software (SAS Institute, Inc., Cary, NC) for significance using either one-way
ANOVA for a factorial experimental design or paired t test.
The multicomparison significance level for the one-factor ANOVA was
0.05. If significance was achieved by one-way analysis, post-ANOVA
comparison of means was performed using Scheffé’s F tests
(18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A–D). In addition to
E2, Cyr61 was up-regulated by 10 nM
DHT, but not by DEX or the progestin, R5020, in MCF-7 cells (Fig. 2, A and B). Analysis of Cyr61 mRNA
levels in MCF-7 cells at 0, 0.5, 1.0, 2.0, 4.0, and 8.0 h of
either DEX or R5020 treatment did not result in increased expression
(data not shown). However, in T47D cells that overexpress PR, R5020,
but not DEX, stimulation induced Cyr61 (Sampath, D., et al.,
manuscript in preparation). In MCF-7 cells, DHT induction occurred in
an immediate-early fashion, with maximal expression occurring within
1 h and was completely inhibited by the androgen receptor (AR)
antagonist, 2-hydroxyflutamide, indicating that the up-regulation of
Cyr61 was dependent on AR activity (data not shown). However, the
maximum level of Cyr61 protein up-regulated by 10
nM DHT in MCF-7s was less than half of that
observed with 1 nM E2 (Fig. 2B). Interestingly, in MDA-MB-453 adenocarcinoma cell lines that
overexpress AR, Cyr61 was induced more than 8-fold by DHT in an
immediate-early fashion as well (Sampath, D., et al.,
manuscript in preparation). Therefore, although Cyr61 is up-regulated
by androgens and progestins in cell lines that overexpress AR and PR,
respectively, estrogen is the primary inducer of this gene in MCF-7
cell lines.
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at the transcriptional level and ERß can vary between subclones of
breast tumor cells, we first determined the relative levels of both
receptors by quantitative RT-PCR. In the MCF-7 cells used for our
experiments, ER is exclusively overexpressed, and ERß is
detectable only at very low levels (data not shown) (19).
Induction of Cyr61 by E2 in MCF-7 is primarily
mediated through ER, as the antiestrogen, ICI 182,780, completely
abrogated expression (Fig. 3).
Furthermore, in ER- MDA-MB-231 adenocarcinoma
cells expression of Cyr61 was not enhanced by E2,
but was induced upon EGF or HB-EGF stimulation, demonstrating that ER
was required for Cyr61 induction (data not shown). Additionally,
inhibition of Cyr61 up-regulation was not observed in MCF-7 cells
cotreated with 10 µg/ml Chx and 10 nM
E2, suggesting that de novo protein
synthesis was not required for steroid induction (Fig. 3, A and B). In
contrast, significant overexpression of Cyr61 was observed upon
cotreatment with Chx and E2 compared with
E2 treatment alone (Fig. 3B). These increases in
mRNA levels are often observed with Chx cotreatment, which may be due
to the lack of synthesis of mRNA decay or turnover factors. However,
cotreatment of E2 with 1 µg/ml actinomycin D
completely blocked steroid induction, implying that ER mediates
Cyr61 expression at the transcriptional level (Fig. 3, A and B).
Therefore, Cyr61 is primarily induced by estrogen through ER in an
immediate-early fashion in MCF-7 cells at the transcriptional
level.
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and 4B). Interestingly, Cyr61 levels
were significantly higher in tumors that were
ER+/EGFR+ (n = 20)
compared with those in
ER-/EGFR+ tumors (n =
20; Fig. 4C). However, the overall percentage of Cyr61 positive breast
tumors did not vary significantly between ER+
(75%, 15 of 20) and ER- (65%, 13 of 20) subsets (Fig. 4D).
Furthermore, the results of in situ hybridization studies
revealed that Cyr61 is selectively overexpressed in hyperplastic
luminal and stroma-invading epithelial cells contained within tumors
isolated from patients with invasive ductal carcinoma (Fig. 5, B and F). No expression of Cyr61 was
observed in the ductal epithelium or surrounding paranchymal cells
(i.e. myoepithelial, adipocytes, and fibroblasts) in
adjacent healthy mammary tissue (Fig. 5, A and E).
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, A and
B). However, cotreatment with 10 ng/ml EGF and 10 nM
E2 resulted in sustained expression beyond 2
h compared with that observed for E2 or EGF
treatment alone (Fig. 6, C and D). Thus, the combination of
E2 and EGF appears to have a synergistic effect
in enhancing Cyr61 expression and may in part explain the
overexpression of Cyr61 in those tumors that are estrogen and EGF
receptor positive.
