Abstract
Background: Karyopherin-α 2 (KPNA2) is a member of the importin α family and has recently been reported to play an important role in tumorigenesis and tumor progression. The aim of the current study was to elucidate the clinicopathological significance of immunohistochemical expression of KPNA2 in esophageal squamous cell carcinoma (ESCC). Patients and Methods: KPNA2 expression was investigated by immunohistochemistry in 116 surgically resected ESCC, and the association of KPNA2 expression with clinicopathologic features was also examined. Results: Sixty (51.7%) ESCCs demonstrated positive expression of KPNA2. Positive expression of KPNA2 showed a significant association with poor differentiation (p=0.015), tumor depth (p=0.001), lymphatic invasion (p<0.001), venous invasion (p<0.001), and tumor stage (p=0.008). Positive expression of KPNA2 was also significantly associated with Ki-67 labeling index (p=0.039). Univariate analysis revealed that the prognosis of the ESCC patients whose tumors demonstrated positive expression of KPNA2 was significantly poorer than that of those that did not (p=0.009). Multivariate analysis revealed that only tumor depth and the presence of lymph node metastasis, which are strong prognostic factors in ESCC, were independently associated with poor prognosis in this study. Conclusion: KPNA2 expression is associated with poor differentiation, tumor invasiveness, and tumor proliferation in ESCC.
Esophageal squamous cell carcinoma (ESCC) is one of the most difficult malignancies to cure. Its prognosis remains unsatisfactory despite significant advances in surgical techniques and perioperative management and the introduction of multimodality therapies (1).
Nucleocytoplasmic transport mechanisms have been reported to be involved in many cellular processes, such as gene expression, cell-cycle progression, and signal transduction (2). Modulation of the nuclear import of macromolecules is recognized to be important for changing cellular phenotypes during development and malignant cell transformation (3). Nucleocytoplasmic transport occurs through cylindrical structures spanning the nuclear envelope known as nuclear pore complexes (NPCs) (2). Although ions, small molecules, and small proteins (<20 kDa) can pass through NPCs by diffusion, NPCs restrict the passage of macromolecules (>40 kDa) to those bearing appropriate signals. The direction of transport through NPCs is determined by a signal known as the nuclear localization signal (NLS) (2), and nucleocytoplasmic transport is mediated by soluble receptors that recognize NLS in their cargoes. Most of these transport receptors are members of a large family of homologous proteins known as karyopherins or importins. In human cells, at least twenty-two importin β and six importin α proteins have been identified (2, 4). Proteins transported into the nucleus contain NLS, which are recognized by importin α/importin β heterodimers. Importin α recognizes and binds to the NLS, and importin β allows the passage of the complex through NPC. Importin α can also work independently by binding directly to nuclear cargoes without the help of importin β (5).
Karyopherin-α2 (KPNA2), which is also known as importin-α1 in humans, is a member of the importin α family. Although the function of KPNA2 has not been fully investigated, KPNA2 has recently been suggested to be a transporter of several tumor suppressors (6, 7). In breast cancer, nuclear protein expression of KPNA2 is significantly associated with higher tumor stage, positive lymph node status, higher tumor grade, negative estrogen and progesterone receptor status, and a higher Ki-67 labeling index (LI) (8, 9). Furthermore, KPNA2 expression was found to be significantly associated with poor survival and was demonstrated to be an independent prognostic factor in breast cancer (10). These previous studies indicated that KPNA2 plays a central role in tumor proliferation in breast cancer. In ESCC, the KPNA2 mRNA expression in clinical tissue specimens of primary ESCC tumors was reported to be higher than that in other normal organ cells (11). However, the clinicopathologic significance of KPNA2 expression in ESCC has not yet been determined. The aim of our study was to determine the relationship between KPNA2 expression and the clinicopathological features of ESCC. Additionally, in order to investigate its association with tumor proliferation, we also examined the relationship between KPNA2 expression and Ki-67 LI in ESCC.
Patients and Methods
Patients and samples. Surgical specimens were obtained from 116 ESCC patients (108 males and 8 females) who had undergone potentially curative surgery at the Gunma University Department of General Surgical Science between 1997 and 2007 after obtaining their written informed consent. The ages of the patients ranged from 40 to 83 years with a mean of 62.6 years. The median follow-up period for survivors was 31 months (range: 2-113 months). The pathological features of the specimens were classified based on the 6th edition of the TNM classification of the International Union Against Cancer. None of the patients had received irradiation or chemotherapy prior to surgery, nor did any of them have hematogenic metastases at the time of surgery. Postoperative chemotherapy and/or radiation therapy were not performed until tumor recurrence was confirmed by a radiologic or endoscopic examination. Prior to the analysis, resected specimens were fixed with 10% formaldehyde, embedded in paraffin blocks, cut into 4-μm thick sections, and mounted onto glass slides.
