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Clinical correlation of calpain-1 and glypican-3 expression with gallbladder carcinoma

Gallbladder carcinoma (GBC) possesses a poor prognosis, which is primarily attributed to the lack of early and timely surgical intervention. Calpain-1 and glypican-3 have been implicated in the progression of various types of cancer. The present study aimed to detect the expression of calpain-1 and glypican-3 in GBC, and analyzed whether the expression levels of these proteins correlated with any clinicopathological variables. A total of 100 patients with GBC and 30 patients with cholecystitis who accepted surgical treatment were enrolled in the present study. Pathological and clinical data were obtained from all patients. The expression of calpain-1 and glypican-3 was detected in paraffin-embedded tissues by immunohistochemistry. Calpain-1 expression was manually assessed with an immunohistochemical H-score with a slight modification. Glypican-3 expression was assessed as negative and positive. The correlations between protein expression and clinicopathological characteristics, and the associations between the proteins were analyzed. All patients exhibited positive expression of calpain-1. Notably, the high expression rate of calpain-1 was significantly increased in patients with GBC, compared with patients with cholecystitis (32.0 vs. 6.7%; χ2=7.668; P=0.006), suggesting that calpain-1 expression may be associated with progression from cholecystitis to GBC. In addition, the positive rate of glypican-3 expression was 53.0% in patients with GBC and 63.3% in patients with cholecystitis, with no significant difference (χ2=0.997; P=0.318). Furthermore, the expression of calpain-1 and glypican-3 had no significant correlation with gender, age, degree of tumor differentiation and tumor-node-metastasis classification in patients with GBC. Notably, the expression of calpain-1 and glypican-3 displayed a significant positive correlation in patients with GBC (r=0.517; P<0.01), but a significantly negative correlation (r=-0.856; P<0.01) in patients with cholecystitis. In conclusion, calpain-1 expression may be associated with progression from cholecystitis to GBC. Combined detection of calpain-1 and glypican-3 may be beneficial for prognosis assessment of GBC.


Introduction
Gallbladder carcinoma (GBC) is the most commonly observed malignancy of the biliary tract, representing 80-95% of all the cases of biliary tract cancer worldwide, and is the sixth most frequent malignant neoplasm of the digestive tract (1). In 2012, ~76,844 patients were diagnosed with GBC, and ~60,334 patients succumbed to disease (2). Currently, the overall mean survival time for patients exhibiting GBC is 6 months, and the 5-year survival rate is 5% (3). A positive clinical outcome may depend on early and timely surgical resection of GBC (4). However, >90% of patients do not undergo surgical resection due to the advanced stage of their tumors at the time of diagnosis. Invasion of adjacent organs or distant metastases is observed in patients with advanced GBC, and almost 50% of them exhibit lymph node metastasis (5,6). Thus, it is essential to identify novel prognostic biomarkers and therapeutic targets for the treatment of GBC.
The mammalian calpain protease family comprises intracellular Ca 2+ -regulated cysteine proteases that mediate regulatory cleavage of specific substrates (7). Calpains are involved in various physiological functions, including cell differentiation, transcriptional regulation, cytokine processing,

Clinical correlation of calpain-1 and glypican-3 expression with gallbladder carcinoma
cell cycle, signal transduction, migration, apoptosis and protein renewal during growth and tissue regeneration (7,8).
In addition, the calpain family, including calpain-1, has been observed to be involved in the progression of cancer (8,9). The expression of calpain-1 has been reported to be associated with relapse-free survival in patients with breast cancer treated with trastuzumab following adjuvant chemotherapy (8), and was also correlated with increased malignancy in renal cell carcinoma (10). Glypican-3 is a cell surface protein that attaches to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor (11). Glypican-3 is able to combine with Wnt molecules to form a complex, thereby promoting cancer growth via stimulation of canonical Wnt signaling (12). It has been reported that glypican-3 is able to regulate developmental growth by interacting with the Hedgehog signaling pathway (13). Previous studies have revealed that mutated glypican-3 lacking its GPI anchor domain is able to block Wnt signaling and inhibit the growth of Wnt-dependent tumors (14,15). Additional reports have demonstrated that glypican-3 expression is involved in various human malignancies, including hepatocellular carcinoma (HCC) (16), melanoma (17), ovarian clear cell carcinoma (18), yolk sac tumor (YST) (19), neuroblastoma, hepatoblastoma and Wilms' tumor, among others (20,21).
However, to the best of our knowledge, the expression of calpain-1 and glypican-3 in GBC has not been investigated thus far. In the present study, the expression of calpain-1 and glypican-3 was detected in 100 patients with GBC and 30 patients with cholecystitis by immunohistochemistry, and the correlations between calpain-1 and glypican-3 expression and certain clinicopathological characteristics of the patients were analyzed.

