Colonic carcinoma is one of the most common cancers worldwide. It is the third most common type of cancer and the fourth most frequent cause of death because of cancer, with one million new cases occurring annually (Parkin et al., 2003). According to the Egyptian National Cancer Institute, colonic carcinoma accounts for 6.5% of all cancers in Egypt (El-Bolkainy et al., 2005).
The conventional prognostic factors for patient survival are histologic tumor grade (differentiation) and tumor stage (TNM, tumors/nodes/metastases, stages I–IV) (Stanley and Hamilton, 2000; Green et al., 2002; Kehoe and Khatri, 2006), which is based on the depth of tumor invasion, involvement of regional lymph nodes, and metastatic spread to other organs (Stanley and Hamilton, 2000). If metastasis has occurred, the 5-year survival of a patient after surgery decreases considerably from 90% to less than 10% (Kehoe and Khatri, 2006). Therefore, identification of novel biological markers related to tumor aggressiveness is required to identify high-risk patients who would benefit from adjuvant therapy and to identify new molecular targets for the development of novel treatments.
Disruption of the cell–cell junctions with concomitant changes in the expression of junctional proteins is a hallmark of cancer cell invasion and metastasis (Förster, 2008). Cell–cell adhesion in epithelial cell sheets is maintained mainly through adherens junctions (AJs) and tight junctions (TJs). AJs have been a major focus of cancer studies, and the expression and/or integrity of several AJ components, catenins and E-cadherin in particular, are altered or dysregulated in various carcinomas (Tsukita et al., 2001; Soini, 2005; Sheehan et al., 2007). In contrast to the roles of AJs proteins, the role of TJ proteins in cancer is less well understood. TJs are the most apical cell–cell contacts and are important for barrier function in epithelial and endothelial cells (Robenek et al., 1981; González-Mariscal et al., 2007). A number of integral membrane proteins associated with the TJ have been identified in recent years, including occludin (Jakab et al., 2010), junctional adhesion molecule (Jiang et al., 2004), and the claudin family consisting of at least 24 members (Swisshelm et al., 2005). Claudins are recently identified members of the tetraspanin family of proteins, which are integral to the structure and function of TJs (Robenek et al., 1981; Jiang et al., 2004; Swisshelm et al., 2005; González-Mariscal et al., 2007; Jakab et al., 2010). Recent studies have shown changes in the expression/cellular localization of claudins during tumorigenesis; however, a causal relationship between claudin expression/localization and cancer has not been established (Jiang et al., 2004; Swisshelm et al., 2005).
Using immunohistochemistry, claudin-1 expression in colonic carcinoma has been investigated and its relation with E-cadherin, proliferation marker kinase inhibitor (Ki-67) protein expressions, and with biological behaviors of colonic carcinoma is discussed in this study.
Materials and methods
A total of 30 patients (20 men and 10 women) with primary colonic carcinoma, who underwent surgery between 2000 and 2010 in the Suez Canal University Hospital, were enrolled in this study [mean±SD (range): 57.9±11.3 (18–82 years)]. None of the patients had received radiation therapy or chemotherapy before the operation. The histological findings, lymph node metastasis, and TNM stage were evaluated on the basis of the WHO classification of tumors.
Specimens were fixed in formalin, embedded in paraffin wax, cut into 4-µm-thick sections, and stained with hematoxylin and eosin.
The sections were immunostained with anti-rabbit polyclonal antibodies for claudin-1 (7.0 ml, ready to use; Labvision), monoclonal anti-E-cadherin (7.0 ml, ready to use; Labvision, USA), and Ki-67 (MB67, ready to use; NeoMarkers, USA) using the EnVision (USA) method. The sections were deparaffinized and heated in a microwave oven for 10 min to retrieve the antigens. After immersion in 3% hydrogen peroxide of 100% methanol for 10 min to block the endogenous peroxidase activity, the sections were incubated with primary antibodies for 60 min at room temperature, with EnVision for 20 min, and then immersed into a DAB solution. The sections were counterstained with hematoxylin, dehydrated, and mounted. Between steps, the sections were washed three times with PBS. As a negative control, PBS was used instead of the primary antibody. Two independent observers without knowledge of the clinical outcomes evaluated the immunohistochemical staining of sections until a complete agreement on the classification.
