Download Clinicopathological Features of Gastric Cancer Cases and

Aberrantly
Expressed Recoverin in Gastric Cancer
Tohoku J. Exp. Med., 2004, 202,
213-219
213
Clinicopathological Features of Gastric Cancer Cases
and Aberrantly Expressed Recoverin
HIROSHI OHGURO, HIROKI ODAGIRI,1 YASUHIRO MIYAGAWA, IKUYO OHGURO, MUTSUO
SASAKI1 and MITSURU NAKAZAWA
Departments of Ophthalmology and 1Department of Surgery (Section 2),
Hirosaki University School of Medicine, Hirosaki 036-8562
OHGURO, H., ODAGIRI, H., MIYAGAWA, Y., OHGURO, I., SASAKI, M. AND NAKAZAWA,
M. Clinicopathological Feature of Gastric Cancer Cases and Aberrantly Expressed
Recoverin. Tohoku J. Exp. Med., 2003, 202 (3), 213-219 ── Cancer-associated
retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby
immunological reactions toward recoverin, a retina-specific calcium binding protein,
and other retinal antigens aberrantly expressed in tumor cells lead to the degeneration of retinal photoreceptor cells. Recently we reported that aberrant expression of
recoverin was identified in more than 50% of tumor cells and their cell lines from
several kinds of cancers, including gastric cancer, lung carcinoma, and other cancers.
To elucidate the clinicopathological significance of aberrantly expressed recoverin in
cancer cells, we performed immunocytochemical analysis using a monoclonal antibody against human recoverin. Within 18 patients with different clinical stages (I-IV)
of gastric cancer, the aberrant expression of recoverin in tumor cells was recognized
in 6 out of 18 patients (2 out of 2 stage IA, 1 out of 1 stage IB, 2 out of 3 stage II, 0
out of 7 stage IIIA, 0 out of 1 stage IIIB, and 1 out 4 stage IV). The present data are
consistent in part with the previous observations that recoverin-expressing cancer
cells induced tumor immunity and provide a favorable prognosis for primary cancer
in CAR patients. ──── cancer-associated retinopathy (CAR); recoverin; gastric
cancer; tumor immunity; immunocytochemistry
© 2004 Tohoku University Medical Press
Cancer-associated retinopathy (CAR) has
been identified as an ocular manifestation of
paraneoplastic syndrome (Sawyer et al. 1976;
Grunwald et al. 1985; Berson and Lessel 1988).
CAR is frequently found in patients with small
cell carcinoma of lung and other malignancies
(Kornguth et al. 1982, 1986; Keltner et al. 1983;
Buchanan et al. 1984; Klingele et al. 1984;
Grunwald et al. 1985), and is clinically characterized with retinitis pigmentosa-like photoreceptor
degeneration caused by an autoimmune reaction
toward recoverin, a photoreceptor specific calcium
Received August 27, 2003; revision accepted for publication February 6, 2004.
Address for reprints: Hiroshi Ohguro, Department of Ophthalmology, Hirosaki University School of
Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
e-mail: [email protected]
213
214
H. Ohguro et al.
binding protein, and other retinal antigens (Ohguro
and Nakazawa 2003). Recoverin is known to
play an important role in the light and dark adaptation by regulating rhodopsin phosphorylation and dephosphorylation in a Ca2+-dependent
manner (Kawamura 1991; Ohguro et al. 1996).
As a mechanism for the apoptotic cell death of
photoreceptor cells observed in CAR, it has been
postulated that anti-recoverin antibody generated
by unknown mechanisms in some cancer patients
penetrates into the photoreceptor cells via the
peripheral circulation and causes misregulation of
the phototransduction pathway by blocking recoverin function (Adamus et al. 1998; Maeda et al.
2001b). In the initial step of CAR pathogenesis,
several studies suggested that aberrant expression
of retina-specific recoverin, serving as a shared
antigen between tumor cells and retina, was critical since such aberrant expression of recoverin
had been identified in tumor cells in CAR patients
but not in non-CAR cancerous patients (Polans
et al. 1995; Matsubara et al. 1996; Yamaji et al.
