A Calcium-Binding Protein of the S100 Family, in Human Colorectal Adenocarcinomas1

172 Vol. 6, 172–177 January 2000 Clinical Cancer Research Increased Expression of S100A6 (Calcyclin), a Calcium-binding Protein of the S100 Family, in Human Colorectal Adenocarcinomas1 Keiko Komatsu,2 Akiko Andoh, Shingo Ishiguro, INTRODUCTION Noriko Suzuki,3 Hiroko Hunai, A number of S100-related low-molecular-weight calcium- Yoshiko Kobune-Fujiwara, Masao Kameyama, binding proteins have been identified in mammalian cells (1) Jun Miyoshi, Hitoshi Akedo, and and are thought to mediate calcium signals in normal and transformed cells. One such protein, S100A6, is preferentially Hiroyuki Nakamura expressed in proliferating rather than quiescent cells (2, 3). The Departments of Tumor Biochemistry Research Institute [K. K., A. A., human S100A6 gene (2, 4), which is located on chromosome Y. K-F., H. A., H. N.], Pathology [S. I., N. S., H. H.], and Surgery 1q21 (5), encodes an acidic 90-amino-acid protein (M 10.5) [M. K.], Osaka Medical Center for Cancer and Cardiovascular Diseases, r and Takai Biotimer Project ERATO, Japan Science and Technology containing two EF-hand motifs. The gene product has been Corporation [J. M.], Higashinari-ku, Osaka 537-8511, Japan implicated to be involved in growth of hair follicles (6), differ- entiation (7), regeneration (8, 9), secretion (10, 11), and metas- tasis (12, 13) in mammalian cells. ABSTRACT Colorectal cancer shows a clear step-wise progression from The expression of S100A6 (also known as Calcyclin/2A9/ normal through premalignant and malignant stages to the met- 5B10/PRA) in surgically resected human colorectal adenocar- astatic state. There has been progress in molecular genetic cinomas was examined to investigate whether S100A6 plays a analysis of colorectal tumorigenesis (14). In the present study, role in the malignancy of human tumor cells. Western blot we investigated the expression of S100A6 in surgically resected analysis using the lysates from colorectal adenocarcinomas and normal human colonic mucosa, adenomatous polyps, adenocar- adjacent normal mucosa from 10 patients revealed that the cinomas, and metastatic nodules in the liver to clarify its bio- average S100A6 level of adenocarcinomas was significantly logical relevance to the progression of human colorectal adeno- higher (about 2.4-fold) than that of normal mucosa. Immuno- carcinomas. histochemical analysis using formalin-fixed paraffin-embedded surgical specimens and monoclonal anti-S100A6 antibody MATERIALS AND METHODS (mAbA6) demonstrated that 2 (5%) of 42 normal mucosa and Surgical Specimens. Fresh human tissues (primary colo- 6 (46%) of 13 adenoma specimens were mAbA6-positive and rectal adenomatous polyp, primary adenocarcinoma, and adja- showed granular staining localized at the supranuclear regions cent normal colorectal mucosa from specimens resected for of epithelial cells, whereas 23 (55%) of 42 adenocarcinomas carcinoma and liver metastases) were collected from patients and 13 (100%) of 13 carcinoma cells that metastasized to the undergoing surgical resection in our hospital. The primary tu- liver were mAbA6-positive and showed diffuse cytoplasmic mors were staged according to Dukes’ classification system staining. A significant correlation between S100A6 expression (15). Surgical specimens were immediately stored at Ϫ80°C for and Dukes’ tumor stage or lymphatic permeation but not with Western blotting (10 specimens) or fixed with 10% formalin in other clinicopathological factors was shown. S100A6 was PBS for immunohistochemistry (42 specimens). stained more intensely in peripheral portions than in central Preparation of Tissue Extracts. Frozen surgical speci- portions of adenocarcinomas, whereas Ki-67 (a growth mens were thawed, minced with scissors, crushed in a solution marker) was stained equally in these two portions. These re- consisting of 0.05 M Tris-HCl (pH 6.8), 2% (w/v) SDS, 6% (v/v) sults suggest that S100A6 may be involved in the progression ␤-mercaptoethanol, and 10% (w/v) glycerol using polytron, and invasive process of human colorectal adenocarcinomas. and centrifuged at 15,000 ϫ g for 10 min. The supernatant was used immediately or stored frozen at Ϫ80°C for immunoblot analysis. SDS-PAGE and Western Blot Analysis. SDS-PAGE Received 5/11/99; revised 10/25/99; accepted 10/25/99. was performed as described by Laemmli (16). Protein samples The costs of publication of this article were defrayed in part by the were electrophoresed on 15% polyacrylamide gel under reduc- payment of page charges. This article must therefore be hereby marked ing conditions. The resolved proteins were electrophoretically advertisement in accordance with 18 U.S.C. Section 1734 solely to 4 indicate this fact. transferred to PVDF membrane (17). S100A6 and actin were 1 Supported in part by a grant-in-aid from the Ministry of Health and detected using monoclonal antibodies against pig S100A6 Welfare for a New 10-Year Strategy for Cancer Control, Japan. (mAbA6; Sigma, St. Louis, Mo) and pan-actin (Anti-Actin, 2 To whom requests for reprints should be addressed, at Department of Tumor Biochemistry Research Institute, Osaka Medical Center for Can- cer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan. Phone: 81-6-6972-1181; Fax: 81-6-6972-7749. 