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), differentiation (7), regeneration (8, 9), secretion (10, 11), and metastasis (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.

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 metastasized to the liver [13 (100%) of 13 cases] were mAbA6- t test. positive (Table 1; Fig. 3D). In normal colorectal tissues, smooth muscle of blood vessel (in most but not in all cases) and nerve bundle were strongly stained (Fig. 3F). RESULTS Comparison of the Staining Pattern between S100A6 Western Blotting of S100A6 in Human Colorectal Ad- and Ki-67 in Human Colorectal Adenocarcinomas. Fig. 4 enocarcinomas. We first subjected recombinant rabbit shows S100A6 and Ki-67 staining in a serial section of colo- S100A6, recombinant mouse S100A4, and human colorectal rectal adenocarcinoma. Ki-67 is a growth marker which is adenocarcinoma lysate to SDS-PAGE to examine whether the present in the nuclei (especially nucleoli) of growing normal and mAbA6 used in this study cross-reacts with human S100A4 or tumor cells, although its function is still obscure (18). S100A6 not. As shown in Fig. 1, A and B, S100A6 ran faster than staining was more intense in peripheral portion than in central S100A4 in SDS-PAGE, and mAbA6 reacted with human portion of the carcinoma (Fig. 4A), whereas Ki-67 staining S100A6, but it did not cross-react with human S100A4. pattern did not show such a tendency (Fig. 4B). Thirty-four Fig. 2A shows S100A6 and actin protein expression of the (89%) of 38 colorectal adenocarcinoma specimens were stained matched colorectal adenocarcinomas (T) and adjacent normal as mAbA6-positive in the peripheral portions, whereas 11 (29%) colorectal mucosa (N) from 10 patients. In 9 of 10 adenocarci- of 38 specimens were stained as mAbA6-positive in the central nomas, S100A6 levels were higher than those of normal mu- portions of the carcinomas (Table 3). This staining pattern was cosa. Average S100A6 level of adenocarcinomas was signifi- statistically significant (P Ͻ 0.0001). On the other hand, the

Fig. 2 Expression of S100A6 in human colorectal adenocarcinomas and adjacent normal mucosa. A, Western blot analysis of the S100A6 protein expression. Tissue extracts (1 ␮g) were run on SDS-PAGE under reducing conditions and blotted onto PVDF membrane. S100A6 immunoblot in normal mucosa (N) and adenocarcinoma (T)in matched samples from 10 patients (1—10) is shown (upper row). Actin immunoblot is also shown (lower row). B, comparison of average S100A6 expression levels in human normal colorectal mucosa and adenocarcinoma. “Normal” and “Tu- mor” indicate the means of densitometric ratio (S100A6:actin) of N and T samples, respectively, from 10 patients shown in A.

Table 1 mAbA6 reactivity in human colorectal normal and neoplastic cells (20)], it is premature to conclude that the higher expression epithelial cells examined immunohistochemically of S100A6 protein in adenocarcinoma specimens indeed reflects Normal and neoplastic epithelial tissues with more than 50% the expression in carcinoma cells themselves. To evaluate this stained cells were defined as mAbA6 positive. point and to examine the expression of S100A6 in adenocarci- mAbA6 reactivity noma cells more closely, we performed immunohistochemical Positive/Total % analyses using mAbA6. Normal colorectal mucosa and adenoma cells showed gran- Normal mucosa 2/42 5 Adenoma 6/13 46 ular staining localized at the supranuclear regions (Fig. 3, A and Adenocarcinoma 23/42 55 B). Furthermore, we also observed such a staining pattern in Liver metastasis 13/13 100 normal small intestinal mucosa (data not shown). A role for S100A6 in the process of mucus secretion in the epithelia that lines the gastrointestinal, respiratory, and urinary tracts and in the process of insulin release from the pancreatic ␤ cells was staining pattern of Ki-67 in peripheral portions of adenocarci- suggested (10, 11). Therefore, S100A6, which we observed in noma specimens was similar to that in central portions (Table 3). normal colorectal mucosa, may play a role in mucus secretion. In adenocarcinoma cells, S100A6 was stained more in- DISCUSSION tensely and diffusely in the cytoplasm (Fig. 3C). Such a staining Altered expression of S100A6 has been reported in several pattern was also observed in the tumor cells permeated in human neoplastic cells (19–23). No functional implications of lymphatic vessels and metastasized to the liver (Fig. 3, D and E), S100A6 in tumor development, however, have been established, whereas normal liver cells were not stained at all. As our results although biochemical studies have shown to specifically interact indicate in relation to clinicopathological factors, S100A6 ex- with annexins, tropomyosin, caldesmon, and other proteins (24– pression in colorectal adenocarcinomas was significantly asso- 29). Another link between S100 family members and tumorige- ciated with Dukes’ tumor status (i.e., nodal status) and lym- nicity comes from the location of the S100 gene cluster, because phatic permeation (Table 2). These results suggest that S100A6 the chromosome region 1q21 is frequently rearranged in various expression may be linked to the progression of colorectal neo- tumors, especially in human breast carcinomas (30). plasms. In the present study, S100A6 levels in human colorectal When normal and neoplastic epithelial tissues containing adenocarcinoma and matched normal mucosa were quantita- more than 50% stained cells were defined as mAbA6-positive, tively measured by Western blotting (Fig. 2A). The expression there was not so much difference in immunoreactivity between level was about 2.4-fold higher in adenocarcinomas than in adenoma (46%) and adenocarcinoma (55%) cells (Table 1). normal mucosa (Fig. 2B). Because S100A6 has been reported to However, when more than 10% stained cells were defined as be expressed in a variety of cell types [such as fibroblasts and positive, immunoreactivity was as follows: (a) normal mucosa 4 epithelial cells (31), nerve bundles, and blood vessel endothelial (10%) of 42; (b) adenoma 6 (46%) of 13; (c) adenocarcinoma 39