Materials and methods
Clinical samples. The present study was performed according to REporting recommendations for tumor MARKer prognostic studies (REMARK) criteria (22). A total of 100 patients with GBC and 30 patients with cholecystitis who accepted surgical treatment between January 2007 and December 2011 in The First Affiliated Hospital, School of Medicine, Zhejiang University (Hangzhou, China) were enrolled in the study. Written informed consent was obtained from the patients prior to commencement of the study. The present study was approved by the Ethics Review Committee of The First Affiliated Hospital, School of Medicine, Zhejiang University (reference number 2014-332). The inclusion criteria of the patients with GBC were set as follows: i) The postoperative pathological diagnosis was GBC; ii) no radiotherapy or chemotherapy had been administered prior to surgery; iii) no comorbid diseases were present; and iv) complete pathological and clinical information was available, including age, gender, degree of tumor differentiation, tumor-node-metastasis (TNM) classification (23) and presence of distant metastases. The detailed clinicopathological variables of the patient cohort are presented in Table I.
Assessment of staining was conducted by scanning the slides with an inverted microscope (BX41; Olympus Corporation, Tokyo, Japan) at magnification, x200. The expression of calpain-1 in tumor and cholecystitis cells was manually assessed by immunohistochemical H-score, with a slight modification to the method previously described (8). Staining intensity was assessed as negative (0), weak (1), medium (2) or strong (3) over each stained area. The stained area score was assessed as <50% (1) or ≥50% (2). H-scores were calculated by multiplying the stained area score by the staining intensity score (H-score range, 0-6). Calpain-1 H-score was dichotomized into low and high immunoreactivity groups using X-Tile Software (http://medicine.yale.edu/lab/rimm/research/software. aspx), and correlated with clinicopathological criteria. The expression of glypican-3 in tumor and cholecystitis cells was manually assessed as negative ( -) or positive ( + ). A total of 50% of the slides were examined by a second independent assessor who was blinded to the scores and clinicopathological criteria, and good concordance existed between the two scorers (single measure intraclass correlations, >0.8). An average H-score was generated by calculating the mean of 10 random high-power fields. Average scores were utilized for analysis due to the relatively small sample size.
Statistical analysis. The data distribution was assessed using the Kolmogorov-Smirnov test for goodness of fit. The correlation between protein expression and clinicopathological characteristics was analyzed with Pearson's χ 2 test of association or Fisher's exact test in a 2x2 table. Spearman's rank-order correlations were performed to investigate the associations between various proteins. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS software version 17.0 (SPSS Inc., Chicago, IL) for Windows (Microsoft Corporation, Redmond, WA, USA).

Results
Positive immunohistochemistry results are observed for calpain-1 and glypican-3. Tissue expression of calpain-1 and glypican-3 was investigated in patients with GBC and patients with cholecystitis. Representative staining patterns of calpain-1 expression in GBC and cholecystitis tissues are presented in Fig. 1. Cytomembrane and cytoplasmic staining was observed for calpain-1, with certain granularity and heterogeneity between adjacent tumor cells and adjacent cholecystitis cells, varying from weak to strong staining. According to the computational formula of H-scores, the different intensities of calpain-1 expression (ranging from weak to strong) corresponded to distinct H-scores (ranging from 2 to 6) in the GBC

Differential distribution of calpain-1 and glypican-3 expression is observed in patients with GBC and patients with cholecystitis.
H-score cut-offs were as follows: 0, negative expression; 1-3, low expression; and 4-6, high expression. All 100 patients with GBC and 30 patients with cholecystitis presented positive expression of calpain-1, thus exhibiting a 100.0% positive expression rate for this protein. Of the 100 patients with GBC, 32 exhibited high expression levels of calpain-1 and 68 exhibited low expression levels, resulting in a high expression rate of 32.0%. Of the 30 patients with cholecystitis, 2 exhibited high expression levels of calpain-1 and 28 exhibited low expression levels. Therefore the high expression rate for this protein was 6.7%. Pearson's χ 2 test demonstrated that the high expression rate of calpain-1 was significantly increased in patients with GBC, compared with patients with cholecystitis (χ 2 =7.668; P=0.006; Table II).  Table II).

