Download Alternative Splicing of Cyr61 Is Regulated by

Published OnlineFirst February 24, 2009; DOI: 10.1158/0008-5472.CAN-08-1997
Research Article
Alternative Splicing of Cyr61 Is Regulated by Hypoxia and
Significantly Changed in Breast Cancer
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Marc Hirschfeld, Axel zur Hausen, Herta Bettendorf, Markus Jäger, and Elmar Stickeler
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Department of Obstetrics and Gynecology and 2Institute of Pathology, Freiburg University Medical Center, Freiburg, Germany
Abstract
Hypoxia is known to induce the transcriptional activation of
pathways involved in angiogenesis, growth factor signaling,
and tissue invasion and is therefore a potential key regulator
of tumor growth. Cyr61 (cysteine rich 61) is a secreted,
matricellular protein with proangiogenic capabilities and is
transcriptionally induced under hypoxic conditions. High
expression levels of Cyr61 were already detected in various
cancer types and linked to tumor progression and advanced
stages in breast cancer. Besides hypoxia, there is some
evidence that posttranscriptional pre-mRNA processing could
be involved in the regulation of Cyr61 expression, but was thus
far not investigated. We studied the expression pattern of
Cyr61 mRNA and protein in breast cancer cell lines as well as
in matched pairs of noncancerous breast tissue, preinvasive
lesions, and invasive breast cancers, respectively. In addition,
we analyzed the potential regulatory capability of hypoxia on
Cyr61 expression by functional tissue culture experiments.
Our study revealed a stage-dependent induction of Cyr61
mRNA and protein in breast cancer tumorigenesis and for the
first time alternative splicing of the Cyr61 gene due to intron
retention. Breast carcinogenesis was accompanied by a shift
from an intron 3 retaining toward an intron 3 skipping mRNA
phenotype consecutively leading to processing of the biological active Cyr61 protein. The functional analyses strongly
emphasize that hypoxia serves as a specific inducer of
alternative Cyr61 splicing toward the intron skipping mRNA
isoform with potential biological consequences in tumor cells.
[Cancer Res 2009;69(5):2082–90]
Introduction
Alternative splicing occurs in the vast majority of human genes
and plays a major role in the regulation of gene expression and the
generation of proteomic and functional diversity (1, 2). This
complex nuclear process creates an average of more than three
mRNA isoforms for each gene. Besides skipping and inclusion of
variable exons and usage of alternative splice sites (3), intron
retention is one possible pattern of alternative splicing, where a
variable intron sequence is maintained or skipped within the
mature mRNA transcript (4–6). Intron retention potentially affects
mRNA transport to the cytoplasm (7) or can insert premature stop
codons into the mRNA (4). The latter process is known to induce
mRNA degradation by a mechanism called nonsense-mediated
decay (NMD; refs. 4, 8). However, there is also evidence for intronretaining mRNAs that are encoding biologically active protein
Requests for reprints: Elmar Stickeler, Hugstetterstr. 55, 79106 Freiburg, Germany.
Phone: 49-761-270-3148; Fax: 49-761-270-3148; E-mail: [email protected].
I2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-1997
Cancer Res 2009; 69: (5). March 1, 2009
isoforms (4). These observations emphasize the potent role of
alternative splicing as a strong posttranscriptional regulator of
gene expression with vast functional consequences. A striking
example for this biological importance is given by observed
changes in alternative splicing pattern of genes and alterations in
splicing factor expression under pathologic conditions especially
in human cancers (9–11).
Cyr61/CCN1 (cysteine rich 61) is a secreted, growth factor–
inducible immediate-early gene (12) representing together with
CTGF/CCN2 (connective tissue growth factor) and NOV/CCN3
(Nephroblastoma overexpressed) one of the prototype members of
the CCN family (13). The CCN proteins share a common
multimodular organization with four discrete and conserved
structural domains, each encoded by separate exons (14, 15). The
structural basis results in the remarkable diversity of multiple
cellular activities and biological functions of these matricellular
proteins (14, 16). They either localize intracellularly or associate
with the extracellular matrix and the cell surface and play
important roles in the regulation of cell adhesion, migration,
proliferation, differentiation, and survival (15, 17–21). Cyr61 is
involved in significant physiologic and pathologic processes like
development, wound repair, angiogenesis, inflammation, cell
survival, vascular diseases, and endometriosis (14, 15, 18–24). In a
multitude of various human cancer types, Cyr61 was found to be
overexpressed and is supposed to be a promoter of tumor
progression (25, 26), particularly with regard to breast cancer
(16). O’Kelly and colleagues (16) revealed a significant correlation
between Cyr61 protein levels and tumor size, stage of disease,
and lymph node involvement. Although Cyr61 does not include a
classic nuclear localization sequence like transcription factors,
it could be localized in the cytoplasm and nucleus, respectively,
demonstrating the multifunctional capabilities of this protein (17).