Fig. 3 Immunohistochemical S100A6 staining of human normal colorectal mucosa, adenomas, adenocarcinomas, metastatic nodules in the liver, and adenocarcinoma cells in a lymphatic vessel. Immunostaining was performed as described in “Materials and Methods.” A, normal mucosa; B, adenoma. Granular staining, localized at the supranuclear regions of epithelial cells, is seen in normal mucosa and adenoma. C, adenocarcinoma; adenocarcinoma cells are intensely and diffusely stained. D, metastatic nodule in the liver. E, adenocarcinoma cells in a lymphatic vessel (ly). F, nerve bundles (n) and blood vessels (b). A, B, C, D, and F: ϫ20; E: ϫ25; insets of A, B, and C: ϫ100.

(93%) of 42; (d) liver metastasis 13 (100%) of 13. Thus, the Interestingly, S100A6 was more intensely stained in pe- immunoreactivity in adenoma (46%) was less than in adenocar- ripheral portions with structural atypia or with deeply invaded cinoma (93%) and more than in normal mucosa (10%). In portions than in central portions with differentiated structure of colorectal tissues, several types of cells were stained with colorectal adenocarcinomas (Table 3; Fig. 4A). On the other mAbA6; smooth muscle cells of blood vessels and nerve bun- hand, the Ki-67 staining pattern was similar in these two por- dles invariably showed strong staining (Fig. 3F). The variation tions (Table 3 and Fig. 4B). These results were unexpected of S100A6 contents in normal mucosa specimens by Western because expression of S100A6 has been thought to be involved blotting may be derived from these immunopositive cells. in cell growth. However, a dissociation between S100A6 ex-

Fig. 4 Immunohistochemical staining of S100A6 and Ki-67 in human colorectal adenocarcinoma. Staining was performed as described in “Materials and Methods.” A, adenocarcinoma specimen stained with mAbA6; B, adenocarcinoma specimen stained with anti-Ki-67 antibody. A and B are closely adjacent serial sections. S100A6 is stained more intensely in the peripheral portion than in the central portion of the carcinoma, whereas Ki-67 does not show such a staining pattern. ϫ20.

Table 2 Relationship between S100A6 expression and Table 3 Relationship between S100A6 and Ki-67 staining in central clinicopathological factors and peripheral portions of human colorectal adenocarcinomasa S100A6 levela S100A6 Ki-67 Portion of Reduced Positive P tumor Reduced PositiveP Reduced Positive P Age (mean Ϯ SD, years) 62.8 Ϯ 8.3 60.1 Ϯ 10.3 NSb Central 27 11 23 15 Gender (male/female) 16/3 16/7 NS Peripheral 4 34 Ͻ0.0001 26 12 NS Dukes’ classification a Colorectal adenocarcinoma tissues with more than 50% stained A, B 17 9 cells were defined as positive and others as reduced. C 2 14 0.0025 b NS, not significant. Lymphatic permeation Absent 8 0 Present 11 23 0.0022 Vascular permeation Absent 6 4 stronger S100A6 staining in a higher percentage of positive cells Present 13 19 NS Tumor size (mm) is observed in the more advanced vertical growth phase of Ͻ40 9 12 human primary melanoma as compared with the early growth м40 10 11 NS phase of the melanoma (20). Our present observation showed Histological differentiation that all of the carcinoma cells in metastatic nodules in the liver Well 10 10 Moderate, poor 9 13 NS were mAbA6-positive (100%) when compared with primary a adenocarcinomas (55%) (Table 1). These results suggest the Colorectal adenocarcinoma tissues with more than 50% stained involvement of S100A6 in the progression and invasive process cells were defined as mAbA6 positive and others as reduced. b NS, not significant. of human colorectal adenocarcinoma cells. The immunohistochemical staining pattern of S100A6 in colorectal adenocarcinomas was similar to that of S100A4 reported by Takenaga et al. (33). They reported that the incidence pression and cell growth was reported by Gong et al. in human of S100A4 immunopositive cells increases according to the endometrial carcinoma cell lines (32). In these cell lines, phor- depth of invasion. However, in our study, S100A6 showed a bor esters inhibit cellular proliferation but enhance S100A6 granular staining pattern localized at the supranuclear regions of expression, which may result from the activation of protein epithelial cells in normal colorectal mucosa and adenomas, kinase C. We also observed that the deeply invaded adenocar- whereas Takenaga et al. reported that these epithelial cells are cinoma cells consisting of single cells were intensely stained not stained at all with anti-S100A4 antibody. Mandinova et al. with mAbA6 but not with anti-Ki-67 antibody at all. The infil- also showed a distinct subcellular localization of S100A6 and trating neutrophils and macrophages in the stroma were also S100A4 in human vascular smooth muscle cells (34). These mAbA6-positive (data not shown). Furthermore, Guo et al. observations suggest that S100A6 and S100A4 play different found elevated S100A6 expression in metastatic H-ras-trans- roles in the physiology of normal colorectal epithelial cells. formed NIH 3T3 cells as compared with nonmetastatic ones Although the staining pattern of these 2 proteins seemed to be (12). Weterman et al. showed that S100A6 expression is ele- similar in adenocarcinomas, their functional roles in the tumor vated in highly metastatic human melanoma cell lines as com- remain to be elucidated. Further work is required to provide pared with low metastatic ones (13). They also reported that a evidence for a causative role of S100A6 in invasive and meta-

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Keiko Komatsu, Akiko Andoh, Shingo Ishiguro, et al.

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