1996). However, in contrast, we have recently
found that aberrant expression of retina-specific
recoverin occurred in more than 50% of several
kinds of cancer cells, and their cell lines obtained
from non-CAR cancerous patients (Maeda et al.
2000; Maeda et al. 2001a). Furthermore, we have
made the following experimental observations: 1)
immunofluorescence labeling by affinity purified
recoverin antibody revealed the immunoreactivity toward recoverin as a granular pattern within
the cancer cells (Maeda et al 2001b; Miyagawa
et al. 2003); 2) transfection with human recoverin
cDNA of A549 cells from human lung adenocarcinoma, which did not express recoverin, exhibited a significant reduction in cell proliferation
(Maeda et al. 2000); 3) significant changes were
observed in protein phosphorylation patterns in
cytosol of A549 cells upon addition of purified
recoverin in a calcium-dependent manner (Maeda
et al. 2001b) recoverin-specific cytotoxic T lymphocytes (CTL) exist in the peripheral circulation
of CAR patients, and these CTL can recognize
tumor-expressing recoverin (Maeda et al. 2001a).
These observations correspond well with the
favorable prognosis characteristic of malignant
tumors underlying CAR. Therefore, it is reasonable to think that aberrantly expressed recoverin
may play significant roles in the regulation of cell
proliferation of the tumors.
In the present study, to elucidate the clinical
significance of aberrant expression of recoverin in
patients with malignancies, the presence of recoverin in tumors was compared with clinical factors,
including clinical stages, presence or absence of
metastasis and prognosis. For this purpose, gastric cancer was studied since it is one of the commonest malignancies in Japan and its clinical and
pathological aspects have been most extensively
studied. Therefore, the data obtained herein will
provide a novel insight into the abenently expressed recover in other malignancies.
PATIENTS AND METHODS
The studies were performed in accordance with our institution’s guidelines and the
Declaration of Helsinki on Biomedical Research
Involving Human Subjects and protocols were
approved by the institution’s Committee for the
Protection of Human Subjects. After immunization of mice with human recoverin, prepared using
a pET-3d vector inserted human recoverin generously provided from Prof. Satoru Kawamura
(Osaka University, Osaka), anti-human recoverin
monoclonal antibody was raised by fusion of the
immune spleen cells harvested from mice with
highest titiers to human recoverin and screening the antibody-producing cells to get stable
hybridoma clones. Anti-human recoverin monoclonal IgG was purified by protein G Sepharose
column chromatography before use (Miyagawa
et al. 2003). The Kato III originated from gastric
cancer purchased from American Type Culture
Collection was basically established as described
previously (Maeda et al. 2000) and was maintained in RPMI 1640 containing 10% fetal bovine
serum and antibiotics.
Aberrantly Expressed Recoverin in Gastric Cancer
215
Patients
Gastric carcinoma tissues were obtained
from 18 patients who underwent surgery at
Hirosaki University Hospital. No retinopathy has
been associated with these patients. The definitions of stage grouping were made according to
the 13th edition of the Japanese classification
of Gastric Carcinoma (Japanese Gastric Cancer
Association 1999)
Western blot
Cancer cell line (Kato III) was solubilized in
0.5% Tween 20 in PBS and subjected to Western
blot analysis as described previously (Ohguro et
al. 1993).
Immunocytochemistry
Tumors, which had been surgically removed,
were fixed with 4% paraformaldehyde in 0.1
M phosphate-buffered saline (PBS) (pH 7.4),
embedded in paraffin and sectioned at 3-µm
thickness, mounted on subbed slides, and dried.