Immunohistochemical analysis of claudin-1, E-cadherin, and the Ki-67 labeling index
Claudin-1 was expressed in the cell membrane and/or the cytoplasm. The intensity of staining in the cell membrane and cytoplasm and the percentage of immunoreactive cells over the total tumor cells were evaluated. Immunoreactivity was scored according to the percentage of immunoreactive cells over the total tumor cells counted as 0 if less than 5% cells were stained, 1 if 5–25% cells were immunoreactive, 2 if 26–50% cells were immunoreactive, and 3 if more than 50% cells were immunoreactive.
Distinct membranous staining for E-cadherin was considered to be positive, irrespective of the staining intensity. Loss of expression was classified into three grades: mild, less than 1/3 of each tumor compartment, that is, the central part or the invasive front, showing loss of staining; moderate, 1/3–2/3 of each tumor compartment showing loss of staining; and marked, more than 2/3 of each tumor compartment showing loss of staining.
A positive expression of Ki-67 staining was found in the nuclei of carcinoma cells. The Ki-67 labeling index was defined as the ratio of immunoreactive cells over 1000 tumor cells.
The χ-test was used for univariable categorical analysis. All statistical analyses were carried out using SPSS 10.0 (SPSS, Inc., Chicago, Illinois, USA). A P value less than 0.05 was considered statistically significant.
The mean age±SD [range (years)] of the patients was 57.9±11.3 (18–82 years). A total of 20 men and 10 women were included in this study; 12 patients were right sided and 18 patients were left sided, with the reference point being the splenic flexure. No rectal or rectosigmoid cases were included in this study. The degree of tumor differentiation as defined by the AJCC25 was as follows: 14 poorly differentiated, four moderately differentiated, and 12 well differentiated; 12 mucinous; and 18 nonmucinous. Evidence of lymphatic or vascular invasion was present in 10 cases (Table 1).
Relation between claudin-1 expression and clinicopathological parameters of colonic carcinoma
Claudin-1 was mainly expressed in the cell membrane and/or the cytoplasm of colonic carcinoma cells (Fig. 1). The expression of claudin-1 was related to the histological type, degree of invasiveness, and lymph node metastasis of colon cancer (P<0.05). However, the expression of claudin-1 was not significantly related to the sex and age of colon cancer patients (Table 2).
The claudin-1 overexpression rate was the highest in mucinous adenocarcinomas and lower in poorly differentiated carcinomas than in well-differentiated to moderately differentiated carcinomas. It was significantly higher in the mucosa of patients in whom the tumors invaded the muscularis propria and visceral peritoneum, or lymph nodes than in the mucosa of patients in whom the tumors only invaded lamina propria or submucosa, or without lymph node metastasis.
The claudin-1 expression in the invasive front was different from that in the mucosa. The claudin-1 overexpression was the highest in well-differentiated to moderately differentiated carcinomas and the lowest in poorly differentiated carcinomas. The deeper the invasive depth, the higher the claudin-1 overexpression rate. The incidence of claudin-1 overexpression rate was 50% in the invasive front with the tumors invading the visceral peritoneum and significantly higher in patients with lymph node metastasis. The expression of claudin-1 was not related to the proliferation index (Ki-67) (Fig. 2) of colonic carcinoma cells.
Relation between E-cadherin expression and clinicopathological parameters of colonic carcinoma
E-cadherin expression was reduced in 16 (53.3%) of 30 cases (Fig. 3). The reduced expression of E-cadherin correlated with differentiation (P=0.0027), histopathological type (mucinous vs. nonmucinous) (P=0.0106), and advanced stage (P=0.0051). No statistically significant association was observed between reduced expression of E-cadherin and lymph node metastasis or distant metastasis (Table 3).
In the present study, reduced expression of claudin-1 was correlated with poor tumor differentiation, advanced TNM stage, and poor prognosis. In support of our data, reduced claudin-1 expression has been reported earlier in stage II colon cancer with recurrence and poor survival (Resnick et al., 2005). The claudin-1 overexpression rate was lower in poorly differentiated carcinomas than in well-differentiated to moderately differentiated carcinomas. These findings are in agreement with those of Matsuoka et al. (2011), Tzelepi et al. (2008), and Resnick et al. (2005), who reported that decreased expression of claudin-1 is significantly associated with higher tumor grade and poor differentiation. In addition, low-mRNA levels of claudin-1 were reported to be associated with poor tumor differentiation (Gröne et al., 2007; Ersoz et al., 2011). The results of our study together with other reports (Resnick et al., 2005; Gröne et al., 2007; Sheehan et al., 2007; Tzelepi et al., 2008; Ersoz et al., 2011; Matsuoka et al., 2011) suggest that the decrease in claudin-1 expression in colonic carcinoma of higher grade because of its downregulation might contribute toward the more aggressive behavior of the tumors.
