J Cancer Res Clin Oncol (2011) 137:1213–1218 DOI 10.1007/s00432-011-0987-z
ORIGINAL PAPER
Expression of claudin 10 protein in hepatocellular carcinoma: impact on survival G. W. Huang • X. Ding • S. L. Chen L. Zeng
•
Received: 28 December 2009 / Accepted: 23 February 2010 / Published online: 7 June 2011 Ó Springer-Verlag 2011
Abstract Purpose To investigate the expression of claudin 10 in hepatocellular carcinoma (HCC) and the impact on angiogenesis and the postoperative survival of HCC patients. Methods The expression of claudin 10 protein was analyzed on samples from 99 HCC patients undergoing hepatectomy with immunohistochemistry and 31 fresh specimens with Western blotting. We examined the relationship between claudin 10 expression with clinicopathological factors, microvessel density (MVD), and postoperative survival. Results Western blotting and immunohistochemical staining showed that claudin 10 protein was highly expressed in HCC, compared with paraneoplastic liver tissue and normal liver tissue (P \ 0.01). Claudin 10 protein expression levels were significantly higher in HCC specimens with microscopic venous invasion (P \ 0.01). MVD in HCC increased with enhanced claudin 10 expression (P \ 0.01). Kaplan–Meier survival analysis revealed that HCC patients with high claudin 10 expression had significantly shorter overall survival (P = 0.01). Multivariate analysis showed that claudin 10 expression
G. W. Huang (&) Department of General Surgery, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, People’s Republic of China e-mail:
[email protected] X. Ding Department of Organ Transplantation, Xiangya Hospital, Central South University, Changsha 410008, People’s Republic of China S. L. Chen L. Zeng Department of Pathology, Hunan Provincial Tumor Hospital, Changsha 410008, People’s Republic of China
was an independent prognostic indicator for postoperative overall survival of HCC patients (P \ 0.01). Conclusions Claudin 10 protein is highly expressed in HCC tissue and is closely related to angiogenesis. Claudin 10 protein could be a useful marker to predict poor prognosis of HCC patients after hepatectomy. Keywords Prognosis
Claudin 10 Hepatocellular carcinoma
Introduction Hepatocellular carcinoma (HCC) ranks the second leading cause of cancer death among men in China and is also among the five leading causes of cancer death globally (He et al. 2005). Owing to advances in surgical techniques and perioperative care, immediate results of hepatic resection, which is one of the mainstays for curative treatment for HCC, have improved greatly (Fan et al. 1999). However, the long-term prognosis remains unsatisfactory and the postoperative 5-year survival rate remains as low as 20–50% (Hubert et al. 2007; Sherman 2008). Assessment of the prognosis of HCC remains difficult due to the limited prognostic value of conventional clinicopathological parameters. Thus, many different biological markers have been investigated with the aim of enhancing the accuracy of conventional prognostic factors (Wang et al. 2004; Huang et al. 2005, 2009; Mann et al. 2007; Wang et al. 2007). The claudins form the backbone of the tight junction strands, which play a crucial role in the maintenance of permeability of epithelial and endothelial cells. The claudin family is composed of 23 closely related transmembrane proteins. The expression of claudins frequently alters in
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various cancers. For example, claudin 1 has been found to be reduced in breast cancer and colon cancer (Tokes et al. 2005; Resnick et al. 2005). High expression of claudin 3 and claudin 4 in ovarian cancer has also been proven (Rangel et al. 2003). While their functions in cancer progression have not been completely elucidated, the role in angiogenesis has been suggested (Li et al. 2009). Claudin 10 has been shown to be involved in HCC invasion in vitro. Overexpression of claudin 10 conferred malignant phenotypes to HCC cell line, whereas knockdown of claudin 10 in HLE, an invasive cell line with high level of claudin 10 expression, abolished invasion (Ip et al. 2007). However, there has been no large-scale clinical report investigating the relationship between the expression of claudin 10 protein and angiogenesis or the postoperative survival of HCC patients. The present study attempts to show the expression pattern of claudin 10 protein in HCC and to investigate the relationship between claudin 10 expression and the clinicopathological features, including microvessel density (MVD) and the postoperative survival of HCC.