The sections were incubated with anti-human
recoverin monoclonal IgG at 1:100 in PBS for 1
hour and rinsed with PBS three times for 5 minutes each. Then the sections were incubated with
goat anti-human IgG labeled with FITC (Santa
Cruz Biotechnology Inc., Santa Cruz, CA, USA)
at 1 : 500 in PBS containing 0.035% Tween 20
(T-PBS) at room temperature for 1 hour. The
sections were then rinsed three times with PBS
for 5 minutes, and mounted on a glass slide using
Vectashield fluorescence mounting medium (Vector
Laboratories, Inc., Burlingame, CA, USA).
RESULTS
In the present study, we studied aberrant
expression of recoverin, a major CAR antigen in
tumor tissues obtained from18 surgically treated
patients with several clinical stages of gastric
cancer using monoclonal antibody to human
recoverin (anti-human recoverin mAb). In western blot analysis, a 26kDa prominent band was
recognized by this antibody in cell lysate from
Kato III cell lines established from gastric cancer
Fig. 1. Western blot analysis of monoclonal antihuman recoverin IgG.
Cell lysate of Kato III cells obtained by homogenization in 0.5% Tween 20 was subjected to
SDS-PAGE followed by western blot analysis.
Lane 1, protein staining of the cell lysate; lane
2, anti-human recoverin monoclonal IgG, (1st
antibody, 1 : 2000 dilution), and HRP labeled
anti-mouse IgG (2nd antibody, 1 : 3000 dilution).
(Fig. 1). Immunofluorecence labeling identified aberrant expression of recoverin in a total
of 6 patients among these 18 patients’specimens
(Representative positive and negative of the imunofluorescence lebeling in Table 1 are shown in
Fig. 2). In comparison to their clinicopathological features, the recoverin expression was more
often evident in tumor specimens obtained from
patients with early clinical stages (I and II) of
gastric cancer than in specimens from advanced
stages (III and IV) of this cancer (p=0.0011, Chisquare test), and was more often recognized in
tubular carcinoma (4 out of 9) and mucinous
216
H. Ohguro et al.
TABLE 1. Clinicopathological feature of gastric cancer cases and recoverin expression
Patient
Stage
Tumor size
(cm)
Pathology
Invasion
1) 40M
2) 68F
3) 64F
4) 32M
5) 46F
6) 48F
7) 70F
8) 74F
9) 70F
10) 65M
11) 76M
12) 46F
13) 72F
14) 60M
15) 57M
16) 44M
17) 63M
18) 68F
1A
IA
IB
II
II
II
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIB
IV
IV
IV
IV
4×3
1.7×0.7
2.5×2.5
3.5×3.5
6.2×6
7×4.5
4.5×3
3×2.8
4.2×4.5
7×7.5
8×9
6.5×6.5
2.2×2.4
10×7
3.8×3
14×10
8×7
4.5×4
tub2
tub1
tub2
tub2
por2
muc
tub2
tub2
por
por
tub2
por2
tub2
por2
tub2
muc
por
por2
sm
m
ss
mp
ss
se
ss
mp
ss
se
ss
se
sm
ss
se
se
se
se
Lymph node
Metastasis
invasion
0
0
0
N1
N1
0
N2
N2
N2
N1
N1
N2
N1
N2
N3
N2
N3
N3
−
−
−
−
−
Liver
Liver
−
Liver
Liver
−
Liver
−
−
Liver
−
−
−
Prognosis
Recoverin
Expression
Alive
Alive
Unknown
Alive
Alive
Unknown
Dead
Alive
Dead
Alive
Dead
Alive
Dead
Dead
Alive
Unknown
Dead
Alive
+
+
+
+
−
+
−
−
−
−
−
−
−
−
−
−
+
−
Clinical stage, pathology and regional limph node invasion were determined by Japanese Classification
Gastric Carcinoma by Japanese Gastric Cancer Association (The 13th edition). Pathology: tub, tubular
adenocarcinoma (tub 1, well differentiated type; tub 2, moderately differentiated type) ; por, poorly
differentiated adenocarcinoma (por 1, solid type; por 2, non-solid type); muc, mucinous adenocarcinoma.
