Abstract
Background: The function of a cysteine-glutamate exchanger (xCT) transporter is to increase the intracellular concentration of glutathione in order to protect cells from oxidative stress. In several types of cancer, xCT is thought to play a role in the onset of resistance to chemotherapy and radiotherapy. xCT is stabilized on the tumor cell surface after combining with cluster of differentiation 44 variant (CD44v). Materials and Methods: We examined the xCT and CD44v6 expression in 304 primary tumor samples obtained from patients with colorectal cancer using immunohistochemical analysis. Results: Immunoreactivity for xCT was observed in 208 (68.4%) tumors. Among 218 patients with stage I-III disease who underwent curative surgery, the postoperative recurrence rate was 32.9% in those with xCT-positive tumors, which was significantly (p=0.003) higher than in those with xCT-negative tumors (10.7%). Immunoreactivity for CD44v6 was observed in 101 cases (33.2%), although the rate of postoperative recurrence in patients with CD44v6-positive tumors did not exhibit any significant correlation. Multivariate analyses revealed increased xCT expression to be an independent significant predictor of disease recurrence, in addition to depth of tumor invasion, lymph node metastasis and venous invasion.
Colorectal cancer (CRC) is a major cause of morbidity and mortality worldwide. This type of lesion accounts for over 9% of all cancers (1) and is the third most common type of cancer worldwide and the fourth most common cause of cancer-related death (2). Novel useful independent prognosticators for CRC have long been investigated; however, none have yet been integrated into routine practice, and prognosis remains unresolved with respect to CRC management. Consequently, although some improvements in survival of patients with CRC have recently been achieved due to advances in drugs used in chemotherapy, survival rates remain poor. Even after curative resection, tumor recurrence occurs in 17% of patients (3).
Currently, the prognosis of CRC is determined primarily based on tumor stage. According to the National Comprehensive Cancer Network (NCCN) guidelines for CRC, pathological high-risk factors for CRC recurrence include a tumor depth greater than pT4, dissection of fewer than 12 lymph nodes and poorly differentiated tubular adenocarcinoma; however, these factors do not exhibit adequate reliability for predicting recurrence (4).
xCT is the transporter subunit of the Na+-independent heterodimeric amino acid transport system xc. The xc system consists of xCT and a regulatory heavy chain component (4F2hc) and its function is to exchange cystine/glutamate with cystine entering cells in association with the release of glutamate in a 1:1 ratio. Cystine taken into the cell by xCT subsequently mediates the maintenance of the intracellular glutathione (GSH) level, which is essential for protecting cells from oxidative stress (5,6).
In contrast, cluster of differentiation 44 (CD44) is a major adhesion molecule in the extracellular matrix that functions in a wide variety of physiological processes, including leukocyte homing and activation, wound healing and cell migration, as well as tumor cell invasion and metastasis (7-9). CD44 exists in numerous variant isoforms generated through alternative mRNA splicing (10). Among them, variant type 6 (CD44v6) has been reported to be a cancer stem cell marker of CRC and its level is correlated with poor survival (11).
Ishimoto et al. showed that xCT is stabilized by combining with CD44 variants on the tumor cell surface (12). In human cancer cells, cystine uptake is largely mediated by the xc system, and a high expression of xCT has been demonstrated to be associated with a poor prognosis in a variety of human carcinomas, including Kaposi sarcoma, lymphoma, glioma and liver, breast, prostate, ovarian, pancreatic, gastric and esophageal cancer (13-17).
Although some studies have demonstrated a relationship between xCT expression and the prognosis of patients with various types of solid tumors, as far as we are aware, none investigated the prognosis in patients with CRC. Although CD44v6 has been reported to exhibit a relationship with prognosis in patients with CRC, its effects remain unclear (11). The aim of this study was, therefore, to investigate whether xCT and CD44v6 expression is associated with the prognosis of CRC and assess the potential of these variables as risk factors for CRC recurrence.
Patients and Methods
Patients. We enrolled 304 patients with CRC who underwent surgery at the Osaka City University Hospital, Japan, between 2001 and 2008. The characteristics of the patients are shown in Table I. The pathological diagnosis and classifications were made according to the seventh edition of the UICC TNM Classification of Malignant Tumors (18). The patients consisted of 171 males and 133 females, with a mean age of 65.8±10.0 years. Fifty-five patients were identified to have tumor of stage I, 84 of stage II, 79 of stage III and 86 of stage IV. For these patients, curative resection was performed in 218 (71.7%).