Cyr61 transcription is inducible by multiple factors, like growth
factors [e.g., EGF (27), VEGF, and TGF-b (28)], chemokines (29, 30),
estrogen, TNF-a (30), and hypoxia (16). The 42 kDa protein
interacts within the cell or with surrounding cells through an
autocrine-paracrine mechanism, exerting its functions mediated by
at least five integrins as well as heparan sulfate proteoglycans (17).
The Cyr61 receptor interaction results in the activation of several
signal transduction pathways, including Wnt (31), nuclear factornB (32), tyrosine kinase, and Akt and the consecutive initiation of
transcription of target genes (17). It is likely that overexpression of
Cyr61 causes an up-regulation of its own receptors in an autocrine
signaling loop, resulting in the promotion of tumorigenesis and
cancer progression (16, 33).
Hypoxia is a key regulatory factor in tumor growth and induces a
transcriptional cascade that promotes an aggressive cancer
phenotype. Experimental studies support its tumorbiological
importance with regard to angiogenesis, growth factor signaling,
immortalization, genetic instability, tissue invasion, and apoptosis,
especially for breast cancer (34). Interestingly, Cyr61 expression
is transcriptionally induced under hypoxic conditions possibly
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Cyr61 Alternative Splicing
regulated by hypoxia-inducible factor-1a (HIF1-a; refs. 35, 36). As a
consequence, Cyr61 promotes adhesion, migration, and survival of
vascular endothelial cells (35, 37–39). Martinerie and colleagues
(40) detected two different mRNA phenotypes of human Cyr61 in a
Northern blot analysis of tumor cells from the nervous system,
concluding that alternative splicing might be the potential
underlying molecular mechanism for this phenomenon. However,
this hypothesis was never further investigated.
In the present study, we analyzed the expression pattern of
human Cyr61 in human breast cancer in cell lines and matched
pair tissue samples, respectively. We describe for the first time the
occurrence of alternative splicing of this tumorbiologically
important gene by the detection of a specific intron 3 retention,
leading most likely to NMD of the intron-retaining isoform. Further
functional experiments revealed that hypoxia regulates alternative
Cyr61 pre-mRNA splicing by inducing skipping of intron 3, leading
to the only thus far known active isoform of this protein. With the
additional knowledge of hypoxia-induced Cyr61 transcription and
the proangiogenic capability of this protein, we postulate that
hypoxia is a key regulator of Cyr61 expression and acts as on/off
switch for its biological activation and its known tumor-promoting
capabilities.
Materials and Methods
Patients and tissue samples. Matched-pair tissue samples of invasive
breast cancers and corresponding nonneoplastic tissue from six consecutive
patients with invasive primary breast cancer who were treated at the
Department of Obstetrics and Gynecology, University Medical Center
Freiburg, were analyzed. Approval by the local ethics committee (no. 313/
2001) and written informed consent of each patient was obtained. The
tissue samples were obtained at the time of surgery, examined by a
specialized pathologist, and freshly frozen ( 80jC). In a matched-pair
analysis, we tested the specimen for Cyr61 expression by reverse
transcription-PCR (RT-PCR). In addition, for immunohistochemical analyses, the corresponding paraffin-embedded tissues were analyzed by the
same pathologist.