Lymph node invasion. Prognosis was evaluated at 6 years after last surgical operation. Invasion: m, mucosa;
mm, muscularis mucosae; sm, submucosa; mp, muscularis propria; ss, subserosa; se, serosa exposed.
adenocarcinoma (1 out of 2) than poorly differentiated carcinoma (1 out of 7). Six years after
the surgical operations, 3 out of 4 and 6 out of
11 patients with recoverin positive and negative
tumors, respectively, were still alive.
DISCUSSIONS
It was identified that recoverin is aberrantly
expressed in the cancer cells or their cell lines
obtained from CAR patients, and this may trigger the autoimmune reaction causing progressive
retinal degeneration (Ohguro and Nakazawa
2003). Preliminary studies had revealed that such
aberrant expression of retinal specific recoverin is
not identified in cancer cells without retinopathy.
These observations suggested that aberrant expres-
sion of recoverin in cancer cells is an initial and
critical step in the cause of retinopathy. However,
in contrast, we found aberrant expression of recoverin in approximately 50% of cancer tissues
from non-CAR cancerous patients. This high rate
of recoverin expression, which is beyond the incidence rate of retinopathy, allowed us to speculate
that this aberrant expression must have additional
underlying roles that have been heretofore unknown. To elucidate functional roles of the aberrant expression of recoverin in cancer cells, we
transfected recoverin cDNA in A549 cells, which
is a lung adenocarcinoma cell line not originally
containing recoverin expression, and found that
this caused their proliferation to slow down
(Maeda et al. 2000). Furthermore, we also found
Aberrantly Expressed Recoverin in Gastric Cancer
217
Fig. 2. Immunofluorescence labeling for recoverin of gastric cancer tissue (Table 1, case 1, upper panel;
case 5, lower panel).
Sections were probed with anti-human recoverin mAb and FITC labeled anti-mouse IgG, and photographed with an immunofluorescence microscope as described in “Patients and Methods”.
that recoverin-specific cytotoxic T lymphocytes
(CTL) exist in the peripheral circulation of CAR
patients, and these CTL can recognize tumor-expressing recoverin (Maeda et al. 2001a). These
observations correspond well with the favorable
prognosis characteristic of malignant tumors
underlying CAR. Therefore, it is reasonable to
theorize that aberrantly expressed recoverin may
play significant roles in the regulation of cell proliferation of the tumors. So far, however, no study
has been available that examines the relationship
between recoverin expression and tumor prognosis. Here, to elucidate the clinicopathological significance of the recoverin expression in malignant
tumor, we studied recoverin expression in tumor
tissues obtained from18 surgically treated patients
with several clinical stages of gastric cancer, and
found aberrant expression of recoverin in a total
of 6 patients within this group (Fig. 1 and Table
1). This rate (33.3%) of recoverin expression in
gastric carcinoma is less than that (50%) recognized in several tumor cell lines. We assumed
218
H. Ohguro et al.
that the rate of former should be more clinically
relevant than the latter because natures of tumor
cell lines may be modified from the nature of the
original tumors during their cell culture steps.
Clinicopathologically, recoverin expression was
more often evident in tumor specimens obtained
from patients with earlier clinical stages (I and
II) and in tumors of lower malignancy pathology
type, tubular carcinoma type of gastric cancer.
The mechanisms responsible for the aberrant
expression of recoverin in cancer cells are still
unknown at present. However, functional roles of
recoverin, regulation of rhodopsin phosphorylation (Ohguro et al. 1996) in a calcium dependent
manner in photoreceptor cells, allowed us to speculate that recoverin may effect calcium-dependent
protein phosphorylation of some G-protein
coupled receptors by their specific kinases, GRK
(G-protein coupled kinase) homologous to RK
in cancer cells. In fact, we have recently found
that recoverin was coupled with caveolin-1 and
GRK 2, 5 and 6 within tumor cells, and suggested
that recoverin may be linked with caveolin-1 dependent regulations, such as tumor progression,
metastasis and drug resistance (Miyagawa 2003).
