Abstract
Calcyclin-binding protein (CacyBP)/Siah-1 interacting protein (SIP), a component of ubiquitin-mediated proteolysis, could bind the Skp1-Cul1-F box protein complex. Although CacyBP/SIP was implicated in p53-induced β-catenin degradation, its exact function was still unknown. Our previous studies showed that CacyBP/SIP could modulate the multidrug-resistant phenotype of gastric cancer cells and was highly expressed in gastric cancer tissues compared with that in non-cancerous tissues. In this study, CacyBP/SIP protein expression profile in a broad range of human normal tissues and carcinomas was analyzed by immunohistochemistry staining with anti-CacyBP/SIP monoclonal antibody first produced in our laboratory. CacyBP/SIP was generally localized in the cytoplasm/nucleus. Positive staining of CacyBP/SIP was found in brain, heart, lymph node, and esophagus. Weak staining was shown in the rectum and kidney. No CacyBP/SIP was detected in other normal tissues. However, CacyBP/SIP was ubiquitously detected in all kinds of tumor tissues and was highly expressed in nasopharyngeal carcinoma, osteogenic sarcoma, and pancreatic cancer. To our knowledge, this is the first study on the CacyBP/SIP expression pattern in a broad range of human normal and tumor tissues. The data presented should serve as a useful reference for other investigators in future studies of CacyBP/SIP functions. Hopefully, this knowledge will lead to discovery of more roles of CacyBP/SIP in tumorigenesis. (J Histochem Cytochem 56:765–772, 2008)
Keywords: calcyclin-binding protein/Siah-1 interacting protein, immunohistochemistry, tissue distribution, tumor tissue, normal tissue
The calcyclin-binding protein (CacyBP) was named because it could interact with S100A6 (calcyclin) at a physiological range of Ca2+ concentration when it was first found in ascites tumor cells (Filipek and Kuznicki 1998). Three years later, Matsuzawa and Reed (2001) found that the human analog of mouse CacyBP interacted with Siah-1, and called this protein Siah-1 interacting protein (SIP). Therefore, it was named CacyBP/SIP.
Although CacyBP/SIP was initially identified as a novel target protein of S100A6, it seemed that it could bind other S100 proteins, such as S100A1, S100A12, S100B, and S100P, but not all of them (Filipek et al. 2002a).
Interestingly, CacyBP/SIP could be translocated into the nucleus and phosphorylated when Ca2+ concentration was changed in neurons and neuroblastoma NB-2a cells (Filipek et al. 2002b). This phenomenon has also been observed in retinoic acid–induced neuronal differentiation of neuroblastoma SH-SY5Y cells (Wu et al. 2003). However, the significance of CacyBP/SIP nuclear translation was unknown.
Matsuzawa and Reed (2001) found that p53 could induce β-catenin degradation through the Siah-CacyBP/SIP-Skp1/Cullin1/F-box (SCF) complex pathway, in which CacyBP/SIP associated with Skp1 and regulated its function. Unlike the SCF complex, where β-catenin was ubiquitinated in phosphorylated form, CacyBP/SIP changes the level of non-phosphorylated β-catenin. This suggested that CacyBP/SIP might be a component of a ubiquitination complex.
In previous studies, we found that CacyBP/SIP was overexpressed in the multidrug-resistant gastric cancer cells SGC7901/ADR compared with their parental cells SGC7901 (Zhao et al. 2002). Upregulation of CacyBP/SIP could enhance the resistance ability of gastric cancer cells to multiple chemotherapeutic drugs, whereas downregulation of CacyBP/SIP could partially reverse the drug-resistant phenotypes of gastric cancer cells (Shi et al. 2004). This was the first report of CacyBP/SIP associated with tumors. However, it was still unknown whether CacyBP/SIP was correlated with tumor development and how CacyBP/SIP was distributed in tumor tissue. To explorer its role in tumor development, monoclonal antibodies against CacyBP/SIP were produced in our laboratory, which possessed higher sensitivity and specificity (Zhai et al. 2006).
In this study, we conducted an immunohistochemical analysis of the CacyBP/SIP protein expression profile in a broad range of human normal tissues and cancers to further explore the possible role of CacyBP/SIP in the tumorigenesis of human tumors.
Materials and Methods
Tissue Microarrays
Commercially available adult human normal tissue arrays were obtained from Cybrdi (Xi'an, China). The tissue array (nc00-11-001) contained 33 points of a 1.5-mm-diameter disk of formalin-fixed, paraffin-embedded tissues representing histological normal organs from individuals 25–75 years of age. The following samples were absent from in the arrays, and additional cases were obtained and examined as standard histological sections: lymph node (five cases) and esophagus (six cases).