Cell lines and culture conditions. The established human tumor cell
lines HeLa, MDA-MB-231, MDA-MB-453, and T47D were cultured in a
humidified incubator (37jC in 5% CO2, 95% air) and maintained in DMEM
(Life Technologies, Invitrogen), supplemented with 10% fetal bovine serum
‘‘Gold’’ (PAA Laboratories GmbH), 1% of 1 mol/L HEPES buffer (Life
Technologies), and 100 units/mL penicillin/streptomycin (Sigma-Aldrich
Chemie GmbH). For hypoxia experiments, cultures were transferred to
hypoxic culture conditions (1% O2; mentioned as hypoxia) in a hypoxic
chamber placed in the same incubator. Cells were cultured in parallel
experiments under normal oxygen conditions (95% air, mentioned as
normoxia, used as control), and for different time periods under hypoxic
(6, 12, 18, and 24 h in 1% O2) and ‘‘rescue’’ conditions (3, 6, 12, and
24 h normoxic conditions following 24 h hypoxia). Experiments concluded
with the immediate harvesting of treated cultures for RNA extraction. For
the immunocytochemical detection of Cyr61 protein HeLa, MDA-MB-231,
MDA-MB-453, and T47D cells were cultured under normoxic and hypoxic
conditions for 24 h, as well as under rescue conditions (12 h normoxia
following 24 h hypoxia), using four-chamber culture slides (BD Falcon, BD
Biosciences Europe) as culture vessels. Experiments concluded with the
immediate fixation of treated cultures for immunocytochemistry.
RNA extraction and RT-PCR. Breast tissue specimens were minced on
ice by treatment with a tissue homogenizer (IKA Werke) in TRIzol reagent
(1 mL per 100 mg tissue, thrice for 10 s; Invitrogen) and RNA was isolated
following the manufacturer’s protocol. Purified RNA was dissolved in
RNase-free water and stored at 80jC for further analysis. Total RNA from
cultured cells was isolated similarly using the TRIzol method.
In total, 4 Ag RNA, as determined by optical densitometry, were used for
cDNA synthesis using Moloney murine leukemia virus reverse transcriptase
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(Promega), RiboLock RNase inhibitor (Fermentas GmbH), and random
hexamer primers (New England Biolabs GmbH) followed by PCR (35 cycles)
using Taq polymerase (Fermentas) and primers specific for exons 3-4
of Cyr61 (5¶-primer, 5¶-GGCAGACCCTGTGAATATAA-3¶; 3¶-primer,
5¶-CAGGGTTGTCATTGGTAACT-3¶) and 18S RNA as internal control
(5¶-primer, 5¶-AACTCACTGAAGATGAGGTG-3¶; 3¶-primer, 5¶-CAGACAAGGCCTACAGACTT-3¶). Expected amplicon sizes were 481 bp for
Cyr61 and 305 bp for 18S RNA, respectively. All results from RT-PCR for
Cyr61 products were normalized using the corresponding 18S RNA
expression as a comparative value.
Cloning and sequencing. cDNA from malignant breast specimen
displaying an amplification of both Cyr61 mRNA isoforms were used for
assembly in an effectual quantity. The two Cyr61 variants were cloned into
One Shot Top10 chemically competent Escherichia coli (Invitrogen) using
the pJET1.2 cloning vector (Fermentas). Sequences of both Cyr61 mRNA
isoforms were analyzed by Genterprise Genomics (Genterprise Genomics
Gesellschaft für Genanalyse und Biotechnologie mbH).
Immunohistochemistry. Routinely formalin-fixed and paraffin-embedded specimens were studied for the expression of human Cyr61 by using a
polyclonal Cyr61 (H78) antibody (Santa Cruz Biotechnology, Inc.). For
visualization of Cyr61 expression, antigen retrieval and indirect immunoperoxidase technique were applied. Antigen retrieval was performed by
cooking the dewaxed sections for 10 min in a 10 mmol/L sodium citrate
buffer (pH 6.0). Inhibition of endogenous peroxidase was performed by
5-min incubation with 3% H2O2. Endogenous avidin-biotin was blocked by
the use of a commercial biotin blocking system (DAKO, Dako GmbH) for
10 min. After two washes in trissaline buffer, slides were incubated with 1%
goat serum for 30 min to block unspecific staining. The sections were
exposed to the Cyr61 antibody (1:200) overnight at room temperature.
Slides were incubated with biotinylated anti-rabbit immunoglobulins for
60 min at room temperature and treated with streptavidin-peroxidase
(DAKO). Staining was achieved by 3,3¶-diaminobenzidine (DAB; Vectastain,
Vector Laboratories, Inc., Linaris GmbH) and the slides were counterstained
with hemalaun.
Immunocytochemistry. Immunocytochemistry was carried out with
previously treated cell cultures on special culture slides (BD Falcon).
