Expression of CXCL12/CXCR4 and its correlation to prognosis in esophageal squamous cell carcinoma

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1 [Chinese Journal of Cancer 28:2, ; February 2009]; 2009 Sun Yat-sen University Cancer Center Clinical Research Paper Expression of CXCL12/CXCR4 and its correlation to prognosis in esophageal squamous cell carcinoma Dao-Feng Wang, 1,2 Ning Lou, 1,2 Can-Guang Zeng, 1,3 Xu Zhang 1,3 and Fu-Jin Chen 1,4, * 1 State Key Laboratory of Oncology in South China; 2 Department of Intensive Care Unit; 3 Department of Thoracic Surgery; 4 Department of Head and Neck Surgery; Cancer Center; Sun Yat-sen University; Guangzhou, Guangdong P.R. China Key words: esophageal neoplasm, CXCL12, CXCR4, prognosis Background and Objective: CXCL12/CXCR4 is expressed in many kinds of tumors, which is associated with tumor proliferation and invasion. This study was to investigate the expression of CXCL12/CXCR4, and explore its correlation to prognosis and clinicopathologic factors of esophageal squamous cell carcinoma (ESCC). Methods: The expression of CXCL12/CXCR4 protein in 186 specimens of ESCC was assessed by immunohistochemistry. Results: The positive rates of CXCL4 and CXCR12 protein in ESCC tissues were 67.2% and 63.4%, respectively. CXCL4 and CXCR12 were not expressed in 20 specimens of normal esophageal epithelium. PTNM stage and positive expression of CXCR4 were independent prognostic factors of ESCC (p < 0.05). The five-year survival rates of CXCL12-positive and CXCL12-negative groups were not significantly different (21.0% vs. 18.8%, p > 0.05), while the five-year survival rate was significantly higher in CXCR4-negative group than in CXCR4-positive group (28.5% vs. 2.2%, p < 0.05). The expression of CXCR4 was higher in the group with lymph node metastasis and pathological T3 stage than in the group without lymph node metastasis and with pathological T1 T2 stages (p < 0.05). The expression of CXCR4 was not correlated with the expression of CXCL12 in ESCC. Conclusions: CXCL12/CXCR4 is intensively expressed in esophageal squamous cell carcinoma. The level of CXCR4 is positively correlated to progression and prognosis of ESCC. Chemokines are a family of cytokines which regulate chemotaxis. Among them, C-X-C chemokine ligand 12 (CXCL12) and its receptor c-x-c chemokine receptor 4 (CXCR4) play an important *Correspondence to: Fu-Jin Chen; State Key Laboratory of Oncology in South China; and Department of Head and Neck Surgery; Cancer Center; Sun Yat-sen University; Guangzhou, Guangdong P.R. China; Tel.: ; Fax: ; chenfuj@mail.sysu.edu.cn Submitted: 06/16/08; Revised: 08/08/08; Accepted: 08/20/08 This paper was translated into English from its original publication in Chinese. Translated by: Beijing Xinglin Meditrans Center ( and Hua He on 11/30/08. The original Chinese version of this paper is published in: Ai Zheng (Chinese Journal of Cancer), 28(2); Previously published online as a Chinese Journal of Cancer E-publication: role in migration of stem cells and many immunological reactions. The signaling pathway consisting of CXCL12 and CXCR4 is closely correlated to growth, invasion and metastasis of tumors. Esophageal cancer possesses a high occurrence rate and low survival rate in China. Lymph node metastasis and exterior expansion of esophageal cancer are the major reasons for incomplete incision of the tumor and postoperative failure. This study was to investigate the expression of CXCL12/CXCR4, and explore its correlation to prognosis and clinicopathologic factors in 186 cases of esophageal squamous cell carcinoma (ESCC). Patients and Methods Patient selection. Inclusion criteria of patient enrollment were as follows: (1) patients with pathologically confirmed ESCC after radical surgery; (2) patients at pathological stages II (IIA, IIB) and III (pt3n1); (3) patients who underwent a left or a right thoracotomy, including anastomosis through the left neck, upper aortic arch or lower aortic arch, as well as routine lymph node dissection in the fields of the chest and abdomen; (4) patients with no history of radiotherapy or chemotherapy prior to surgery; (5) patients with follow-up information. General information. In total 188 patients with ESCC treated at Sun Yat-sen University Cancer Center from January 1998 to March 2003 were enrolled. The ESCC tissues from 188 patients were pathologically identified. Two patients died before receiving surgery, and 186 patients with complete clinical, pathological and follow-up information were included in the research. Patients were followed up until death or April 2008 ( months). All patients had no primary tumors in other sites, did not