Tissue Specimens
Formalin-fixed, paraffin-embedded tumor tissue blocks were obtained from the archives of the Department of Pathology, Xijing Hospital, which is affiliated with the Fourth Military Medical University. The following tumors were studied: gastric adenocarcinoma (80 cases), colon adenocarcinoma (33 cases), rectum adenocarcinoma (20 cases), hepatocellular carcinomas (10 cases), lung carcinoma (40 cases), esophagus cancer (20 cases), thyroid carcinoma (13 cases), pancreas adenocarcinoma (10 cases), renal carcinoma (10 cases), prostate adenocarcinoma (10 cases), urothelial carcinoma (6 cases), ovarian carcinoma (6 cases), osteogenic sarcoma (8 cases), uterine cervix cancer (6 cases), mesoglioma of brain (10 cases), nasopharyngeal carcinoma (7 cases), melanoma (5 cases), and breast carcinoma (8 cases).
Antibodies and Reagents
Mouse monoclonal antibody (MAb) against CacyBP/SIP (clone BD1) was raised in our laboratory by immunizing with full-length recombinant hCacyBP/SIP expressed in E. coli and standard cell fusion techniques. The MAb BD1 could recognize CacyBP/SIP protein in both native and denatured forms (Zhai et al. 2006). The SP immunostaining kit (PV-6002 Power Vision Two-Step Histostaining Reagent) was from DAKO (Carpinteria, CA).
Immunohistochemistry
Immunohistochemistry was performed using the Histostain PV kit. Negative controls were conducted by replacing the primary antibody with preimmune mouse serum. Tissue microarray and tissue histological sections were deparaffinized in xylene and dehydrated through a graduated alcohol series before endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 10 min. Normal goat serum served as the blocking reagent for 1 hr at room temperature. Tissue sections were incubated with the anti-CacyBP/SIP antibody (1:150, initial concentration 2.1 mg/ml) at 4C overnight in a moist box; sections were exposed to PBS and treated with goat anti-mouse antibody–horseradish peroxidase (HRP) for 1 hr at room temperature, followed by additional washes with PBS. After washing, antibody binding was visualized by incubation with DAB for 5 min at room temperature. The slides were counterstained with hematoxylin and then counterstained with hematoxylin, dehydrated in a graded series of ethanol, cleared in xylene, and coverslipped.
The immunohistochemical stains were independently evaluated by two pathologists. Cytoplasm/nuclear staining was considered positive, and it was scored on the following basis: 0 (no detectable staining); 1+ (<25% positive cells); 2+ (25–49% positive cells); 3+ (50–74% positive cells); 4+ (>75% positive cells). In general, cases showing 3+and 4+staining also had strong intense staining, so intensity was not factored into the score. The list of tumors is shown in Table 1.
Table 1.
Immunohistochemical staining of cancers
| Staining pattern | 0 | 1+ | 2+ | 3+ | Subcellular localization |
|---|---|---|---|---|---|
| Gastric adenocarcinoma | 55 | 25 | 0 | 0 | Cytoplasm/nuclear |
| Colon adenocarcinoma | 16 | 17 | 0 | 0 | Cytoplasm/nuclear |
| Rectum adenocarcinoma | 15 | 5 | 0 | 0 | Cytoplasm |
| Hepatoma | 10 | 0 | 0 | 0 | No staining |
| Lung carcinoma | |||||
| Squamous carcinoma | 10 | 10 | 0 | 0 | Cytoplasm/nuclear |
| Adenocarcinoma | 11 | 9 | 0 | 0 | Cytoplasm/nuclear |
| Esophagus squamous carcinoma | 12 | 8 | 0 | 0 | Nuclear |
| Thyroid papillary carcinoma | 10 | 3 | 0 | 0 | Cytoplasm |
| Pancreatic adenocarcinoma | 3 | 4 | 3 | 0 | Cytoplasm |
| Renal clear cell carcinoma | 4 | 6 | 0 | Cytoplasm | |
| Prostatic adenocarcinoma | 7 | 3 | 0 | 0 | Cytoplasm |
| Bladder/ureter transitional cell carcinoma | 4 | 2 | 0 | 0 | Nuclear |
| Ovarian mucinous adenocarcinoma | 6 | 0 | 0 | 0 | No staining |
| Osteogenic sarcoma | 3 | 2 | 3 | 0 | Nuclear |
| Uterine cervix squamous carcinoma | 6 | 0 | 0 | 0 | No staining |
| Mesoglioma of brain | 7 | 3 | 0 | 0 | Cytoplasm/nuclear |
| Nasopharyngeal carcinoma | 2 | 1 | 2 | 2 | Cytoplasm |
| Melanoma | 5 | 0 | 0 | 0 | No staining |
| Breast adenocarcinoma | 3 | 5 | 0 | 0 | Cytoplasm/nuclear |
Results
CacyBP/SIP Immunohistochemical Staining in Normal Human Tissues
The degrees of CacyBP/SIP protein expression were determined by immunohistochemistry. Strong diffuse CacyBP/SIP staining was seen in neuron and neuralgia cells of the brain, myocardial cells of the heart, and squamous cells of the esophagus. Positive immunoreactions were also observed in the germinal center of the lymph nodes; the surrounding cells of the trabecula, postcapillary venule endothelia, and lymphocytes were negative. Weak staining was shown in the epithelium of the rectum and proximal and distal convoluted tubule epithelia of the kidney, but the cells of the glomerular epithelium and collecting tubule epithelia of the kidney were negative. No other normal tissues had detectable CacyBP/SIP staining, including the stomach, colon, liver, lung, testicle, prostate, and spleen. Figure 1 shows examples of CacyBP/SIP immunohistochemistry in brain and other normal tissues.