Immunohistochemical staining. Immunohistochemical determination of the xCT and CD44v6 levels in the colorectal cancer cells was carried out according to the manufacturer's instructions. In brief, the slides were de-paraffinized in xylene and hydrated in decreasing concentrations of ethyl alcohol. The sections were then de-paraffinized and incubated with 3% hydrogen peroxide in methanol for 15 min to block the endogenous peroxidase activity. The tissues were subsequently heated for 10 min at 105°C by autoclaving in Target Retrieval Solution (Dako, Carpinteria, CA, USA), and the sections were washed in phosphate-buffered saline (PBS) and incubated in 10% normal rabbit serum for 10 min to reduce non-specific antibody binding. The specimens were incubated with antibodies to xCT or CD44v6 overnight at 4°C, followed by two washes with phosphate buffer saline (PBS). The primary antibody used for the immunohistochemical detection of xCT was goat polyclonal antibody to xCT, and that for CD44v6 was mouse monoclonal antibody to CD44v6 (VEF18) (both Abcam, Cambridge, UK). The sections were incubated with biotinylated rabbit anti-goat immunoglobulin G for 10 min, followed by two washes with PBS. The slides were then treated with streptavidin-peroxidase reagent for five minutes and washed with PBS twice. Finally, the slides were incubated with diamino benzidine (DAB) kit (Histofine SAB-PO Kit; Nichirei, Tokyo, Japan) for 150 sec for xCT antibodies and 180 sec for CD44v6 antibodies, then counterstained with Mayer's hematoxylin and mounted.
Evaluation of immunopositivity for xCT and CD44v6. The entire invasive margin available for each specimen was analyzed while viewing the margin in a high-power field (×400). Positive xCT staining of the tumor gland duct was observed in the cytoplasm of the carcinoma cells (Figure 1). The amount of immunoreactivity for xCT was quantified based on the estimated percentage of immunopositive cells (0-19%, negative; 20-100%, positive). We used a cut-off value of 20% according to a previous study Shiozaki et al. (16). Positive CD44v6 staining of the tumor gland duct was clearly observed in the cell membrane of carcinoma cells (Figure 2). Immunoreactivity for CD44v6 was quantified as the percentage of immunopositive cells (0-24%, negative; 25-100%, positive). The cut-off value was set at 25% according to a previous study reported by Saito et al. (19). The slides were evaluated by two investigators without any knowledge of the corresponding clinicopathological data, and, when discordance was found, the cases were re-evaluated by both investigators until a consensus was reached.
Characteristics of the 304 patients with colorectal cancer.
Statistical analysis. Comparative analyses of the data were performed using Pearson's Chi-square test or binomial logistic regression analysis. The recurrence-free survival rate was calculated using the Kaplan–Meier method and analyzed according to Wilcoxon's test in order to compare the cumulative recurrence-free survival in the patient groups. Cox's proportional-hazards regression was used to compute the multivariate hazards ratios for the study parameters. A p-value of less than 0.05 was defined as statistically significant. JMP 10.0 software program (SAS Institute Inc., Cary, North Carolina, USA) was used for the analyses.
Results
Correlations between the xCT and CD44v6 expression and clinicopathological findings. Immunoactivity for xCT was observed in 208 (68.4%) tumors. There were no significant differences between the expression of xCT by age, gender, tumor diameter, tumor location, histological type, depth, lymph node metastasis, distant metastasis or clinical stage (Table II).
Lymphatic invasion was detected more frequently in patients with xCT-positive tumors than in those with xCT-negative tumors (74.0% vs. 60.4%, p=0.017). Similarly, venous invasion was more frequently observed in those with xCT-positive tumors than in those with xCT-negative tumors (29.8% vs. 17.7%, p=0.025).
Immunoreactivity for CD44v6 was observed in 101 (33.2%) tumors. However, there were no significant differences in the expression of CD44v6 by clinicopathological features of the patients.
Immunohistochemical evaluation of xCT-positive cells in colorectal cancer. The presence of xCT-positive cells was observed as dark brown staining in contrast to blue nuclear staining. Images are shown at the original magnification (A: ×100; B: ×400).
Correlation between xCT expression and relapse-free survival. Among 218 patients with stage I-III disease who underwent curative resection, postoperative recurrence was observed in 25.2% cases (55/218). Table III shows the correlations between xCT expression, clinicopathological features and disease recurrence. Male gender, greater depth of tumor invasion, lymph node metastasis, lymphatic vessel invasion, venous invasion and a positive xCT expression were significantly associated with disease recurrence.
The recurrence rate was 32.9% (47/143) in patients with xCT-positive tumors, which was significantly (p=0.003) higher than that observed in patients with xCT-negative tumors (10.7%, 8/75). On the other hand, the recurrence rate in patients with CD44v6-positive tumors was not significantly different from that observed in those with CD44v-negative tumors.
Figure 3 shows the recurrence-free survival sub-divided according to the expression of xCT, The recurrence-free survival was significantly shorter in the xCT-positive group than in the xCT-negative group (p=0.0004).