No significant associations exist between calpain-1 and glypican-3 expression and various clinicopathological variables in patients with GBC.
The correlation of calpain-1 expression with clinicopathological variables in patients with GBC was analyzed using Fisher's exact (2x2) test, as shown in Table III   The correlation of glypican-3 expression with clinicopathological variables in the patients with GBC presented a similar pattern to that mentioned above for calpain-1 expression (Table III) Fisher's exact test indicated no significant difference (χ 2 =1.474; P=0.225). With regard to TNM classification, 11 patients presented positive glypican-3 expression and 6 negative for stages I+II, while 42 patients were positive and 41 were negative for stages III+IV. Fisher's exact test indicated no significant difference (χ 2 =1.127; P=0.288). The results of the present study suggested that the expression of glypican-3 had no significant correlation with gender, age, tumor differentiation degree and TNM classification in patients with GBC. Table IV,     3 patients demonstrated independent high expression of calpain-1 and 24 patients demonstrated independent positive expression of glypican-3. Spearman's rank-order correlations indicated that the expression of calpain-1 and glypican-3 in patients with GBC presented a significantly positive correlation (r=0.517; P<0.01).

Discussion
GBC always results in advanced disease with invasion of adjacent organs or distant metastases at the time of presentation, primarily attributed to its relatively low incidence and unclear symptomatology, thereby leading to poor prognosis and reduced survival rates (24). Previous reports have demonstrated that several risk factors including age, parity, gender, obesity and ethnicity may be associated with GBC (24,25). The initiation and development of GBC may be due to a wide range of etiologies, including infectious and environmental exposure to chemical carcinogens, mechanical obstruction via gallstones, autoimmune disease, polyps, adenomas and anatomical variations such as pancreaticobiliary malfunction (24,25). Although significant progress has been made to identify potential prognostic biomarkers for GBC, this disease remains an uncommon and challenging malignancy with an overall poor prognosis (25).
Carbohydrate antigen 19-9 and carcinoembryonic antigen are the most commonly used clinical biomarkers in GBC (26). However, they are frequently increased in the advanced stages of the disease with a low specificity, and therefore, they are not generally used independently for GBC prognosis (25). Previous studies have demonstrated that calpain-1 may be involved in the progression of certain types of cancer, including breast cancer (8) and renal cell carcinoma (10). Furthermore, a number of reports have indicated that glypican-3 expression may be involved in various types of cancer, including HCC (16), melanoma (17), ovarian clear cell carcinoma (18) and others (20,21). However, to the best of our knowledge, the expression of calpain-1 and glypican-3 in GBC has not been investigated to date.
In the present study, the expression of calpain-1 and glypican-3 was detected in 100 patients with GBC and 30 patients with cholecystitis by immunohistochemistry, and the correlations between calpain-1 and glypican-3 expression and the clinicopathological characteristics of the patients were analyzed. It was identified that all patients presented positive expression of calpain-1. Notably, the high expression rate of calpain-1 in patients with GBC was markedly increased compared with that observed in patients with cholecystitis. Furthermore, the expression of calpain-1 and glypican-3 had no significant correlation with gender, age, degree of tumor differentiation and TNM classification in patients with GBC. Notably, calpain-1 and glypican-3 expression presented a significantly positive correlation in patients with GBC, but a significantly negative correlation in patients with cholecystitis.
The results of the present study are interesting in the light of previous studies highlighting the role of calpain-1 in the progression of cancer (8,27,28). Kulkarni et al (27) investigated the role of calpain-1 in trastuzumab-treated human epidermal growth factor receptor 2 (HER2) + breast cancer in vitro. The authors demonstrated that calpain-1 was activated following trastuzumab treatment, and subsequently cleaved HER2, thus disrupting signaling, while trastuzumab-resistant cells were dependent on calpain-1 activity for survival (27). Storr et al (8) reported that calpain-1 expression was associated with relapse-free survival, and proposed that calpain-1 expression may be a useful biomarker for the prediction of relapse-free survival in patients with breast cancer treated with adjuvant trastuzumab and chemotherapy. An additional study by Storr et al (28) demonstrated that the expression of calpain-1 and calpastatin were associated with various clinicopathological features, including tumor grade and estrogen receptor expression, which was verified