Figure 1.
Examples of calcyclin-binding protein (CacyBP)/Siah-1 interacting protein (SIP) immunohistochemistry in a normal tissue microarray. Arrows indicate sites of CacyBP/SIP expression. (A) Expression in brain sample. (Inset) Staining the neuron and neuralgia cells (arrows). (B) Expression in heart sample. (C) Strong expression in lymph node sample. (Inset) Staining the lymph cell (arrow). (D) Strong expression in the esophagus sample. (Inset) Staining the squamous epithelium (arrow). (E) Expression in the rectum sample. (Inset) Staining the rectal epithelium (arrow). (F–H) Examples of tissues where CacyBP/SIP was not expressed, including stomach, colon, and prostate. Bar = 50 μm.
CacyBP/SIP Immunohistochemical Staining in Human Tumor Tissues
Adenocarcinomas with cytoplasm/nuclear CacyBP/SIP staining included gastric adenocarcinomas (25 of 80, 31%), colon adenocarcinomas (17 of 33, 51%), rectum adenocarcinomas (5 of 20, 25%), prostatic adenocarcinomas (3 of 10, 30%), breast carcinomas (5 of 8, 63%), thyroid carcinomas (3 of 13, 23%), and lung adenocarcinomas (9 of 20, 45%). Pancreas adenocarcinomas showed strong diffuse immunoreactivity (7 of 10, 70%). Extensive adenocarcinomas staining for CacyBP/SIP were most commonly seen in nasopharyngeal carcinomas (five of seven, 71%). In other adenocarcinomas such as ovarian mucinous adenocarcinomas, there was no staining available.
CacyBP/SIP staining could also be seen in squamous carcinomas such as lung squamous carcinomas (10 of 20, 50%) and esophagus squamous carcinomas (8 of 20, 40%), but could not be seen in uterine cervix squamous carcinomas.
In other cell resource tumors, CacyBP/SIP could be stained in tumors of the transitional epithelium genesis, such as bladder/ureter transitional cell carcinomas (2 of 6, 33%), mesoglioma of the brain (3 of 10, 30%), and osteogenic sarcomas (5 of 8, 63%; Figures 2 and 3).
Figure 2.
Examples of CacyBP/SIP immunohistochemistry in cancers. (A) Nasopharyngeal carcinoma, showing diffuse cytoplasm staining. (B) Pancreas adenocarcinoma, showing diffuse cytoplasm staining. (C) Rectum adenocarcinomas, showing diffuse cytoplasm staining. (D) Thyroid carcinoma, showing diffuse cytoplasm staining. (E) Gastric adenocarcinoma, well differentiated, showing diffuse cytoplasm/nuclear staining. (F) Colon adenocarcinoma, somewhat differentiated, showing diffuse cytoplasm/nuclear staining. (G) Gastric adenocarcinoma, showing diffuse cytoplasm/nuclear staining. (H) Colon adenocarcinoma, showing diffuse cytoplasm/nuclear staining. Arrows indicate sites of inset. Bar = 50 μm.
Figure 3.
Examples of CacyBP/SIP immunohistochemistry in cancers. (A) Osteogenic sarcoma, showing diffuse nuclear staining. (B) Urothelial carcinoma, showing diffuse nuclear staining. (C) Mesoglioma of brain, showing diffuse cytoplasm/nuclear staining. (D) Melanoma, showing no staining. (E) Lung carcinoma, showing diffuse cytoplasm/nuclear staining. (F) Uterine cervix cancer, showing no staining. A–F are samples from tissue samples. Arrows indicate sites of inset. Bar = 50 μm.