Cells were fixed using paraformaldehyde fixation reagent (4% paraformaldehyde in PBS) and incubated with the Cyr61 (H78) antibody (1:500
in blocking buffer, Santa Cruz Biotechnology, Inc.) overnight, followed
by incubation with anti-rabbit immunoglobulin and treatment with
streptavidin-peroxidase (DAKO). Staining was achieved by DAB (Vectastain)
and cells were counterstained with hemalaun.
Equipment for documentation and evaluation. Documentation of
electrophoresis gels was performed using DIANA II (CCD Camera System,
Raytest Isotopenmessgeräte GmbH) and evaluation with AIDA (2D
densitometry) software (Raytest). Microscopic examination of cells and
tissues was performed using Axioplan 2 microscope (KARL ZEISS Microimaging GmbH), documentation with Canon Powershot G5 and Adobe
Photoshop CS2 Version 9.
Statistical analysis. Expression difference of the Cyr61 mRNA isoforms
between tumorous and noncancerous breast tissue was calculated.
Percentages of Cyr61 mRNA were normalized against the corresponding
internal control (18S RNA). The expression differences of Cyr61 mRNA
isoforms in samples originating from functional in vitro experiments (cells)
were calculated in an analogous manner. The Kolmogorov-Smirnov test
showed a nonnormal distribution of the results, which was not improved
by logarithmic transformation. Therefore, the Mann-Whitney U test for
unpaired groups was used. The Statistical Package for the Social Sciences
software version 15.0.1 (SSPS 15.0.1.) was used for statistical analysis.
Results
Cyr61 is alternatively spliced in human breast tissue. Using
RT-PCR analysis, alternative splicing of Cyr61 was identified in
malignant and noncancerous human breast tissue. Instead of the
expected 481-bp amplicon, a different distinct mRNA phenotype,
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f130 bp bigger in amplicon size, was detected in all examined
tissue specimens. In contrast, the expected 481-bp product was
only detectable in some samples (Fig. 1).
The mRNAs isolated from malignant breast tissue, expressing
both mRNA isoforms, were used in parallel to amplify the two
distinct Cyr61 mRNA fragments. They were separated and purified
before cloning. Selected clones were tested by PCR and plasmids
containing one distinct fragment were isolated for sequencing.
Repeated sequence analyses revealed that the two distinct Cyr61
mRNA isoforms were due to retention of the 131-nt intron 3
separating exon 3 and exon 4 of the Cyr61 gene, leading to an
intron-retaining phenotype (IR) with a total of 612 bp and the
intron skipping phenotype (IS) with the thus far published 481 bp
mRNA isoform (Fig. 1). More interestingly, further sequence
analysis revealed that intron 3 contains two stop codons
(Fig. 1B), highly suggesting that this mRNA isoform is not capable
of generating a functional full-length protein and only the shorter
intron-skipping mRNA phenotype encodes the active Cyr61.
Alterations in Cyr61 alternative splicing pattern in breast
cancer. Although the majority of examined breast tissue specimen
exhibited high expression levels of the Cyr61 IR mRNA isoform,
the IS phenotype was differentially expressed. By normalizing the
Cyr61 mRNA levels against 18S mRNA, it became evident that in
the matched-pair analysis, expression levels of the functional IS
phenotype was markedly increased in invasive breast cancer
compared with the corresponding noncancerous breast tissue by
nearly 50% (Fig. 2A). Besides the box plots, we performed an
additional statistical analysis that revealed a significant difference
in the expression of the Cyr61 IS mRNA isoform between tumor
and noncancerous breast tissue (P = 0.004, two-sided, Mann-
Whitney U test). Expression levels of the Cyr61 IR mRNA isoform
did not show a significant difference between malignant and
noncancerous tissue. To further determine the alternative splicing
activity and the consecutive activity level of Cyr61, we analyzed the
given ratio of IS to IR (IS/IS+IR) in these matched pairs of tumors
and noncancerous tissues. In parallel to the above-mentioned
findings, a strong shift toward an induced alternative splicing of the
IS mRNA phenotype in breast cancer versus noncancerous tissue
from 30% to 46% was detectable (Fig. 2B).