receive radiotherapy or chemotherapy before surgery, and had definite causes of death. According to PINM classification of malignant tumors (UICC2002 version), 84 cases were at stage IIa, 21 at stage IIb and 81 at stage III. Thirty-six cases received radiotherapy or chemotherapy after surgery and 150 cases received neither. Among the patients, 145 were males and 41 females. The patients were ranged from 33 to 76 years old, with a median age of 58 years. One hundred and seven patients were < 65 years and 79 patients were 65 years. The tumors were located in the lower third (29 cases), middle third (115 cases) and upper third (42 cases) of the esophagus. The tumors were < 5 cm in diameter in 118 patients, and 5 cm in the other 68 patients. Eighty-nine patients were 154 Chinese Journal of Cancer 2009; Vol. 28 Issue 2

2 classified at stage N0 and 97 at stage N1; 63 were classified at stage T1-T2, and 123 at stage T3. There were 38 well differentiated, 81 moderately differentiated and 67 poorly differentiated ESCC. Severn cases had positive cutting edge, whereas 179 cases showed negative cutting edge. Main agents. The primary CXCR4 antibody was purchased from R&D Systems, Inc. (Minneapolis, MN USA). The secondary CXCL12 antibody was bought from Abcam Inc. (Cambridge, MA USA). The SP9000 kit was provided by Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd (Beijing, P.R. China). Experiment methods. The labeled streptavidin biotin (LSAB) method was applied in the research. The paraffin wax sections were cut into 5 µm thick. Sections were cleared in xylene and dehydrated through graded ethanol. H 2 O 2 (3%) was used to eliminate the activity of endogenous peroxidase. EDTA (ph 8.0) was applied to repair the sections. After the antigen was exposed, goat serum was added, followed by addition of the primary antibody, secondary antibody and tertiary antibody sequentially. The sections were stained with diaminobenzidine (DAB), re-stained with hematine and mounted after being dried (the working concentration for both CXCL12 and CXCR4 antibodies was 1:100). The primary and secondary antibodies were replaced by PBS in the control group. Judgment of results. The staining results of CXCR4 and CXCL12 were semi-quantitatively assessed based on the method previously reported 1,2 and our preliminary data. Five different vision fields were observed under high magnification (x 400). The staining intensity was categorized as follows: no color (score 0), pale yellow (score 1), brown yellow (score 2), dark brown (score 3). According to the percentage of positive cells, cells were graded into < 5.0% (score 0), % (score 1), % (score 2), 51% to 75% (score 3) and >75% (score 4). The product of the above two scores was recorded as the final score of each section. The final score of 1 was regarded as negative, and >1 was positive. All results were identified by experienced pathologists. Statistical analysis. The SPSS 10.0 Statistical Package was used to perform statistical analyses. Comparisons between positive and negative CXCR4 or CXCL12 groups were performed by Kaplan- Meier Survival Analysis. Intergroup comparison was conducted using the log-rank test. Multivariate analysis was performed using the Cox regression model. The chi-square test was applied to analyze the correlation of CXCR4/CXCL12 expression and clinicopathological factors of ESCC. p < 0.05 was considered significantly different. Results Expression of CXCR4 and CXCL12 in ESCC and normal esophageal epithelial tissues. Positive staining of CXCR4 (brown yellow) was mainly located in the membrane and cytoplasm of ESCC (Fig. 1A), with an expression rate of 67.2%. Positive staining of CXCL12 (light brown) was primarily located in the membrane and cytoplasm of ESCC (Fig. 1B), with an expression rate of 63.4%. No expression of CXCR4 and CXCL12 was detected in 20 specimens of normal esophageal epithelial tissues (Fig. 1C and 1D). Multivariate survival analysis of ESCC patients. The following factors were enrolled for analysis: blood type (type B and nontype B), pathological differentiation (high differentiation and moderate to low differentiation), PTNM classification (class II and class III), age (< 60 years and 60 years), tumor length (< 5 cm and 5 cm), tumor location (upper, middle and lower third of the esophagus), cutting edge (positive and negative) and postoperative therapy (radiotherapy/chemotherapy, and without radiation or chemotherapy). Results showed that PTMN classification and expression of CXCR4 were the independent prognostic factors of ESCC patients receiving radical surgery (Table 1). Survival analysis for ESCC patients. The overall five-year survival