Other tumor tissue types showed no or obscure staining of CacyBP/SIP, including hepatocellular carcinomas, melanomas, and ovarian carcinomas. Table 1 shows the results of CacyBP/SIP immunohistochemical findings for the complete set of tumors examined.
Discussion
In this study, we investigated the expression pattern of CacyBP/SIP protein in a large set of the human normal and tumor tissues. We found that CacyBP/SIP was expressed only in a few human normal tissues. Our results were consistent with data reported by Filipek and Kuznicki (1998) and Cheng et al. (2004), who showed that CacyBP/SIP was highly expressed in the brain and heart, whereas the lowest expression levels were in the stomach, liver, and spleen.
Unlike its distribution in normal tissues, CacyBP/SIP was widely expressed in tumor tissues. The immunohistochemistry staining showed that most tumor tissues exhibited positive staining, whereas negative staining was detected in the minority of tumors. Furthermore, CacyBP/SIP was weakly expressed or barely detected in most normal tissues, including the stomach and colon, but many tumor tissues exhibited positive staining such as gastric cancer and colon cancer cells. Therefore, we presumed that CacyBP/SIP functioned universally in most human tumors tissues, or it was satellite phenomena that needs further research to confirm. In addition, strong expression of CacyBP/SIP only appeared in nasopharyngeal carcinomas, osteogenic sarcomas, and pancreatic adenocarcinomas. Increased CacyBP/SIP expression in these tumor tissues might play important roles in tumorigenesis and might serve as a tumor marker in these tumors.
Because CacyBP/SIP is a target protein of S100A6, the ubiquitous distribution of CacyBP/SIP as an effecter of the S100A6 signal transduction pathway was reasonable because S100A6 was widely expressed in human tumor tissues and played a crucial role in cell survival, growth, and differentiation. Like CacyBP/SIP, calcyclin is mostly located in the cytoplasm and nuclear envelop and is often highly expressed in gastric cancer cells, hepatic cellular cancer cells, malignant melanomas, and neurogliomas (Wojda and Kuźnicki 1993). Moreover, its expression had a positive correlation with metastasis of malignant melanoma (Weterman et al. 1992). Apart from that, a new study found that CacyBP/SIP was a component of proteasome-mediated degradation. CacyBP/SIP could bind with SKP1, a component of F-box protein (SCF). Now, many studies had produced a large amount of evidence that SCF E3 ligases played an integral part in the highly ordered progression of the cell cycle and that their deregulation contributed to tumorigenesis (Bai et al. 1996). Therefore, combined with our results that CacyBP/SIP expression was increased in tumor tissues, we speculated that CacyBP/SIP may participate in tumor development through the S100A6 or ubiquitin degradation pathway. However, this speculation needs further study.
Our immunohistochemical results showed that CacyBP/SIP occurred in both the cytoplasm and nucleus and was further accumulated in the nucleoli. This was one of the prominent features of CacyBP/SIP distribution shown in this study. By analyzing the gene consequence, we found that CacyBP/SIP contained a non-classical nuclear localization signal (kkvktdt vlilcrkkve, aa residues 143–159). Filipek et al. (2002b) and Wu et al. (2003) confirmed that CacyBP/SIP could translocate to the nucleus on increased intracellular calcium concentrations. These phenomena agreed with our results that CacyBP/SIP translocated into the nucleus in gastric and colon cancer cell lines on increased intracellular calcium concentrations (data not shown). Because determining the distribution of CacyBP/SIP was the major purpose of this study, the subcellular location of CacyBP/SIP was not described here. However, the significance of CacyBP/SIP nuclear translocation evoked our interest and is now under study.
In conclusion, CacyBP/SIP protein expression was found in a variety of tissues, indicating that it was not a tissue-specific protein. We began the process of illuminating the function of this gene by describing here, for the first time, the expression pattern of CacyBP/SIP protein in a wide range of normal and tumor tissues. The data suggested that CacyBP/SIP may play important roles in tumorigenesis. Further studies will be necessary to examine the physiological and pathological roles of the CacyBP/SIP protein in these tissues. The data presented should serve as a useful reference for other investigators in future studies of CacyBP/SIP function. Hopefully, this knowledge will lead to the discoveries of more roles for CacyBP/SIP in tumorigenesis.
Acknowledgments
This work was supported by National Natural Science Foundation of China Grants 30530780 and 30572113.
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