Immunohistochemical detection of Cyr61 protein in breast
tissue. To investigate the expression and localization pattern of
Cyr61 under pathologic conditions, tissue sections from patients
with primary invasive breast cancer were examined. The polyclonal
Cyr61 antibody (H-78, Santa Cruz Biotechnology) used is directed
against amino acids 163-240, the so-called ‘‘hinge region’’ connecting the NH2- and COOH-terminal halves of the protein. Therefore,
this antibody is only capable to detect functional Cyr61 protein.
A clear and specific Cyr61 staining pattern was visible in the
examined breast specimen. Protein expression was highly induced
in malignant compared with noncancerous epithelial cells (Fig. 3).
Interestingly, specific and strong cytoplasmic and perinuclear Cyr61
staining was detectable in tumor cells of invasive ductal carcinoma,
whereas the noninvasive cells of ductal carcinoma in situ (DCIS)
displayed a weaker and more heterogeneous expression pattern.
In these cases, more single foci of cells with strong Cyr61 expression
were identified. In contrast to DCIS, a more homogenous expression
profile of Cyr61 was seen in lobular carcinoma in situ with a more
pronounced perinuclear expression pattern (Fig. 3C).
Hypoxia induces a significant shift of Cyr61 alternative
splicing in vitro. Cyr61 mRNA expression was detectable in all
Figure 1. mRNA isoforms of human Cyr61.
A, expression of Cyr61 mRNA isoforms in breast
tissue. The alternatively spliced pre-mRNA isoform
Cyr61 IR characterized by the retention of intron 3
(amplicon size 612 bp) and the constitutive pre-mRNA
isoform Cyr61 IS (intron 3 skipping, amplicon size
481 bp) were detected. RT-PCR analysis with equal
amounts of total RNA (4 Ag) in matched pairs of
malignant (CA ) and corresponding noncancerous
tissues (N ) originating from patients with primary
adenocarcinoma of the breast. HeLa-cDNA served as
control (C); 18S RNA expression served as a
comparative value. Box, Cyr61 alternative splicing;
genomic structure of both Cyr61 mRNA isoforms
(Ex1–Ex5 exons, In1–In4 introns). B, nucleotide
sequence of exons 3 and 4 (uppercase characters ) and
intron 3 (lowercase characters ) of the human Cyr61
gene. The retention of intron 3 creates two stop codons
(in boxes ) within the intronic sequence.
Cancer Res 2009; 69: (5). March 1, 2009
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Cyr61 Alternative Splicing
Figure 2. Statistical analysis: matched pairs of malignant
(tumor tissue) and corresponding noncancerous specimen
(normal tissue) of the breast. A, expression of Cyr61 IS (intron
skipping) mRNA isoform (P = 0.004, Mann-Whitney U test).
Normalized on 18S RNA. B, Cyr61 alternative splicing.
Percentage of the Cyr61 IS mRNA isoform compared with total
Cyr61 expression [IS/(IR+IS)]. Thick lines, median (50%
percentile); gray boxes, 25% to 75% percentile; thin lines,
minimal and maximal value.
examined cell lines (HeLa, MDA-MB-231, MDA-MB-453, T47D).
Because Cyr61 is a hypoxia-inducible proangiogenic factor, we
examined the expression levels of this gene under normoxic (5%
CO2, 95% air) and hypoxic (1% O2) conditions, and in ‘‘rescue’’
experiments with normoxia following 24 hours of hypoxia.
The IR mRNA isoform was detectable under normoxic as well as
under hypoxic conditions. In contrast, the IS mRNA isoform,
resulting from intron 3 skipping, was only hardly detectable by
RT-PCR in cells cultivated under normoxia. However, a strong
shift in alternative splicing toward the IS phenotype emerged with
extended phases of hypoxia and this shift was furthermore
reproducibly decreased in a stepwise fashion with reversion to
normoxia following hypoxia (Fig. 4).
These observations in different cell lines strongly suggest that
oxygen deprivation causes a reversible shift of the alternative
splicing pattern of Cyr61 in a time-dependent manner toward the
intron 3 skipping mRNA isoform, presumably the only Cyr61
mRNA phenotype resulting in a functional Cyr61 protein.
Therefore, we hypothesize that hypoxia serves as an on/off
switch for the processing of the full-length protein, acting as an
tumorbiologically pertinent factor.