rate was 19.9%. The five-year survival rates for the groups with positive and negative expression of CXCL12 were 18.8% and 21.0%, respectively, without significant difference (p = ). The five-year survival rates for the groups with positive and negative expression of CXCR4 were 2.2% and 28.5%, respectively, which were significantly different (p = ). (Figs. 2 and 3). Correlation of CXCR4/CXCL12 expressions to pathological characteristics of ESCC. The expression of CXCR4, but not that of CXCL12, was positively correlated with T staging and N staging (p < 0.05)(Table 2). Discussion Chemokines are a group of cytokines which can induce and promote inflammation. They are a class of small protein polypeptides of the cytokine superfamily. The chemokine receptors are a class of G protein-coupled receptors (GPCR) which mediate the functions of chemokines. CXCL12, also named as stromal cellderived factor-1 (SDF-1), is a chemokine of the CXC subfamily. It was firstly discovered among the chemokines secreted by mouse bone marrow stromal cells, and CXCR4 is its specific receptor. It was previously thought that CXCL12 interacted with its specific receptor CXCR4 to form a coupled molecular pair, which could pass information to each other and control oriented migration of immunocytes. They play an important role in cell growth, adhesion and migration in collaboration with some other chemokines. However, recent researches have found that CXCL12/CXCR4 is expressed not only in immunocytes, but also in many tumor cells. 3-7 The combination of CXCL12 and CXCR4 could induce conglomeration and re-allocation of cytoskeletal protein in breast cancer cells, thus to mediate metastasis of breast cancer cells. 8 Addition of 100 nmol/l CXCL12 into breast cancer cells in vivo could increase the quantity of actins in a short time, thus to form apparent pseudopodia and promote metastasis and invasion of breast cancer cells in a dose-dependent manner. Moreover, CXCL12/CXCL4 could activate many signaling pathways in tumor cells, such as MAPK, PI3K and camp protein kinase A, and actively involved in invasion, development and metastasis of cancer cells. 9,10 Differential expression of the CXCL12/CXCR4 axis results in different development directions and prognosis of tumors individually. Primary tumors with positive CXCR4 expression show higher metastatic potential than those with negative CXCR4 expression. 11 Kaifi et al. 12 studied 136 cases of esophageal cancer and Chinese Journal of Cancer 155

3 Figure 1. Expression of CXCL12 and CXCR4 in esophageal squamous cell carcinoma (ESCC) and normal esophageal epithelium (SP 200). (A) Strong positive staining of CXCL12 in the cytoplasm and cytomembrane of ESCC. (B) Strong positive staining of CXCR4 in the cytoplasm and cytomembrane of ESCC. (C) Negative staining of CXCL12 in normal esophageal epithelium. (D) Negative staining of CXCR4 in normal esophageal epithelium. found that patients with positive expression of CXCR4 had poorer prognosis than those without CXCR4 expression, suggesting an important role of CRCR4 in early metastasis of esophageal cancer. We showed that PTNM classification and expression of CXCR4 were independent prognostic factors of ESCC, whereas CXCL12 was not. Additionally, the survival rate was significantly lower in patients with positive expression of CXCR4 than in those with negative expression of CXCR4. However, Gockel et al. 13 did not observe such a correlation of CXCR4 to prognosis in either ESCC or esophageal adenocarcinoma. Their study implied that the expression of CXCR4 was not an independent prognostic factor of esophageal cancer. The discrepancy may be due to the use of two different pathological types of esophageal cancer and a relatively small simple size in their study. We detected that CXCR4 and CXCL12 were not expressed in 20 cases of normal esophageal epithelium, but in the cytomembrane and cytoplasm of some ESCC tissues. CXCR4 was highly expressed in cases with deep esophageal invasion (r = 0.226, p = 0.002) and in cases with lymph node metastasis (r = 0.477, p = 0.000), which were consistent with the finding performed by Kaifi et al. 12 We propose that the function of the CXCL12/CXCR4 axis might be dependent on CXCR4. With the development of Table 1 Multivariate survival analysis of 186 patients with esophageal squamous cell carcinoma ESCC, CXCL12 selectively binds to cells with positive expression of CXCR4 to promote growth of tumor cells. Therefore cells with high expression of CXCR4 show malignant tendency and strong invasive capability. Uchida 14 et al. and Almotfi et al. 15 discovered that over-expression of CXCR4 could apparently increase the metastatic potential of tumor cells to migrate to regional lymph nodes. Philips et al. 16 studied non-small cell lung cancer and found that cancer cells with positive expression of CXCR4 tend to metastasize to organs with high expression of CXCL12 when they are stimulated by CXCL12. Our results were in line with this finding. We speculate that no or low expression of CXCL12 exists in the site of primary esophageal cancer, while high expression of CXCL12 exists in lymph nodes and other tissues with a tendency towards metastasis; highly malignant esophageal carcinoma cells 156 Chinese Journal of Cancer 2009; Vol. 28 Issue 2