3 Present address: Third Department of Medicine, Osaka City University 4 The abbreviations used are: PVDF, polyvinylidene difluoride; Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan. mAbA6, monoclonal anti-S100A6 antibody. Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2000 American Association for Cancer Research. Clinical Cancer Research 173 Boehringer Mannheim, Mannheim, Germany), respectively. This mAbA6 cross-reacts with human, rabbit, and rat S100A6 and does not react with S100A2, S100a, and S100b. Polyclonal antimouse S100A4 antibody was kindly supplied by Dr. K. Takenaga. Antimouse IgG (H ϩ L) AP conjugate or antirabbit IgG (Fc) AP conjugate (Promega, Madison, WI) and BCIP/NBT Color Substrate (Promega) were used for alkaline phosphatase detection. Both S100A6 and actin protein expression levels were quantitatively estimated by densitometric scanning performed with NIH Image 1.55f. S100A6 protein concentration was nor- malized to actin level and expressed as densitometric ratio. Protein concentration was determined by Protein Assay (Bio- Rad, Richmond, CA). Fig. 1 The specificity of monoclonal anti-S100A6 antibody. The re- Immunohistochemical Staining. Four-␮m sections combinant rabbit S100A6 (50 ng), recombinant mouse S100A4 (50 ng), ␮ from formalin-fixed, paraffin-embedded tissues were mounted and human colorectal adenocarcinoma lysate (1 g) were run on SDS- PAGE under reducing conditions and blotted onto PVDF membrane. on poly-L-lysine-coated slides. They were then air-dried and The membrane was cut in half, and Western blotting was performed. A, deparaffinized. Endogenous peroxidase activity was blocked S100A6 immunoblot with mAbA6 (1:500); B, S100A4 immunoblot with 0.35% hydrogen peroxide in 50% methanol for 15 min at with anti-S100A4 antibody (1:1000). room temperature. The sections were rehydrated and washed with PBS. After blocking nonspecific binding sites with 2% normal horse serum in PBS for 30 min at room temperature, the cantly higher (about 2.4-fold; P ϭ 0.001) than that of normal sections were incubated with mAbA6 or monoclonal anti-Ki-67 mucosa (Fig. 2B). antibody (MIB-1, Immunotech, Westbroak, ME) in PBS con- Immunohistochemical Analysis of S100A6 Expression taining 0.1% BSA overnight at 4°C. After rinsing with PBS, the in Human Colorectal Adenocarcinomas. In order to exam- sections were incubated with biotinylated horse antimouse IgG ine the expression of S100A6 at the histological level, we (Vector, Burlingame, CA) for 30 min at room temperature performed immunohistochemical analysis. The staining was followed by washing with PBS. Immunoreactivity was detected abolished when an adjacent serial section was incubated with Ј with an avidin-biotin system (Vector) using 0.025% 3,3 -diami- mAbA6 that had been previously absorbed with excess recom- nobenzidine tetrahydrochloride as a chromogen for 2.5 min. The binant S100A6 protein and further abolished by incubating with sections were lightly counterstained with Mayer’s hematoxylin. normal mouse IgG1 (data not shown). Evaluation of Degree of Antibody Reactivity. The de- Two (5%) of 42 normal mucosa and 6 (46%) of 13 ade- gree of monoclonal anti-S100A6 or anti-Ki-67 reactivity with noma specimens showed mAbA6-positive and granular staining each tissue section was scored by the percentage of stained localized at the supranuclear regions of epithelial cells (Table 1; normal or neoplastic epithelial cells in the section. In this study, Fig. 3, A and B). In adenocarcinomas, 23 (55%) of 42 cases were normal and neoplastic epithelial tissues with more than 50% mAbA6-positive and diffusely stained in whole cytoplasms (Ta- stained cells were defined as “positive” and others (Ͻ50%) as ble 1; Fig. 3C). There was a significant correlation (P Ͻ 0.01) “reduced”. Three persons (K. K., A. A., and H. N.) independ- between S100A6 level and Dukes’ tumor stage or lymphatic ently judged the stained cells. permeation but no other clinicopathological factors (Table 2). Statistical Analysis. Correlations between positive ex- The carcinoma cells that invaded into lymphatic vessels were pression and clinicopathological factors were tested by the ␹2 immunopositive (Fig. 3 E). All of the carcinoma cells that test except for age parameter, which was assessed by Student’s

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