Looking in more detail into the examined cell lines, we were able
to assess slight differences in their alternative splicing alterations
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in response to altered oxygen conditions. The basic levels of IS/
IS+IR showed similar low levels of splicing activity in the examined
cell lines. Changing the cultivation conditions to oxygen deficiency
resulted uniformly in a marked shift toward the highest expression
levels for IS after 24 hours (Fig. 4). Hypoxia induced a dramatic
change in Cyr61 mRNA expression with an induction of the Cyr61
IS phenotype compared with total Cyr61 mRNA (IS/IS+IR) from
8.5% up to 62% in HeLa and from 9.3% to 60% in MDA-MB-231
cells, respectively. Statistical analysis revealed a highly significant
increase in the intron-skipping Cyr61 isoform expression between
cells cultivated under normal oxygen conditions and hypoxia
(24 hours; P < 0.0001, two-sided, Mann-Whitney U test). These data
were reproducible in all examined cell lines. Rescue experiments
showed a fast and continuous decrease in expression of this splice
product after recovery of normal oxygen conditions and the shift
toward predominant expression of the IR phenotype. MDA-MB-453
and T47D cells featured a decelerated adaptation to altered oxygen
concentrations compared with the other cell lines, resulting in a
prolonged higher expression rate of the functional active Cyr61
mRNA isoform (Fig. 4).
Immunocytochemical detection of Cyr61 protein under
altered oxygen conditions. The immunocytochemical detection
of Cyr61 protein was performed using HeLa, MDA-MB-231,
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membrane, respectively, of cells displaying proximate amitosis.
After dissolution of the nuclear membrane, the protein was also
detectable in low concentrations in the cytoplasm of dividing cells.
During the whole process of mitosis, the Cyr61 protein was present
in the nuclear elements.
Oxygen deficiency (1% O2) triggered a specific increase of Cyr61
protein in all cells, independently from the current stage of cell
cycle and caused nuclear as well as very strong cytoplasmic Cyr61
expression. Nevertheless, the highest concentrations were still
detectable in the nuclear elements (Fig. 5).
In parallel to the mRNA findings, the rescue experiment revealed
the reversibility of the on/off switch of Cyr61 alternative splicing
induction by hypoxia. This was shown by a markedly decreased
nuclear as well as cytoplasmic Cyr61 protein expression (Fig. 5C).
This experimental part of the immunocytochemical examination
displayed the most notable differences among the various cell lines.
Some cell types seemed to react toward the oxygen conditions with
regard to their Cyr61 protein expression faster than others. In
particular, HeLa and MDA-MB-231 returned faster to their original
Cyr61 expression pattern than the other cell types.
Discussion
Figure 3. Immunohistochemical localization of the Cyr61 protein in breast
tissue. A, invasive ductal carcinoma. Invasive carcinoma cells on the left side
(arrows ) show significant cytoplasmic and perinuclear overexpression of Cyr61
in contrast to nonneoplastic ductal epithelium on the lower right side, which
reveals no expression of Cyr61 in the epithelial or myoepithelial cell layer. B, in
DCIS, Cyr61 expression is weaker and heterogenous. Single foci of cells with
strong Cyr61 expression are identified (arrows ). C, lobular carcinoma in situ
shows a more homogenous expression pattern than DCIS and a more
perinuclear localization (arrows ). Cyr61 antibody H-78 (Santa Cruz
Biotechnology) counterstained with hemalaun. Magnification, 400.
MDA-MB-453, and T47D cells, cultivated under normoxic and
hypoxic conditions for 24 hours, as well as rescue conditions
(12-hour normoxia following 24-hour hypoxia). The H-78 (Santa
Cruz Biotechnology) antibody was used for the exclusive detection
of functional Cyr61 protein. The subsequent propositions apply
consistently to all examined cell lines.
Under normal oxygen conditions (5% CO2, 95% air), the Cyr61
protein was only measurable in mitotic active cells. High protein
concentrations were located in the nuclear matrix and the nuclear
Cancer Res 2009; 69: (5). March 1, 2009
Hypoxia is a key regulator in tumor growth and induces
transcriptional activation of pathways involving angiogenesis,
growth factor signaling, immortalization, and tissue invasion and
was already identified as a tumorbiologically important factor in
breast cancer (34). Thus far, it is known that Cyr61 expression is
transcriptionally induced under hypoxic conditions, which are
frequently observed in rapidly growing tumors and their metastases (35, 36). In this scenario, Cyr61 displays a proangiogenic activity
by promoting vascular endothelial cell survival, adhesion, and
migration (35, 37–39). Aberrant expression of Cyr61 was already
linked to tumor development and elevated expression levels were
associated with advanced tumor stages in breast cancer (17, 25, 41).