Considering the specific binding of caveolin and
G proteins, GRK and other proteins, the association of tumor-expressed recoverin with caveolin-1
and GRKs observed in our present study allowed
us to speculate that recoverin may have important
functional roles within tumor cells. In terms of
pathological roles of caveolin in cancer, Yang
et al. (1998) reported that caveolin-1 gene was
identified as a metastasis-related gene in the
mouse prostate cancer model using differential
display-polymerase chain reaction method. It
was also reported that elevated expressions of
caveolin-1 are recognized in specimens of human
patients with prostate and breast cancer (Lavie et
al. 1998). Thus, caveolin-1 is suggested to be a
novel prognostic marker for clinically confined
human prostate cancer. Furthermore, recent studies demonstrated a potential role of caveolin-1 in
the resistance of various malignant tumors against
multiple neoplastic agents (Lavie et al. 1998;
Yang et al. 1998). Although recoverin function in
tumor cells is unknown at present, recoverin may
potentially be linked with caveolin dependent
regulations of tumor progression, metastasis and
drug resistance with GRKs. Therefore, to better
understand the clinicopathological roles of aberrant expression of recoverin in malignant tumors,
further investigations, such as identification of
what G-protein coupled receptors are linked with
recoverin and GRKs will be next our project.
References
Adamus, G., Machnicki, M., Elerding, H., Sugden, B.,
Blocker, Y.S. & Fox, D.A. (1998) Antibodies
to recoverin induce apoptosis of photoreceptor
and bipolar cells in vivo. J. Autoimmun., 11,
523-533.
Berson, E.L. & Lessell, S. (1988) Paraneoplastic night
blindness with malignant melanoma. Am. J.
Opthalmol., 106, 307-311.
Buchanan, T.A., Gardiner, T.A. & Archer, D.B. (1984)
An ultrastructural study of retinal photoreceptor
degeneration associated with bronchial carcinoma. Am. J. Ophtahlmol., 97, 277-287.
Grunwald, G.B., Klein, R., Simmonds, M.A. & Kornguth, S.E. (1985) Autoimmune basis for visual
paraneoplastic syndrome in patients with smallcell lung carcinoma. Lancet, 1, 658-661.
Kawamura, S. (1991) Rhodopsin phosphorylation as
a mechanism of cyclic GMP phosphodiesterase
regulation by S-modulin. Nature, 362, 855-857.
Keltner, J.L., Roth, A.M. & Chang, R.S. (1983) Photoreceptor degeneration. Possible autoimmune
disorder. Arch. Ophthalmol., 101, 564-569.
Klingele, T.G., Burde, R.M., Rappazzo, J.A.,
Isserman, M.J., Burgess, D. & Kantor, O. (1984)
Paraneoplastic retinopathy. J. Clin. Neurol.
Ophthalmol., 4, 239-245.
Kornguth, S.E., Klein,R., Appen, R. & Choate, J.
(1982) Occurrence of anti-retinal ganglion cell
antibodies in patients with small cell carcinoma
of the lung. Cancer, 50, 1289-1293.
Kornguth, S.E., Kalinke, T., Grunwald, G.B., Schtta, H.
& Dahl, D. (1986) Anti-neurofilament antibodies in the sera of patients with small cell carcinoma of the lung and with visual paraneoplastic
syndrome. Cancer Res., 46, 2588-2595.
Lavie, Y., Fiucci, G. & Liscovitch, M. (1998) Up-regulation of caveolae and caveolar constituents in
multidrug-resistant cancer cells. J. Biol. Chem.,
Aberrantly Expressed Recoverin in Gastric Cancer
273, 32380-32383.
Maeda, A., Ohguro, H., Maeda, T., Wada, I., Sato, N.,
Kuroki, Y. & Nakagawa, T. (2000) Aberrant
expression of photoreceptor-specific calciumbinding protein (recoverin) in cancer cell lines.
Cancer Res., 60, 1914-1920.
