ANTICANCER RESEARCH 33: 4103-4110 (2013)
Aberrant Expression of CXCR4 and β-Catenin in Pancreatic Cancer ZHENG WANG, QINGYONG MA, PEI LI, HUANCHEN SHA, XUQI LI and JUN XU
Department of Hepatobiliary Surgery, the First Affiliated Hospital, Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi, P.R. China
Abstract. Aim: The stromal cell-derived factor-1 (SDF1)/C-X-C chemokine receptor type 4 (CXCR4) axis and Wingless and INT-1 (Wnt)/β-catenin pathway has been related to cancer progression. The aim of this study was to investigate the expression of CXCR4 and β-catenin in pancreatic cancer. Patients and Methods: A total of 48 pancreatic cancer samples and 8 normal pancreatic tissues were selected to detect CXCR4 and β-catenin expression by an immunohistological technique. Spearman and Chi-square analyses were used to study the relation between the protein expression and clinical characteristics. Survival analysis was evaluated by the Kaplan–Meier product limit method. Results: The proportions of CXCR4 and β-catenin expression on pancreatic cancer cells were significantly higher than in normal pancreas tissues. There was a significant difference in CXRC4 expression levels, lymph node metastasis and TNM stage. Clinical Significance was observed for β-catenin expression and lymph node metastasis; Kaplan-Meier curves suggested that clinical prognosis is poor for patients expressing CXCR4. Multivariate analysis showed that CXCR4 expression was an independent prognostic factor for pancreatic cancer. Conclusion: Both CXCR4 and β-catenin are abnormally expressed in pancreatic cancer. CXCR4 may be an important marker for pancreatic cancer progression. Pancreatic cancer is the fourth leading cause of death from malignant cancers (1). At the time of diagnosis, more than 80% of patients with pancreatic cancer have locally advanced or metastatic lesions and fewer than 5% of patients survive five years after the diagnosis (2). The mechanism of metastasis remains unclear; however, molecular research has indicated an
Correspondence to: Qingyong Ma or Jun Xu, Department of Hepatobiliary Surgery, The First Affiliated Hospital, Medical College of Xi’an Jiaotong University, Xi’an 710061, Shaanxi, P.R. China. Tel: +86 2985323899, e-mail:
[email protected] or
[email protected] Key Words: Pancreatic cancer, tumor progression, CXCR4, β-catenin.
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important relationship between cancer progression and chemokine receptors, particularly C-X-C chemokine receptor type 4 (CXCR4) (3-6). It has been demonstrated that stromal cell-derived factor-1 (SDF-1)/CXCR4 interaction is a critical molecular determinant for events such as embryo development, mediation of immune inflammatory reaction, and modulation of the hematopoietic system (7). Recently, the SDF-1/CXCR4 pathway has been proven to be involved in simulating the metastatic process of different neoplasms (8, 9). The chemokine receptor CXCR4, which is expressed in as many as 23 different tumor types, is a particularly exciting new target (10-14). Research potentially suggests a critical role of CXCR4 on cancer progression. β-Catenin is a key factor in the canonical Wingless and INT (Wnt) pathway and plays critical roles in embryonic development, inflammation cancer proliferation and metastasis (15-17). It has potentially dual functions: cell adhesion and cell signaling. The deregulation of Wnt/βcatenin pathway leads to a high metastatic ability of cancer cells and influences cell adhesion, migration, and transcription through interactions with different protein complexes in the development of cancer (16, 18, 19). Deregulation of Wnt signaling is a well-established hallmark of certain types of human cancer. In one study of neural progenitors, it seemed that SDF-1 was able to modulate β-catenin to influence the canonical Wnt pathway and finally influence cell differentiation (20). There are limited reports on the relation between the SDF1/CXCR4 and the Wnt pathways in cancer progression. Our previous in vitro study showed that CXCR4 knockdown inhibited Wnt/β-catenin pathway activity and its target gene expression, which resulted in the decrease of pancreatic cancer cell growth, cell tumorigenesis and invasive ability (21). With this knowledge, we were able to gain insight into the relationship between SDF-1/CXCR4 and the Wnt/βcatenin pathway in the progression of pancreatic cancer (22). Therefore, we were eager to investigate in vivo the expression of CXCR4 and β-catenin in pancreatic ductal carcinoma, and analyze the relationship between the expression of these proteins and clinical prognosis.
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ANTICANCER RESEARCH 33: 4103-4110 (2013) Patients and Methods Patients and tissue samples. The patients included in the study were those who underwent curative or palliative surgery for pancreatic cancer at the Department of Hepatobiliary Surgery, the First Affiliated Hospital of Medical College, Xi’an Jiaotong University, P.R. China, from August 2006 to August 2010. Forty-eight tissue samples of pancreatic ductal carcinoma were collected. Eight tissue samples of normal pancreatic tissue were obtained from patients operated on pancreatic trauma, and were verified by pathological techniques. All samples were fixed with 10% formalin, uniformly embedded with paraffin, and stained with hematoxylin-eosin (HE). The mean age of patients overall was 58.5±10.3 years, with a range of 34-76 years. Informed consent was obtained from the patients or their legal representatives before the study. The study was approved by the Institutional Ethics Committee of Xi’an Jiaotong University (No. 001003226). The clinicopathological features of patients according to the tumor-node-metastasis (TNM) stage classification of the International Union Against Cancer (UICC) are shown in Table I (23). Postoperative survival was defined as the time that elapsed from the surgery to cancer-related death. The median follow-up time was 16.2 months. Cell line and culture conditions. Different grade human pancreatic cancer cell lines: poorly differentiated (Panc-1, Miapaca-2), moderately differentiated (BxPC-3, AsPC-1), and well-differentiated (PC-2, Capan1) were stored at the Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi’an Jiaotong University. The cells were cultured in Dulbecco's Modified Eagle’s Medium (DMEM, Invitrogen Corporation, Carlsbad, CA. USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and penicillin (100 units/ml), streptomycin (0.1 mg/ml) at 37˚C in an environment of 95% air and 5% CO2. Immunohistochemical staining. Four-micrometer tissue sections from each tissue sample on glass slides were deparaffinized three times in xylene for 8 min each, rehydrated three times in a graded series of ethanol, and washed with phosphate-buffered saline (PBS) (pH 7.4). The slides were treated with 3% H2O2 for 15 min to block endogenous peroxidase activity. These sections were heated in 0.01 M sodium citrate buffer to retrieve antigen activity and then cooled at room temperature. Primary antibodies to CXCR4 and β-catenin were obtained from eBioscience (San Diego, CA. USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. The sections were first incubated with antibody to CXCR4 (1:50) and to βcatenin (1:200) at 4˚C overnight, then incubated with biotin-labeled secondary antibody for 30 min, and finally incubated with streptavidin-peroxidase for 30 min at room temperature. The tissue sections were developed with diaminobenzidine (DAB), lightly counterstained with hematoxylin, and mounted. Negative controls were obtained by substituting the primary antibody with PBS. The evaluation of target protein was performed simultaneously by two pathologists who had no knowledge of the patients’ clinicopathological features. All slides were observed under a microscope, and representative photographs were taken. Evaluation of immunostaining and semiquantitative analysis. A refined scoring system was used for the evaluation of cytoplasmic staining results for CXCR4 and β-catenin. The immunoreactive score (IRS) was calculated according to a previous study by determining the product of the intensity of immunostaining (none=0, weak=1, moderate=2, strong=3) and the percentage of
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Table I. Relationship between clinicopathological features and expression of C-X-C chemokine receptor type 4 (CXCR4) and β-catenin. Features
Overall Gender Male Female Age, years ≥65