Immunohistochemistry. Immunohistochemistry was performed by the standard streptavidin-biotin peroxidase complex (S-ABC) method, as described previously (12). Each 4-μm thick section was deparaffinized, rehydrated, and incubated with fresh 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature to block endogenous peroxidase activity. After rehydration through a graded series of ethanol treatments, antigen retrieval was carried out in 10 mM citrate buffer (pH 6.0) at 97°C, and then the sections were cooled to 30°C. After rinsing the sections in 0.1 M phosphate-buffered saline (PBS; pH 7.4), non-specific binding sites were blocked by incubation with 10% normal rabbit serum for 30 minutes. The sections were then incubated with the goat anti-KPNA2 polyclonal antibody (SC6917; Santa Cruz Biotechnology, USA) at a dilution of 1:100 in PBS containing 1% bovine serum albumin at room temperature for 1 hour. Negative controls were obtained by replacing the specific primary antibody with PBS. The sections were washed in PBS, incubated with biotinylated anti–goat IgG for 30 minutes at room temperature, and finally incubated in streptavidin-biotin peroxidase complex solution (Nichirei Co., Tokyo, Japan). As a chromogen, 3,3′-diaminobenzidine tetrahydrochloride was applied as a 0.02% solution containing 0.005% hydrogen peroxide in 50 mM ammonium acetate-citrate acid buffer (pH 6.0). The sections were then lightly counterstained in Mayer's hematoxylin and mounted. Immunohistochemistry for Ki-67 was performed as described previously (13).
Evaluation of immunostaining for KPNA2 and Ki-67. As KPNA2 labeling index (LI), the percentage of nuclear stained cells was calculated by examining at least 500 cells in four representative and intensely stained areas. After calculating the KPNA2 LI (median 10.7%; range 0-44.3%), all cases were classified into positive (KPNA2 LI ≥10.7%) or negative (KPNA2 LI <10.7%) categories. The Ki-67 LI was calculated as the percentage of nuclear-stained cells for each section on the basis of 1,000 tumor cell nuclei and was counted in the area with the maximum number of positive nuclei, as previously described (13).
Statistical analysis. Fisher's exact test, the chi-square test, Wilcoxon's signed-rank test, and the Kruskal-Wallis test were used to compare the clinicopathologic data. Kaplan-Meier curves were generated for overall survival, and statistical significance was determined using the log-rank test. Univariate and multivariate survival analyses were carried out using the Cox proportional hazards regression model. A probability value of <0.05 was considered statistically significant. All statistical analyses were performed using the JMP5.0 software (SAS Institute Inc., Cary, NC, USA).
Results
Expression of KPNA2 in ESCC tissues. The expression of KPNA2 was investigated in 116 ESCC by immunohistochemistry. Sixty (51.7%) ESCCs showed positive expression of KPNA2, and 56 (48.3%) did not. Representative results of the immunohistochemistry for KPNA2 in normal and ESCC tissue samples are shown in Figure 1. In normal esophageal epithelia, no KPNA2 expression was apparent (Figure 1A). Although, weak to moderate cytoplasmic staining was found in 26 (22.4%) ESCCs, KPNA2 immunostaining showed a predominantly nuclear staining pattern in ESCC (Figure 1B,C), as described in other organs (8).
Association of KPNA2 expression with the clinicopathological features of ESCC. The relationships between KPNA2 expression and ESCC clinicopathological features are shown in Table I. KPNA2 expression showed significant association with poor differentiation (p=0.015), tumor depth (p=0.001), lymphatic invasion (p<0.001), venous invasion (p<0.001), and tumor stage (p=0.008). To assess the association between KPNA2 expression and proliferation, we examined the association between KPNA2 expression and the Ki-67 LI. The mean KI-67 LI in the KPNA2-positive patients was significantly higher that that in the KPNA2-negative patients (p=0.039).
Immunohistochemical staining of normal esophageal epithelia and esophageal squamous cell carcinomas for KPNA2. A: KPNA2 expression in the normal epithelium (original magnification: ×200). B: Negative expression of KPNA2 in well-differentiated ESCC (original magnification: ×200). C: Positive expression of KPNA2 in poorly differentiated ESCC (original magnification: ×200).
Relationship between KPNA2 expression and clinicopathological features.
Prognostic significance of KPNA2 expression in ESCC patients. The overall survival rate of the ESCC patients whose tumors demonstrated positive expression of KPNA2 was significantly lower than that of the ESCC patients that did not (p=0.009; Figure 2). The five-year overall survival rate of the ESCC patients with tumors which demonstrated positive expression of KPNA2 was 41.6%, whereas that of those with negative KPNA2 expression was 62.3%. In univariate analysis, positive expression of KPNA2 was a significant prognostic factor for poor survival (p=0.009) in addition to tumor depth, the presence of lymph node metastasis, and distant metastasis. However, multivariate analysis of the four factors found to be significant in univariate analysis showed that positive expression of KPNA2 was not an independent prognostic factor for poor survival (p=0.252) (Table II).