Maeda, A., Ohguro, H., Nabeta, Y., Hirohashi, Y.,
Sahara, H., Maeda, T., Wada, Y., Sato, T., Yun,
C., Nishimura, Y., Torigoe, T., Kuroki, Y. &
Sato, N. (2001a) Identification of human antitumor cytotoxic T lymphocytes epitopes of
recoverin, a cancer-associated retinopathy antigen, possibly related with a better prognosis in
a paraneoplastic syndrome. Eur. J. Immunol.,
31, 563-572.
Maeda, T., Maeda, A., Maruyama, I., Ogawa, K.,
Kuroki, Y., Sabara, H., Sato, N. & Ohguro, H.
(2001b) Mechanisms of photoreceptor cell
death in cancer-associated retinopathy. Invest.
Ophthalmol. Vis. Sci., 42, 705-712.
Matsubara, S., Yamaji, Y., Sato, M., Fujita, J. & Takahara, J. (1996) Expression of a photoreceptor
protein, recoverin, as a cancer-associated retinopathy autoantigen in human lung cancer cell
lines. Br. J. Cancer., 74, 1419-1422.
Miyagawa, Y., Ohguro, H., Odagiri, H., Maruyama, I.,
Maeda, T., Maeda, A., Sasaki, M. & Nakazawa,
M. (2003) Aberrantly expressed recoverin is
functionally associated with G-protein coupled
receptor kinases in cancer cell lines. Biochem.
Biophys. Res. Commun., 300, 669-673.
Ohguro, H., Chiba, S., Igarashi, Y., Matsumoto, H.,
Akino, T. & Palczewski, K. (1993) Betaarrestin and arrestin are recognized by autoantibodies in sera from multiple sclerosis patients.
Proc. Natl. Acad. Sci. USA, 90, 3241-3245.
Ohguro, H., Rudnicka-Nawrot, M., Buczylko, J., Zhao,
X., Taylor, J.A., Walsh, K.A. & Palczewski,
219
K. (1996) Structural and enzymatic aspects
of rhodopsin phosphorylation. J. Biol. Chem.,
271, 5215-5224.
Ohguro, H. & Nakazawa, M. (2003) Pathological
roles of recoverin in cancer-associated retinopathy. In: Photoreceptor and Calcium, edited by
W. Baehr & K. Palczewski, Landes Bioscience,
Texas, USA, pp.109-124.
Polans, A.S., Witkowska, D., Haley, T.L., Amundson,
D., Baizer, L. & Adamus, G. (1995) Recoverin, a photoreceptor-specific calcium-binding
protein, is expressed by the tumor of a patient
with cancer-associated retinopathy. Proc. Natl.
Acad. Sci. USA, 92, 9176-9180.
Sawyer, R.A., Selhorst, J.B., Zimmerman, L.E. &
Hoyt, W.F. (1976) Blindness caused by photoreceptor degeneration as a remote effect of
cancer. Am. J. Ophthalmol., 81, 606-613.
Sobin, L.H. & Wittekind, C. (1997) International
union against cancer: TNM classification of
malignant tumors, 5th ed., NewYork, WileyLiss.
Yamaji, Y., Matsubara, S., Yamadori, I., Sato, M.,
Fujita, T., Fujita, J. & Takahara, J. (1996)
Characterization of a small-cell-lung-carcinoma
cell line from a patient with cancer-associated
retinopathy. Int. J. Cancer, 65, 671-676.
Yang, C.P., Galbiati, F., Volonte, D., Horwitz, S.B. &
Lisanti, M.P. (1998a) Upregulation of caveolin-1 and caveolae organelles in Taxol-resistant
A549 cells. FEBS Lett., 439, 368-372.
Yang, G., Truong, L.D., Timme, T.L., Ren, C.,
Wheeler, T.M., Park, S.H., Nasu, Y., Bangma,
C.H., Kattan, M.W., Scardino, P.T. & Thompson,
T.C. (1998b) Elevated expression of caveolin
is associated with prostate and breast cancer.
Clin. Cancer Res., 4, 1873-1880.