Cancer Therapy Vol 1, 103-107, 2003.
Expression of RB1CC1, a novel tumor suppressor
gene, is inversely correlated with the Ki-67 proliferation index in primary
breast cancers
Research Article
Koji Teramoto1, Tokuhiro Chano2,3,
Yoshitomo Ozaki1, Satoru Sawai1, Noriaki Tezuka1,
Keiichi Kontani1*, Shozo Fujino1, Hidetoshi Okabe2
Departments of 1Surgery and 2Clinical
Laboratory Medicine, Shiga University of Medical Science, Seta-tsukinowa, Otsu,
Shiga 520-2192, Japan, 3PRESTO, Japan Science and Technology Corporation
(JST),
*Correspondence: Keiichi Kontani, Department of Surgery, Shiga University of Medical
Science, Seta-tsukinowa, Otsu, Shiga 520-2192, Japan. Tel.: +81-77-548-2244;
Fax: +81-77-544-2901; e-mail address: konbat@belle.shiga-med.ac.jp
Key words:
RB1CC1, RB1, Ki-67, tumor suppressor
gene, breast cancer
Received:
14 May 2003; Accepted: 21 May 2003; electronically published: May 2003
Summary
Retinoblastoma 1 (RB1)-inducible coiled-coil 1 (RB1CC1)
is a key regulator of RB1, and is frequently mutated in breast cancer. We
examined RB1CC1 expression using immunohistochemical means and compared it with
RB1 and Ki-67 labeling indices as well as estrogen receptor (ER) status in 54
primary breast cancers. RB1CC1
protein expression was absent in 8 cancers (15%), and RB1 protein was
significantly decreased or absent in all of the cases lacking RB1CC1. These 8
cases showed no loss of heterozygosity (LOH) at the RB1 locus. Importantly, a loss of RB1CC1 was
significantly correlated with a high Ki-67 index (p < 0.0001) and low ER status
(p = 0.0123). RB1CC1 expression predicts tumor progression, and its prognostic
value should to be established.
I. Introduction
Inactivation of the retinoblastoma 1 (RB1) gene is
considered to play a central role in the pathogenesis of many human malignant
neoplasms (Kamb, 1995; Taya, 1997; Kaelin et al, 1999).
RB1 is also involved in tumorigenesis and tumor progression (Lemoine, 1994; TÕAng,
1998; Kaelin et al, 1999), and its expression
is inversely correlated with its proliferative activity in breast cancer (TÕAng
and Fung, 1991). The RB1 locus is genetically altered in 3 to 37% of breast
cancers (Varley et al, 1989; Thorlacious et al,
1991; Lemoine, 1994;
TÕAng, 1998; Kaelin et al, 1999; Bieche and
Lidereau, 2000). However, it is not always responsible for the absence
of RB1 expression under such conditions (Varley et
al, 1989; Bieche and Lidereau, 2000), and other factors are probably
involved in this phenomenon.
RB1-inducible coiled-coil 1 (RB1CC1) is thought to be
a transcription factor because it is in fact localized
to the nucleus and it contains a nuclear localization signal, a leucine-zipper
motif and a coiled-coil structure (Chano, 2002a,b).
RB1CC1 is co-expressed with RB1 in various cancers and normal tissues (Chano, 2002a,b), where it functions as an inducible
regulator of RB1 expression (Chano, 2002a). We frequently observed RB1CC1 mutations in breast
cancer, suggesting that the functional loss of RB1CC1 results in insufficient
RB1 expression, which promotes dysregulation of the RB1CC1-RB1 pathway and
subsequent tumorigenesis (Chano, 2002c).
To clarify the incidence of RB1CC1
anomalies in primary breast cancers, we analyzed RB1CC1 expression by
immunohistochemical methods and examined its relationship with various
biopathological parameters in breast cancers from 54 patients. The important findings
of the present study suggest that a loss of RB1CC1 expression accelerates cell
proliferation.