The mechanisms underlying the decreased claudin-1 expression in colonic carcinoma remain unknown. It is not quite as clear as to how downregulation of claudins may contribute toward neoplastic progression (Higashi et al., 2007). However, it has been proposed that decreased expression of claudin-1 with the loss of cell polarity is followed by an abnormal influx of different growth factors, which induce autocrine and paracrine stimulations of neoplastic epithelia and thus provides nutrition factors and other factors necessary for tumor cell growth (Qun et al., 2009; Akasaka et al., 2010). Also, it has been proved that the injured structure and function of TJs might disturb the cell proliferation and differentiation and thus promote the carcinogenesis (Chang et al., 2010).
The effect of claudin loss on neoplastic progression through TJ dismantling is easy to comprehend. It has been shown that claudin-1 re-expression in vitro leads to apoptosis in breast cancer spheroids, suggesting yet another mechanism whereby claudin loss may lead to tumor progression (Dhawan et al., 2005). The role of TJ proteins in the development of cancer is not clear. Carcinoma cells, especially those with a higher potentiality of metastasis, frequently show loss of functional TJs, such as ZO-1, ZO-2, and occludin is decreased in tumor and its metastasis (Mullin, 1997; De Oliveira et al., 2005). The exact action of claudin on cancers is not clear. It has been reported that the expression of claudin-1 is decreased in invasive duct carcinoma of breast (Tokés et al., 2005), whereas the expression of claudin-3 and claudin-4 is increased in some other carcinomas (Morin, 2005; Sheehan et al., 2007). The expression of claudin-1 may promote the activation of pro-MMP-9 (Chao et al., 2009; French et al., 2009), suggesting that the expression of claudin-1 may involve the invasiveness and metastasis of adenocarcinoma. Caludin-1 is considered as a target site of β-catenin/Tcf signals, which supports that claudin-1 downregulates the formation of colorectal carcinomas (Takehara et al., 2009).
In the current study, E-cadherin expression was reduced in 14 (46.7%) of 30 cases. The reduced expression of E-cadherin correlated with differentiation (P=0.0027), histopathological type (mucinous vs. nonmucinous) (P=0.0106), and advanced stage (P=0.0051). No statistically significant association was observed between the reduced expression of E-cadherin and invasion depth or lymph node metastasis or distant metastasis. Reduced expression or loss of E-cadherin at invasive fronts was associated with poor tumor differentiation, consistent with previous studies of a large number of colorectal carcinoma cases (Miwa et al., 2000; Qun et al., 2009). Significant relationships have also been reported between loss of E-cadherin expression and poorly differentiated tumors and metastasis. In contrast, no apparent association has been reported between loss of E-cadherin expression and tumor grade, Dukes’ stage, and prognosis in other studies (Dhawan et al., 2005). The discrepancies might be attributed to the differences in the methodology or antibodies used.
In the current study, reduced expression of both claudin-1 and E-cadherin was observed in nonmucinous tumors, whereas overexpression of both was detected in the mucinous tumors. In concordance with a study that showed decreased claudin-1 and E-cadherin expressions in gastric carcinoma, diffuse type than intestinal type, it may be related to loss of cohesion (Soini et al., 2006). In terms of the correlation between claudin-1 and E-cadherin, some studies have reported that E-cadherin has been shown to influence the formation of TJs and desmosomes, although it mainly mediates the assembly of AJs (Gumbiner et al., 1988; Wheelock and Jensen, 1992). However, another study assumed that claudin-1 downregulates E-cadherin expression by upregulating the expression of ZEB-1. Claudin-1 activates Wnt and phosphotidylinositol-3-kinase/Akt signaling. ZEB-1 mediates claudin-1-regulated changes in cell invasion (Gröne et al., 2007). The expression of claudin-1 correlated with that of ZEB-1 in human colon tumor samples. In the progression from normal colonic epithelium to colon adenocarcinoma, the levels of E-cadherin decreased, whereas the levels of claudin-1 and ZEB-1 increased. Downregulation of E-cadherin and upregulation of ZEB-1 in colon tumors were associated with shorter survival times (Nollet et al., 1999; Lugli et al., 2007).
Disruption of cell adhesion molecules correlates with tumor differentiation and progression in colonic carcinomas. Specific marker profiles (reduced claudin-1 and E-cadherin) were identified here as independent prognostic indicators.
Conflicts of interest
There are no conflicts of interest.
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