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, A
and B, and data not shown). In addition, anti-Cyr61 antibodies blocked
EGF-induced DNA synthesis in MDA-MB-231 cells (data not shown).
Controls for the BrdU assay included cotreatment with 10 µg/ml
nonimmune IgG, which had no effect, and 10 µg/ml blocking peptide,
which completely reversed the neutralizing effects of anti-Cyr61 (Fig. 7, A–C). Therefore, Cyr61 appears to be necessary for initiation of
the cell cycle by effecting steroid- and growth factor-mediated entry
into S phase. Therefore, E2- and EGF-dependent
MCF-7 cell proliferation was monitored to directly determine the role
of Cyr61 in cell growth. Increased MCF-7 cell proliferation induced by
E2 was observed in a dose-response fashion, with
an EC50 of 10 pM and maximum cell
growth achieved at 300 pM in a 5-day treatment period (data
not shown). Anti-Cyr61 polyclonal antibodies (10 µg/ml) inhibited
E2-dependent cell growth by 72% and
EGF-dependent cell growth by 40% over a 5-day treatment period (Fig. 7, A and B). Furthermore, continued exposure of MCF-7 cells to 10 and
300 pM E2 over a 5-day period
resulted in significant increases (3- to 4-fold) in Cyr61 protein
levels compared with basal levels when detected by Western analysis
(see inset in Fig. 7B). These results indicate that
prolonged treatment of MCF-7 cells with E2
increases the sensitivity of Cyr61 induction and/or augments protein
stability. It also implies that submaximal levels of Cyr61 protein are
adequate for optimal tumor cell growth when driven by
E2 under these in vitro conditions.
The antiproliferative effect of the neutralizing antibodies was not due
to cytotoxicity, because treated MCF-7 cells did not take up trypan
blue (data not shown). Thus, Cyr61 is required for
E2- and EGF-mediated cell proliferation of MCF-7
cells in vitro.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß,
platelet-derived growth factors A and B, and FGF (reviewed in Ref.
3). Thus, ovarian sex steroid and/or growth factor target
genes in mammary epithelial cells that act as local mediators may be
critical for neoplastic generation and subsequently tumorigenesis.
Given that, we provide evidence that Cyr61, a serum-inducible
immediate-early gene, is an estrogen target gene that is
transcriptionally regulated and required for hormone-dependent cell
proliferation in human MCF-7 mammary adenocarcinoma cells. The
significance of these results is underscored by the observation
that in 70% of breast tumor biopsies isolated from patients with
stage II invasive ductal carcinoma, Cyr61 was up-regulated in
hyperplastic ductal/luminal epithelial cells compared with autologous
healthy mammary tissue controls. Moreover, the levels of Cyr61 protein
were greater in those tumors that were
ER+/EGFR+ than in those
that were ER-/EGFR+,
suggesting that estrogen in conjunction with growth factors may
regulate Cyr61 expression in vivo and during carcinogenesis.
It is noteworthy that other steroid hormones, such as progesterone and
DHT in mammary adenocarcinoma cell lines that overexpress PR and AR,
respectively, induce Cyr61 as well, implying that it may be a general
mediator of hormone-dependent tumorigenesis. Based on previous studies, the role of Cyr61 in mammalian cell development (13, 20) suggests that aberrant expression may predispose cells toward dysregulated growth, such as in tumorigenesis. Indeed, overexpression of Cyr61 has been observed in several human cell lines derived from human bladder papilloma, colon adenocarcinoma, melanoma, and meduloblastoma (14). In some instances Cyr61 is thought to promote tumorigenesis. For example, transfection of a Cyr61 expression vector into the gastric adenocarcinoma cell line RF-1, which does not express Cyr61, increases these cells’ tumorigenicity in vivo (14). Conversely, Cyr61 is shown to be down-regulated in the epithelium of prostate cancer biopsies, uterine smooth muscle cells of leiomyomas, and the embryonal-rhabdomyosarcoma cell line, RD, suggesting that it may also function as a tumor suppressor or regulate cellular differentiation (21, 22, 23). Interestingly, rCop1, a recently identified member of the CCN family that shares a high degree of homology to Cyr61, has been shown to be a negative regulator of cell transformation when overexpressed and thus behaves similarly to tumor suppressors (24). Therefore, depending on the cell and tumor types, variable expression of Cyr61 may result in either positive or negative selection for dysregulated cell growth.