II. Materials and methods
A. Specimens
We analyzed formalin-fixed and
paraffin-embedded primary breast cancer tissues resected from 54 patients
at the Shiga University of Medical Science Hospital between July 1996 and
December 2001. The Ethics Committee of our
institution approved the use of these specimens,
which were histologically classified according to the World Health
Organization (WHO) guidelines.
B. Immunohistochemical analysis
Antigens were retrieved by
autoclaving at 120”C for 1 min. Then RB1CC1, RB1, Ki-67 and estrogen
receptor (ER) were immunohistochemically stained using a Streptavidin-Biotin
Immunoperoxidase Complex System (DAKO Japan, Kyoto, Japan). Primary antibodies were anti-human
RB1CC1 rabbit antiserum (anti-RBICC-642) diluted 1:5000, and mouse monoclonal
antibodies to RB1 (G3-245, Pharmingen, San Diego, CA) diluted 1:500, to Ki-67
(NCL-Ki87-MMI, Novocastra, New Castle, UK) diluted 1:100, and to ER
(NCL-ER-6F11, Novocastra) diluted 1:100, respectively. Labeling indices of
Ki-67 and RB1 were determined as the ratio (%) of labeled compared with total
neoplastic cells in each section.
C. Loss of heterozygosity (LOH)
at RB1 locus
We extracted tumor DNA from the
samples without RB1CC1 expression using DNAzol reagent (Gibco-BRL, Paisley, Scotland)
according to the manufacturerÕs protocols. We analyzed them using the AFM058xd6
microsatellite
markers (UniSTS: 13158). The PCR products were resolved using 7.5% denaturing
PAGE and visualized by silver staining.
D. Statistical analysis
We evaluated relationships between
RB1CC1 expression and the RB1 and Ki-67 labeling indices using StudentÕs t-test. FisherÕs exact test determined
the relationships between RB1CC1 status, ER status and clinicopathological
factors including tumor size, lymph node involvement, histological subtype and
post-menopausal status. Statistical significance was assumed at p < 0.05.
III. Results
A. Positive Correlation between RB1CC1 and RB1
RB1CC1 protein was undetectable in 8
(15%) of 54 primary breast cancers and RB1 positive cells were absent or
significantly depleted in all of them (Figs. 1b and d). In these 8 tumors lacking RB1CC1
expression, they showed no LOH at RB1 locus (Fig. 2). Among the 46 specimens expressing
RB1CC1, RB1 was co-expressed in 45 (Figs. 1a and c) and significantly decreased in
only one. RB1CC1 expression was
significantly correlated with the RB1 labeling index (RB1CC1-positive versus
RB1CC1-negative specimens, 78.6 ± 13.9 versus 13.6 ± 12.1%, p < 0.0001; StudentÕs t-test) (Fig. 3a).
RB1CC1 (Figs. 1e and f), and the Ki-67 labeling index was
significantly higher in RB1CC1-negative tumors (RB1CC1-positive versus
RB1CC1-negative specimens, 20.3 ± 12.8 versus 65.0 ± 12.2%, p < 0.0001; StudentÕs t-test) (Fig. 3b).
RB1CC1
RB1
Ki-67
Case
11 Case
15
Figure 1. Immunohistochemical analysis of RB1CC1, RB1 and Ki-67 in
breast cancer. Both RB1CC1 (a) and RB1 (c) proteins are expressed in the nuclei of breast cancer
cells from Patient 11, but barely detectable in those from Patient 15 (b and d). Ki-67-positive nuclei are abundant
in specimens from Patient 15, whose tumor did not express RB1CC1 (f), but less profuse in tumor samples
from Patient 11 (e).
(Immunoperoxidase staining with hematoxylin counterstain. Magnification, «200.)
Figure 2. Loss of heterozygosity at RB1 locus. DNA samples from 8
tumors without RB1CC1 expression and from matching blood DNAs of Patient 15 and
46 were amplified using primer pairs for the AFM058xd6 microsatellite marker.
PCR products were resolved using 7.5% denaturing PAGE and visualized by silver
staining. All DNA samples showed
no LOH at RB1 locus. M, FX174/Hae III marker; P, genomic DNA sample from matching
blood leukocytes.