Overall survival of ESCC patients according to their KPNA2 expression status. The survival rate of the ESCC patients whose tumors demonstrated positive KPNA2 expression was significantly lower than that of those that did not.
Discussion
In the present study, we investigated the clinicopathological significance of KPNA2 expression in ESCC based on the proposition that nucleocytoplasmic transport mechanisms might be associated with the biological behavior of ESCC. In terms of the protein expression of KPNA2 in human tumors, a few studies have demonstrated its association with clinicopathological features and patient survival (8-10). To the best of our knowledge, this is the first study to investigate KPNA2 protein expression in ESCC.
The role of KPNA2 in cancer remains unclear. However, KPNA2 has recently been suggested to be a transporter of several tumor suppressors (6, 7). NBS1 is the product of the NBS (Nijman breakage syndrome) gene. Nijman breakage syndrome is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, and growth retardation (14-16). In addition to its role in DNA double-strand break repair in the nucleus (15, 16), cytoplasmic NBS1 expression is reported to be directly activated by c-MYC and might contribute to transformation and tumorigenesis through the activation of phosphatidylinositol 3-kinase (PI3K)/Akt in head and neck squamous cell carcinoma (17-19). Thus, NBS1 has a dual role as a tumor suppressor and a promoter of tumorigenesis based on its subcellular localization. KPNA2 is thought to decide the role of NBS1 by determining its cellular localization (20). Chk2 is a nuclear protein kinase involved in checkpoint arrest in response to DNA damage. Activation of Chk 2 and phosphorylation of its substrates induces checkpoint arrest at multiple cell cycle phases (21-25). KPNA2 is reported to interact with Chk2 and contribute to its nuclear import (7). The possible interaction of KPNA2 with these molecules indicates that KPNA2 might be involved in antitumor activity.
On the other hand, KPNA2 has recently been suggested to be associated with tumorigenesis. In a previous investigation of KPNA2 protein expression in 83 breast tissue samples, the proportion of KPNA2-positive cases increased successively in a comparison of adjacent histologically benign breast tissues, ductal carcinoma in situ (DCIS), and invasive carcinomas (8). Furthermore, KPNA2 expression is significantly less frequent in cases involving low-grade DCIS compared to those cases displaying adjacent high-grade DCIS (8). In the aforementioned investigation, KPNA2 was suggested to have a potential role in tumorigenesis. Additionally, an association between KPNA2 expression and tumor progression has also been reported in several tumors. KPNA2 overexpression is significantly associated with poor prognosis in melanoma (26). In breast cancer, several studies have reported an association between KPNA2 expression and tumor stage, lymph node status, higher tumor grade, negative estrogen, progesterone receptor status, and poor outcome (8-10). KPNA2 expression has a significant association with the basal-like and Her-2/neu molecular subtype (9), which is thought to have higher malignant potential and is associated with worse prognosis in patients with breast cancer (27). In the current study, positive expression of KPNA2 was significantly associated with poor tumor differentiation, tumor depth, lymphatic invasion, venous invasion, and tumor stage in ESCC patients. In Ki-67 immunohistochemistry, our study showed that the proliferative activity of tumors with positive KPNA2 expression was significantly higher than that of tumors that demonstrated negative KPNA2 expression, which is consistent with the findings of a previous study (9). Poor differentiation is associated with poor prognosis in ESCC (28, 29). Furthermore, it is well known that the proliferation of tumor cells plays important roles in tumor progression. Thus, KPNA2 expression might be related to progressive behavior of ESCC.
In our present study, positive expression of KPNA2 was associated with poor survival in patients with ESCC. However, multivariate analysis revealed that KPNA2 expression was not an independent prognostic factor. The close association between KPNA2 expression and tumor depth might have resulted in KPNA2 showing no independent prognostic significance for ESCC. It is also presumed that the presence of lymph node metastasis, which is a strong prognostic factor for ESCC but has no significant association with KPNA2 expression, greatly influenced the prognosis of the patients in this study.
Univariate and multivariate analysis of survival in 116 patients with ESCC.
As the functional role of KPNA2 in human cancer, including ESCC, has not been clarified, the use of clinically targeting KPNA2 remains unclear. Previously, KPNA2 expression was shown to be a marker of chemoresistance in breast cancer (9). As KPNA2 expression in ESCC was found to be related to tumor progression, which is inconsistent with the results of other studies in breast cancer, KPNA2 expression might provide useful information for designing therapeutic strategies for ESCC. However, it is clear that further studies are needed to elucidate the role of KPNA2 and its clinical application for the treatment of ESCC.
In conclusion, immunohistochemical KPNA2 expression is associated with poor differentiation, tumor invasiveness, and tumor proliferation in ESCC. KPNA2 expression is also associated with poor prognosis in ESCC.
Footnotes
- Received September 5, 2009.
- Revision received January 15, 2010.
- Accepted January 15, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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