As our studies demonstrate that anti-Cyr61-neutralizing antibodies can
inhibit E2 as well as EGF-dependent DNA synthesis
and cell proliferation by more than 50%, Cyr61 appears to play a novel
and important role in mammary adenocarcinoma cell growth in
vitro. The immediate-early kinetics of Cyr61 are consistent with
other growth-promoting genes that are regulated by estrogen and
required for epithelial cell proliferation (3). As a
ligand for the integrin receptors
vß3,
IIb3a, and
5ß1
(25, 26, 27), it is plausible that Cyr61 may emit its growth
effects through integrin signal transduction or through an as yet to be
identified cognate receptor that may be expressed on breast tumor
epithelial cells. In addition, as Cyr61 is a growth factor-binding
protein and augments growth factor-dependent DNA synthesis (17, 28), it may promote mammary tumor cell growth by directly
interacting with certain growth factors to enhance their bioactivity.
Thus, up-regulation of Cyr61 in ER+ breast tumors
by E2 may augment the bioactivity of growth
factors that, through a positive feedback, would, in turn, result in
waves of Cyr61 expression and continued cell proliferation.
Invasive ductal carcinoma (IDC) is the most common type of breast
cancer and accounts for 80% of known cases (1).
Overexpression of Cyr61 protein in 70% of the IDC patients analyzed in
this study suggests that it may be an important mediator of breast
tumor development and growth in vivo. Interestingly, a
recent report by Tsai et al. demonstrates that Cyr61
mediates MCF-7 cell migration in an
vß3-dependent manner
and is overexpressed in a subset of IDC tumors, further corroborating
our patient observations (29). Moreover, the increased
levels of Cyr61 protein in ER+ breast tumors
suggest that it may contribute to estrogen-driven tumorigenesis
in vivo. Overexpression of Cyr61 in breast tumors may be due
to the ability of E2 to synergize with growth
factors such as EGF and HB-EGF to sustain Cyr61 expression.
Alternatively, mutations within estrogen response elements or growth
factor-activated response elements that result in aberrant promoter
activity, gene amplification, or increased mRNA stabilization may also
lead to Cyr61 overexpression. Growth factor up-regulation of Cyr61 in
steroid-sensitive breast tumors may also predispose a subset of tumor
epithelial cells toward continued dysregulated proliferation, as their
growth becomes hormone-independent or metastatic in the malignancy
process. To summarize, as illustrated in Fig. 8, Cyr61 expression in breast cancer may
conceptually orchestrate multiple activities to 1) promote tumor cell
proliferation in an autocrine and/or paracrine fashion by augmenting
growth factor activity (17, 28) or transmitting
proliferative signals via integrin receptors (25, 26), 2)
coordinate tumor epithelial migration and progression chemokinetically
(29), and 3) regulate tumor neovascularization in a
paracrine fashion as a proangiogenic factor (14, 25). The
modular domain structure of Cyr61 and its localization to the
extracellular matrix may account for its ability to concomitantly
dictate multiple functions during tumorigenesis, such as cell
proliferation and migration.
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Acknowledgments |
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Received November 16, 2000.
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References |
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vß3. J Biol Chem 271:3090–3096
IIß3.
J Biol Chem 274:24321–243276ß1 and cell surface
heparin sulfate proteoglycans. J Biol Chem 275:24953–24961This article has been cited by other articles:
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D. Xie, D. Yin, H.-J. Wang, G.-T. Liu, R. Elashoff, K. Black, and H. P. Koeffler Levels of Expression of CYR61 and CTGF Are Prognostic for Tumor Progression and Survival of Individuals with Gliomas Clin. Cancer Res., March 15, 2004; 10(6): 2072 - 2081. [Abstract] [Full Text] [PDF]
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H. R. Mason, D. Grove-Strawser, B. S. Rubin, R. A. Nowak, and J. J. Castellot Jr. Estrogen Induces CCN5 Expression in the Rat Uterus in Vivo Endocrinology, February 1, 2004; 145(2): 976 - 982. [Abstract] [Full Text] [PDF]
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A. Selvaraj and R. Prywes Megakaryoblastic Leukemia-1/2, a Transcriptional Co-activator of Serum Response Factor, Is Required for Skeletal Myogenic Differentiation J. Biol. Chem., October 24, 2003; 278(43): 41977 - 41987. [Abstract] [Full Text] [PDF]
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