Materials and methods Patients and tissue specimens Ethics Committee of Central South University approved the study protocol. Ninety-nine patients with HCC who underwent hepatic resection at Xiangya Hospital between March 2001 and September 2007 were included in this retrospective study. No patients received other therapy before the operation. The 99 patients included 89 men and 10 women (median age, 53 years; range 19–75 years). Paraneoplastic liver tissue (PLT) was taken from noncancerous tissue 1 cm away from the tumor margin. Sixteen samples of normal liver tissue (NL) were taken from the liver tissue around the hepatic hemangioma. Among these 99 cases of HCC, matched fresh specimens of HCC and PLT from 31 cases were collected for Western blotting. All the sections were reevaluated and the diagnoses were confirmed. The clinicopathological variables were recorded, including preoperative serum alpha-fetoprotein (AFP) concentration, cirrhosis, Edmondson grading, tumor size, and microscopic venous invasion. Western blotting The total protein was extracted from fresh tissues. Briefly, 100 lg of total protein was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred onto PVDF membrane (Millipore). The blotting membranes were incubated with goat anti-human
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claudin 10 polyclonal antibody (Santa Cruz) and horseradish peroxidase-linked mouse anti-goat antibody (1:5,000 dilution; KPL, Gaithersburg, Md) in order. Beta-actin protein was also determined using the specific antibody (Sigma) as a loading control. All experiments were carried out in triplicate, and the individual level of claudin 10 protein was quantified by densitometry (Strategene) and the relative level of claudin 10 protein was conducted by comparison with the corresponding internal control. Immunohistochemistry Formalin-fixed, paraffin-embedded tissue specimens were obtained. Serial 4-lM sections were prepared, and one is stained with H&E. Sections were stained for claudin 10 and CD105 based on streptavidin–biotin–horseradish peroxidase complex formation mentioned before (Huang and Yang 2002). In brief, after deparaffinization and rehydration, endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxide for 30 min. Slides were treated with target retrieval solution. Primary antibodies included polyclonal anti-claudin 10 antibody (Santa Cruz) and monoclonal anti-CD105 antibody (Neomarkers). The peroxidase reaction was developed using diaminobenzidine, and slides were washed and mounted in mountant. Nuclei were lightly counterstained with hematoxylin. Negative controls were performed using phosphate-buffered saline (PBS; 0.01 M, pH 7.2) instead of the antibody. Two observers (Chen SL and Zeng L) who were blinded to clinical and follow-up data independently evaluated the staining results. The immunohistochemical results for claudin 10 were classified according to the number of positive cells as follows: -, no staining; ?, weak staining (less than 50%); and ??, strong staining (more than 50%). MVD was assessed by immunostaining for CD105. The immunostained sections were scanned at low magnification (940), and three tumor areas with the highest density of distinctly highlighted microvessels (‘hot spot’) within each section was selected for the quantitation of angiogenesis (Olympus microscope, Japan). All brownstained endothelial cells or endothelial cell cluster, which was clearly separate from connective tissue elements, was considered a microvessel. MVD was determined in the hot spot by counting all vessels at a total magnification of 9200. The mean value of the counted three fields from two independent observers was considered as the MVD of an individual tumor. Follow-up The follow-up period was defined as the interval between the date of operation and that of patient’s death or the last visit. Deaths from other causes were treated as censored
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cases; the follow-up time ranged from 60 to 1,400 days, with a median follow-up time of 440 days. Follow-up was performed through letter and telephone. Follow-up rate was 90.9% (90/99). Statistics Potential relationship between claudin 10 expression and clinicopathological variables or CD105-MVD was assessed using the nonparametric Mann–Whitney U test or analysis of variance (ANOVA) test, as appropriate. The Student t test was used to evaluate the results of Western blot analysis between groups. The association between claudin 10 expression and the overall survival was evaluated using survival analysis techniques. Kaplan–Meier survival curves were constructed and compared with the use of the log-rank test. A stepwise Cox proportional hazard model was then used to examine associations between the various prognostic factors and survival. For all tests, a P value of less than 0.05 is considered as significant. All statistics are calculated through SPSS 10.0 software (SPSS Inc., Chicago, Ill).