III. Discussion
The novel human gene, RB1CC1, encodes a putative transcription factor
implicated in the regulation of RB1 (Chano et al, 2002a) and exhibits the
characteristics of a classical tumor suppressor gene (Chano et al, 2002c). RB1CC1
mutations lacking function have been identified in breast cancer and might be
involved in their tumorigenesis (Chano et al, 2002c). The present study
evaluated abnormalities of the RB1CC1-RB1 pathway in primary breast cancers and
examined relationships with various biopathological parameters.
RB1CC1 expression was undetectable
and associated with a significant decrease or absence of RB1 positive cells in
8 (15%) of the 54 primary breast cancers. The 8 cases lacking RB1CC1
expression, showed no LOH at RB1 locus. These findings support those of
previous reports (Chano et al, 2002a,b,c) suggesting that RB1CC1 functions as a
key regulator of RB1 expression and that the dysfunction of RB1CC1 results in
insufficient RB1 expression. The present study found a suspected RB1
abnormality in only one specimen with RB1CC1 expression, but we could not
examine the LOH status at RB1 locus because of no matching tumor DNA. A LOH and
other alterations at the RB1 locus were observed in 3% to 37% of breast cancers
(Varley et al, 1989; Thorlacious et al, 1991; Lemoine, 1994; TÕAng,
1998; Kaelin et al, 1999; Bieche and Lidereau, 2000).
However, irregular RB1 protein expression is not always linked to such RB1 gene
derangement (Varley et al, 1989; Bieche and Lidereau,
2000). Since low or absent RB1 expression was usually associated with a
loss of RB1CC1 expression in the present study, the absence of the latter seems
to be a major cause of depleted expression of RB1 and subsequent breast cancer
tumorigenesis.
The loss of RB1CC1 expression was significantly
correlated with a higher Ki-67 labeling index in our series (p < 0.0001).
Since Ki-67 identifies proliferating cells by recognizing a nuclear antigen
(Gerdes et al, 1987), our findings suggest that a loss of RB1CC1 expression
promotes breast cancer progression through disruption of its downstream
pathways that normally suppress proliferative activity.
Other studies have shown that Ki-67 staining levels are positively
correlated with tumor size and nodal involvement in breast cancer (Wintzer,
1991; Molino et al, 1997). The level of Ki-67 may be an independent prognostic
factor in breast cancer because Ki-67 positivity is correlated with
disease-free and overall survival rates (Rolio, 1993; Molino et al, 1997).
RB1CC1-negative cancers tended to show earlier metastasis than RB1CC1-positive
ones. In our series, however, we could not
conclude the sure relationship between RB1CC1 expression and the prognosis of
breast cancer due to the short period of clinical observation and small numbers
of cases. Therefore, longer-term clinical studies involving larger numbers of
patients are required to confirm this issue.
RB1CC1 expression and ER status were
positively correlated. Estrogen regulates the proliferation and maturation of
normal breast tissue through its receptors. In addition, both RB1CC1 and RB1
may contribute to the development and maturation of human embryonic cells (Chano, 2002d) and the RB1CC1-RB1 cascade may play a
role in the maturation of breast tissues involving functional ER expression. About
70% of primary breast cancers are ER-positive (Andersen and Poulsen, 1989; Harvey
et al, 1999) and such patients respond more favorably to endocrine therapy and
survive longer than those with ER-negative cancer (Andersen and Poulsen, 1989; Harvey
et al, 1999). Evaluating RB1CC1 expression in breast cancer may be helpful in
predicting responses to adjuvant therapy.
In conclusion, our findings suggest
that RB1CC1 plays an important role in the RB1 pathway and that the absence of
RB1CC1 expression accelerates cell proliferation in breast cancer. In addition,
RB1CC1 status may be an important prognostic factor in breast cancer.
Acknowledgements
We would like to thank H. Chen, N. Takashima, H. Honjo
and M. Sugimoto for excellent technical assistance.
This study was partially supported by grant-in-aids
for Scientific Research, the Ministry of Education, Science, Sports and
Culture, Japan (08671356, 10671249, 13470520, 13671380 and 15591340).
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Dr. Keiichi Kontani