in an independent cohort of patients. Furthermore, Storr et al (29) investigated the protein expression levels of calpastatin and calpain-1, -2 and -9 in surgically excised gastroesophageal adenocarcinomas derived from patients treated with neoadjuvant chemotherapy and in tumors that had not been previously exposed to chemotherapy, and identified that expression of the calpain system was associated with poor clinical outcomes. Therefore, the authors proposed that calpain-1, calpain-2 and calpastatin may be clinically relevant prognostic biomarkers in gastroesophageal adenocarcinoma (29). In addition, Storr et al (30) indicated that the expression of these proteins was significantly associated with carcinoma of the pancreas, bile duct and ampulla, and influenced the progression of disease.
The potential mechanisms by which the calpain family participates in cancer progression are associated with a number of interrelated signaling pathways (31). Integrin engagement is able to induce focal adhesion kinase (FAK) phosphorylation, resulting in extracellular signal-regulated kinase activation of calpain-1 to cleave FAK, which subsequently enhances cell motility (32). FAK, like phosphatidylinositol (3,4,5)-trisphosphate 3-phosphatase, may be dephosphorylated by phosphatase and tensin homolog, which is indicative of signaling pathway overlap (32). These interrelated signaling pathways synergistically contribute to the progression of various types of cancer (31,33,34). In the present study, all patients with GBC presented positive expression of calpain-1, suggesting that calpain-1 expression may be associated with GBC. However, the expression of calpain-1 had no significant correlation with gender, age, degree of tumor differentiation and TNM classification in these patients, which suggested that calpain-1 may be a potentially useful biomarker for GBC prognosis. Notably, the results of the present study demonstrated that the high expression rate of calpain-1 in patients with GBC was markedly increased compared with that observed in patients with cholecystitis (32.0 vs. 6.7%; P=0.006). Thus, it may be speculated that the expression of calpain-1 is associated with progression from cholecystitis to GBC.
Glypican-3 is a cell surface protein that is highly expressed in certain types of human cancer, including HCC and melanoma (17,35). It is associated with cell proliferation and survival, possibly due to its interaction with insulin-like growth factor (IGF) 2 (11). Song et al (36) mated glypican-3 knockout mice with insulin receptor substrate 1 nullizygous mice, and demonstrated that glypican-3 regulated organism growth independently of IGF signaling. Notably, glypican-3 knockout mice exhibited changes in Wnt signaling (36). Glypican-3 is able to form a complex with Wnt molecules, thereby promoting cancer growth by stimulation of canonical Wnt signaling (12), and is also able to regulate developmental growth via interaction with the Hedgehog signaling pathway (13). In addition, glypican-3 is able to act as a negative regulator of Hedgehog signaling during mammalian development (13). A number of studies have indicated that mutated glypican-3 lacking the GPI anchor domain was able to block Wnt signaling and inhibit the growth of Wnt-dependent tumors (14,15). Previous studies have reported that glypican-3 expression is associated with various types of cancer, including HCC (16), melanoma (17), ovarian clear cell carcinoma (18), YST (19), neuroblastoma, hepatoblastoma, Wilms' tumor and others (20,21). The present study demonstrated that 53 patients with GBC presented positive expression of glypican-3 (53.0%), which suggested that glypican-3 expression may be associated with GBC.
The combined detection of multiple molecular markers is able to enhance the specificity and sensitivity of tumor prognostic assessment, and has become a potentially effective method for the prediction of tumor prognosis (37,38). The present study identified a significantly positive correlation between the expression of calpain-1 and glypican-3 in patients with GBC, and a significantly negative correlation in patients with cholecystitis via Spearman's correlation analysis. Thus, it may be speculated that the combined detection of calpain-1 and glypican-3 may be beneficial for the prognostic assessment of GBC. Identification and validation of additional patient studies on various human populations at risk of developing GBC, which include clinicopathological variables and prognostic values, is required in order to provide further insight into calpain-1 and glypican-3 detection as a potential prognostic biomarker for GBC.
In conclusion, the present study demonstrated that calpain-1 expression was associated with GBC, and may be a useful potential biomarker for the assessment of prognosis in patients with GBC. Notably, the expression of calpain-1 may be associated with progression from cholecystitis to GBC. Furthermore, combined detection of calpain-1 and glypican-3 may be beneficial for the assessment of prognosis in patients with GBC.