Fig. 1 Expression of claudin 10 in hepatocellular carcinoma (HCC) tissues, paraneoplastic liver tissue (PLT), and normal liver tissue (NL)
Results
Claudin 10 and MVD
Expression of claudin 10
CD105 was expressed in endothelium of HCC tissue in 96/99 (96.9%) specimens, but not in endothelium of PLT and NL (Fig. 2d). MVD in HCC increased with enhanced claudin 10 expression (P \ 0.01) (Fig. 3).
Claudin 10 protein was detected in 41.9% HCC tissues (13/31), 12.9% PLT (4/31) and 16.7% NL (1/6) through Western blotting, and the claudin 10 protein levels in HCC tissues was significantly higher than those in PLT and NL (0.80 ± 0.12 versus 0.29 ± 0.03 and 0.27 ± 0.02, P \ 0.01) (Fig. 1). Immunohistochemical staining of claudin 10 was detected predominantly in cytoplasm of cancer cells, and positive claudin 10 expression (Fig. 2a, b) was detected in 38.4% HCC tissues (38 of 99 samples), compared with faint homogenous expression in 10.1% PLT (10 of 99 samples) and 12.5% (2 of 16 samples) NL (P \ 0.01). (Fig. 2c). Association of claudin 10 expression with clinicopathological variables Correlation between the expression levels of claudin 10 in HCC and various clinicopathological variables are summarized in Table 1. Claudin 10 protein expression level was significantly higher in HCC specimens with microscopic venous invasion (P \ 0.01). There was no significant association between the expression of claudin 10 and other clinicopathological parameters, such as serum AFP concentration, cirrhosis, Edmondson grading, and tumor size (P [ 0.05).
Survival analysis The 1-, 3-, and 5-year overall survival rates of the 90 patients in the study are 85.2, 55.2, and 41.4%, respectively. The 1-, 3-, and 5-year overall survival rates are 90.1, 59.4, and 46.2% in claudin 10 negative group, 74.6, 55.3, and 36.9% in claudin 10 weakly positive group, and 53.0%, zero, and zero in claudin 10 strongly positive group, respectively. Our results indicated that the overall survival rate for the patients with claudin 10 strongly positive expression had significant shorter survival time compared with weakly positive expression and no expression (median survival, 420 days vs. 1,140 days and 1,500 days; P = 0.01, log-rank test; Fig. 4). Multivariate analysis of prognostic value of claudin 10 Table 2 shows the results of the multivariate analysis. Preoperative serum AFP concentration, cirrhosis, Edmondson grading, tumor size, microscopic venous invasion, MVD, and claudin 10 expression were included in the model as potential risk factors. Claudin 10 expression was a strong
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Fig. 2 a Strongly positive staining of claudin 10 in HCC tissue (original magnification 9200); b Weakly positive staining of claudin 10 in HCC tissue (original magnification 9200); c Faint homogenous
staining of claudin 10 in PLT (original magnification 9200); d Positive staining of CD105 in HCC tissue (original magnification 9200)
Table 1 Association of claudin 10 expression with clinicopathological variables in HCC
independent predictor of the patients’ prognosis; hazard ratios were 4.21 for overall survival, which were much higher than those associated with all other independent risk factors. In addition to claudin 10 expression, microscopic venous invasion and MVD were also significant risk factors for overall survival.
n
Claudin 10 expression -
?
P value
??