Moreover, the multivariate analyses using a Cox regression model revealed that xCT expression was an independent significant predictor of disease recurrence in addition to the depth of tumor invasion, lymph node metastasis and venous invasion (Table IV).
Discussion
To the best of our knowledge, there have been no reports investigating the correlations between the xCT expression and clinicopathological features in patients with CRC.
In the present study, we confirmed that xCT is expressed in CRC cells. However, there were no significant relationships between xCT expression and the clinicopathological features, except for lymphatic and venous invasion. However, xCT expression was found to be an independent significant predictor of recurrence in patients with CRC. Several previous studies have also reported that the expression of xCT is associated with poor survival in patients with solid malignancies, including esophageal squamous cell carcinoma (17), hepatocellular carcinoma (16) and glioblastoma (14).
Immunohistochemical evaluation of CD44v6-positive cells in colorectal cancer. The presence of CD44v6-positive cells was observed as dark brown staining in contrast to blue nuclear staining. All images are shown at the original magnification (A: ×100; B: ×400).
Kaplan–Meier estimates of recurrence-free survival by xCT expression in patients with stage I, II and III colorectal cancer. All patients were classified according to the Union for International Cancer Control (UICC) classification (18).
CD44v6 also plays an important role in tumor invasion and metastasis by regulating the extracellular matrix, promoting cell motility and suppressing tumor apoptosis (20). With respect to the correlation between CD44v6 expression and prognosis, Mulder et al. (21) and Todaro et al. (11) reported that a positive CD44v6 expression is indicative of poor prognosis in patients with CRC. Meanwhile Nanashima et al. (22) and Zlobec et al. (23) reported the CD44v6 expression to be a favorable prognostic factor. In the current study, CD44v6 expression was not found to be correlated with relapse-free survival in patients with CRC.
xCT combines with CD44 in order to be stabilized on the cellular membrane, which requires cystine to synthesize glutathione. An increased intracellular glutathione concentration reduces p38 Mitogen-activated Protein Kinase activation by inhibiting reactive oxygen species, thus preventing cell apoptosis. Therefore, xCT protects the cell from oxidative stress and thereby promotes cell growth (24, 25).
Correlations between clinicopathological features and the expression of xCT and CD44v6.
Yae et al. studied breast cancer samples and reported that epithelial splicing regulatory protein 1 (ESRP1), the splicing isoform that switches from CD44 standard to CD44v6, induces an increased cell surface expression of xCT and therefore promotes cancer metastasis (26).
Correlations between clinicopathological features and disease recurrence in the patients with stage I-III colorectal cancer treated with curative resection.
Multivariate analyses of the expression of xCT, clinicopathological findings and relapse-free survival in patients with colorectal cancer.
Sulfasalazine, a well-known drug for ulcerative colitis, is recognized to be a specific inhibitor of xCT-mediated cystine transport. Notably, Chen et al. reported the disruption of xCT by sulfasalazine inhibited metastasis and cell proliferation in nude mice and suppressed cell invasion and adhesion to the extracellular matrix in vitro (27). Based on these results, xCT is associated with cancer cell growth, invasion and metastasis, which result in poor survival. These observations suggest that xCT plays a role in cell growth, invasion and metastasis in the setting of CRC.
There might be contradiction in that xCT expression correlated with prognosis, but CD44v6, which stabilizes xCT on the cell surface, did not. Among the CD44 variant family, CD44v8-10 were particularly reported to combine with xCT (10), but the role of CD44v6 was unclear. CD44v6 may play another important role in cancer cell proliferation or metastasis regardless of xCT transporter. Further experiments are clearly needed.
In the present study, there was a correlation between xCT expression and disease recurrence. The rate of disease recurrence was significantly higher in patients with xCT-positive tumors than in those with xCT-negative tumors. Therefore, more intensive chemotherapy should be considered in cases involving xCT-positive tumors. In the adjuvant treatment of high-risk stage II or III colonic cancer, adding oxaliplatin to fluorouracil-plus-leucovorin (LV5FU2) was shown to significantly improve the prognosis in the MOSAIC trial (28). Such powerful combination therapies containing 5-fluorouracil and oxaliplatin are needed for patients with xCT-positive tumors.
Conclusion
In conclusion, xCT expression is associated with tumor recurrence in patients with CRC. Patients with xCT-positive tumors constitute a high-risk group for disease recurrence compared to those with xCT-negative tumors. Further studies are required to confirm the mechanism by which the xCT transporter functions in the setting of CRC recurrence.
There were no sources of financial support or relationships that may pose a conflict of interest with regard to this study.
- Received October 11, 2014.
- Revision received October 31, 2014.
- Accepted November 4, 2014.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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