Its involvement in several important signaling pathways makes
Cyr61 an interesting focus of cancer research. Besides hypoxia,
there was some evidence that posttranscriptional pre-mRNA
processing might be involved in the regulation of Cyr61 expression
because two different mRNA phenotypes were detected in
Northern blot analyses (40). However, the underlying mechanisms
were never further investigated.
For the first time, our study reveals the occurrence of two
distinct Cyr61 mRNA isoforms, which are generated by alternative
splicing of the Cyr61 pre-mRNA. Sequence analysis revealed that
the two existing mRNA phenotypes are due to alternative
retention of intron 3, which is located between exons 3 and 4
of the Cyr61 gene. The splicing effect on both Cyr61 mRNA
isoforms is independent from any mutations. Repeated analyses
of breast tissue and cell culture specimen under changing oxygen
conditions did not show mutational alterations within the
investigated exonic and intronic sequences of the two Cyr61
mRNA phenotypes.
Alternative pre-mRNA splicing is an important nuclear mechanism for the regulation of gene expression, which affects f45% to
60% of human genes (1, 2, 42, 43) and is frequently altered in cancer
(3, 44). However, the complete retention of an intron in a mature
transcript is one of the least common types of alternative splicing
(4) and is estimated to occur in only 5% of splicing events (45).
Genes representing intron retention are more highly and broadly
expressed than other genes (4). Most probably, intron retention is
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Cyr61 Alternative Splicing
associated with weaker splice sites, shorter intron lengths, and
lower density of intronic splicing enhancers like GGG, respectively
(4, 45, 46). In this regard, Cyr61 fits very well into this scenario,
because it contains relatively short introns (intron 1, 317 nt; intron
2, 349 nt; intron 3, 131 nt; intron 4, 115 nt), compared with the
average intron length of human genes with f5,000 nt (4). The exon
3-intron 3-exon 4 complex with a length of 697 nt still matches
the distribution of lengths of retained intron and flanking exons
Figure 4. Statistical analysis. Expression ratio of Cyr61
intron-skipping isoform (Cyr61 IS) compared with total
expression of Cyr61 mRNA isoforms (IS/IS+IR) in human
tumor cell lines HeLa, MDA-MB-231, MDA-MB-453, and
T47D under (N ) normal oxygen conditions (5% CO2, 95%
air); (H ) hypoxic conditions (1% O2) for 6, 12, 18, and 24 h;
and (R ) rescue conditions (5% CO2, 95% air) for 3, 6, 12,
18, and 24 h following 24 h of hypoxia. There is a highly
significant increase in the Cyr61 IS isoform expression
between cells cultivated under normal oxygen conditions
and hypoxia (24 h; P < 0.0001, Mann-Whitney U test),
reproducible in all examined cell lines. Thick lines, median
(50% percentile); gray boxes, 25% to 75% percentile;
thin lines, minimal and maximal value.
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Figure 5. Immunocytochemical detection
of Cyr61 protein in human tumor cell lines.
MDA-MB-231 (A1 ) and MDA-MB-453 (A2)
cells under normal oxygen conditions
(5% CO2, 95% air). Cyr61 protein is only
detectable in cells currently going through
phases of amitosis, in the process
displaying weak cytosolic (30%) and
stronger nuclear expression (70%).
MDA-MB-231 (B1 ) and MDA-MB-453 (B2)
cells under hypoxic conditions (1% O2) for
24 h. Strong Cyr61 protein expression,
80% cytosolic, 20% nuclear. MDA-MB-231
(C1 ) and MDA-MB-453 (C2 ) cells under
rescue conditions (5% CO2, 95% air) for
12 h following 24 h of hypoxia. Cells display
a markedly decreased Cyr61 expression
after reoxygenation: 70% cytosolic and
30% nuclear. Cyr61 antibody H-78 (Santa
Cruz Biotechnology) counterstained with
hemalaun. Magnification, 400.
described by Sakabe and colleagues (4). In addition, the triplet
sequence GGG acting as an intronic splicing enhancer is only rarely
found in the human Cyr61 gene, especially in the flanking exons 3
and 4. A more detailed sequence analysis revealed that intron 3
includes two stop codons, suggesting that they potentially trigger
the mRNA degradation through the process of nonsense-mediated
decay (46, 47). Thus, intron 3 retention in Cyr61 mRNA is most
likely leading to no functional protein and alternative splicing
might therefore represent a potential regulator of Cyr61 activity.