AFP concentration (ng/ml) 37
21
7
9
[50 Cirrhosis
B50
62
40
18
4
With
71
47
17
7
Without
28
14
8
6
I–II
62
37
19
6
III–IV
37
24
6
7
B50
35
19
9
7
[50
64
42
16
6
With
41
13
18
10
Without
58
48
7
3
0.19
Discussion 0.10
Edmondson grading 0.20
Tumor size (mm) 0.19
Microscopic venous invasion
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\0.01
Tumor cells frequently exhibit abnormal tight junction as well as cell polarity. Claudins form the backbone of the tight junction strands between cells. The expression of claudins has been found to be altered in several human cancers. For example, claudin 1 is upregulated in squamous cell carcinoma, and claudin 3 and claudin 4 are upregulated in ovarian and pancreatic cancer, respectively (Morita et al. 2004; Santin et al. 2004; Choi et al. 2007; Nichols et al. 2004). Because of the high specificity of claudin expression patterns in cancer, claudins have also been viewed as potential useful molecular markers for
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Fig. 3 Correlation between claudin 10 expression and microvessel density (MVD)
Fig. 4 Overall survival curve of patients with different claudin 10 expression
Table 2 Multivariate analysis of overall survival in HCC Factor
Hazard ratio (95% CI)
P value
Serum AFP
0.78 (0.39–1.10)
0.48
Cirrhosis
1.14 (0.81–1.40)
0.40
Edmondson grading
1.04 (0.95–1.12)
0.20
Tumor size
1.18 (0.65–1.74)
0.41
Microscopic venous invasion
2.84 (2.00–3.67)
\0.01
MVD
2.33 (1.82–2.84)
\0.01
Claudin 10 expression
4.21 (3.10–5.35)
\0.01
Claudin 10 is one of important members among claudin family. It has been shown to be involved in several cancers. Claudin 10 is found to be highly expressed in lung cancer cell lines and papillary thyroid carcinoma and downregulated in biliary tract cancer (Sugita et al. 2002; Aldred et al. 2004; Nemeth et al. 2009). Claudin 10 has also been shown to be involved in the progression of HCC. Ip et al. (2007) have shown that claudin 10 is functionally involved in HCC invasion. Overexpression of claudin 10 conferred malignant phenotypes to HCC cell line, whereas knockdown of claudin 10 in HLE, an invasive cell line with high level of claudin 10 expression, abolished invasion. Thus, claudin 10 may also be a candidate for HCC-targeted therapy. However, whether claudin 10 protein expression was prognostic for HCC has not been determined clinically. Sanada et al. (2007) have detected claudin 10 protein expression through immunohistochemistry in five of 12 cases of HCC. In such a small trial, no definite conclusion could be drawn about the prognostic value. Previous report has shown that claudin 10 could be used as a molecular marker for disease recurrence after curative hepatectomy for HCC concluded with quantitative RT–PCR methods (Cheung et al. 2005). The present study enrolled 99 HCC patients and further confirmed that claudin 10 protein is highly expressed in HCC and the expression of claudin 10 protein in HCC tissue was prognostic for poor survival. Thus, claudin 10 protein might be a useful marker for the prediction of poor prognosis of HCC. The role of claudins in cancer has not been fully understood. Traditionally, loss of claudins is interpreted as loss of cell adhesion and an important step in the progression of cancer to metastasis. Upregulation of claudins in many cancers means increase in motility and survival of malignant cells. Recently, claudins have been shown to play proangiogenic roles in certain cancers. In vivo and in vitro assays show that claudin 4-expressing cells produce factors that can stimulate angiogenesis (Li et al. 2009). The role of claudin 10 in regulating angiogenesis in HCC has never been studied before. The present study from clinical specimens showed possible association of claudin 10 with angiogenesis. The exact function of claudin 10 and its role in the regulation of angiogenesis in HCC deserve more exploration. In conclusion, claudin 10 is highly expressed in HCC tissue. Claudin 10 protein might be a useful marker to predict poor prognosis of HCC patients after hepatectomy and play important roles in the regulation of angiogenesis. Conflict of interest
None.
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