These data are in line with earlier findings from our group,
which described the retention of short intron 9 of the extracellular
matrix protein CD44 as a form of aberrant splicing event in breast
cancer (6).
Our observations generated even more oncological effect,
because the analyses of matched pairs of invasive breast cancers
and corresponding noncancerous tissue revealed a specific and
very strong induction of alternative Cyr61 splicing toward the IS
phenotype in invasive breast cancers. Whereas noncancerous
tissue predominantly expressed the Cyr61 IR mRNA, splicing
shifted markedly toward the expression of the IS mRNA in invasive
breast cancer. This strong induction of the IS mRNA isoform will
most likely enhance the activity level of Cyr61 protein in tumor
Cancer Res 2009; 69: (5). March 1, 2009
cells because this isoform encodes for the only active Cyr61
protein. The mRNA findings were strongly supported by immunohistochemical analyses showing a stage-dependent induction of
functional Cyr61 protein expression in breast carcinogenesis.
Although normal breast epithelial cells displayed very low levels
of Cyr61 protein, DCIS precursor lesions showed already a stronger
expression, which was further induced up to high levels in invasive
breast cancer cells of ductal carcinomas (Fig. 3).
With the knowledge of the inducibility of Cyr61 expression by
hypoxia, we speculated if our findings in breast cancer specimen
mirror a possible oxygen deprivation in tumor cells and if hypoxic
conditions might alter Cyr61 alternative splicing. Our functional
experiments in HeLa and several breast cancer cell lines revealed
that hypoxia is reproducibly able to alter alternative Cyr61 premRNA splicing pattern by enhancing the removal of intron 3 and
thereby potentially leading to the generation of the biologically
active protein. An up to 6-fold induction of the IS mRNA phenotype
was observed. Remarkably, this activation, which was time
dependent and reached a maximum after 24 hours of hypoxia,
was completely reversible by restoring normoxic conditions for
the tumor cells (Fig. 4). This phenomenon was also present on the
protein level as detected by immunocytochemistry (Fig. 5) and
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Cyr61 Alternative Splicing
strongly supports the hypothesis of a specific mechanism for the
regulation of Cyr61 expression. With the additional knowledge of
hypoxia-induced Cyr61 transcription, we postulate that hypoxia is
a key regulator of Cyr61 expression. It acts through alternative
splicing alterations as an on/off switch for the biological activation
of Cyr61 by promoting intron 3 skipping, leading to functional
protein and thereby enhancing its proangiogenic activities.
At this point, it has to be speculated if specific trans-acting
factors are involved in the hypoxia-induced changes in Cyr61
mRNA splicing pattern. From the literature, it is known that several
splicing factors can change their intracellular concentration and
localization under the influence of hypoxic conditions and
therefore might cause alterations in alternative splicing (45, 48).
Human Tra2-b1, a transcription factor specifically induced in
breast cancer and regulating alternative splicing of the CD44 gene
(49), could potentially affect the splicing pattern of Cyr61. Exon 3 of
the Cyr61 gene comprises five potential sites displaying the Tra2b1 binding motif GVVGANR (49). Sequence analysis of the exon
3-intron 3-exon 4 system also revealed the existence of several
possible binding motifs (YAGR; ref. 50) for the transcription factor
hnRNP A1. Further functional experiments are warranted.
In summary, our study reveals for the first time alternative
splicing of Cyr61 pre-mRNA, which is due to the alternative
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 5/30/2008; revised 11/5/2008; accepted 12/2/2008; published OnlineFirst
02/24/2009.
Grant support: Deutsche Krebshilfe (nos. 106163 and 107690; E. Stickeler and
A. zur Hausen).
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
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Alternative Splicing of Cyr61 Is Regulated by Hypoxia and
Significantly Changed in Breast Cancer
Marc Hirschfeld, Axel zur Hausen, Herta Bettendorf, et al.
Cancer Res 2009;69:2082-2090. Published OnlineFirst February 24, 2009.
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