4 Figure 2. Survival curves of esophageal squamous cancer patients with positive or negative CXCL12 expression Figure 3. Survival curves of esophageal squamous cancer patients with positive or negative CXCR4 expression Table 2 Correlation of CXCR4 and CXCL12 expression to clinicopathologic characteristics of 186 patients with esophageal squamous cell carcinoma Chinese Journal of Cancer 157

5 with positive expression of CXCR4 metastasize via concentration gradients created by CXCL12. Pils et al. 17 found that the expression of CXCR4 was correlated with CXCL12 in ovarian cancer. However, we did not detect such a correlation, implying no direct interaction between CXCR4 and CXCL12 in esophageal cancer. Helbig et al. 18 and Schioppa et al. 19 also believe that tumor suppression proteins, such as hypoxia-inducible factor-1α (HIF-1α), NF-kB and Von Hippel-Lindau (VHL) upregulate the expression levels of CXCL12 and CXCR4. We investigated the influence of CXCR4 and some pathological factors on the prognosis of ESCC, and the correlation of CXCR4/ CXCL12 to other factors, including gender, age, tumor location, length of tumor, pn-category, pt-category and differentiation. Our results preliminarily reveal the function of the CXCL12/ CXCR4 axis in the development of ESCC, the correlation of CXCR4 expression to regional invasion of ESCC and lymph node metastasis. The expression of CXCR4 may be one of the important factors indicating poor prognosis of ESCC. Intervention therapy targeting the CXCL12/CXCR4 axis may open a new direction for treatment of ESCC. References [1] Falleni M, Pellegrini C, Marchetti A, et al. Survivin gene expression in early-stage nonsmall cell lung cancer [J].J Pathol,2003,200(5): [2] Huo SJ, Tang HH, Wei W, et al. Expressions of chemokine CXCL12 and its receptor CXCR4 in human cholangiocarcinoma and their clinical significance [J]. Xian Dai Sheng Wu Yi Xue Jin Zhsan, 2008,8(4): [in Chinese]. [3] Corcione A, Ottonello L, Tortolina G, et al. Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells [J].J Natl Cancer Inst, 2000,92(8): [4] Zhou Y, Larsen PH, Hao C, et al. CXCR4 is a major chemokine receptor on glioma cells and mediates their survival [J].J Biol Chem, 2002,277(51): [5] Koshiba T, Hosotani R, Miyamoto Y, et al. Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression [J].Clin Cancer Res, 2000,6(9): [6] Su L, Zhang J, Xu H,et al. Differential expression of CXCR4 is associated with the metastatic potential of human non-small cell lung cancer cells [J]. Clin Cancer Res, 2005,11(23): [7] Hu J, Deng X, Bian X, et al. The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma [J]. Clin Cancer Res, 2005,11(13): [8] Muller A, Homey B,Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis [J].Nature, 2001,410(6824): [9] Ganju RK, Brubaker SA, Meyer J, et al. The alpha-chemokine, stromal cell-derived factor-1alpha, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways [J].J Bio Chem, 1998,273(36): [10] Cobb MH. MAP kinase pathways [J]. Prog Biophys Mol Biol,1999,71(3-4): [11] Balkwill F. Cancer and the chemokine network [J]. Nat Rev Cancer, 2004,4(7): [12] Kaifi JT,Yekebas EF,Schurr P, et al. Tumor-cell homing to lymph nodes and bone marrow and CXCR4 expression in esophageal cancer[j]. J Natl Cancer Inst, 2005,97(24): [13] Gockel I, Schimanski CC, Heinrich C, et al. Expression of chemokine receptor CXCR4 in esophageal squamous cell and adenocarcinoma [J]. BMC Cancer, 2006,6:290. [14] Almofti A, Uchida D, Begum NM, et al. The clinicopathological significance of the expression of CXCR4 protein in oral squamous cell carcinoma [J]. Int J Oncol, 2004,25(1): [15] Uchida D, Begum NM, Tomizuka Y, et al. Acquisition of lymph node, but not distant metastatic potentials, by the overexpression of CXCR4 in human oral squamous cell carcinoma [J]. Lab Invest, 2004;84(12): [16] Phillips RJ, Burdick MD, Lutz M, et al. The stromal derived factor-1/cxcl12-cxc chemokine receptor 4 biological axis in non-small cell lung cancer metastases [J]. Am J Respir Crit Care Med, 2003,167(12): [17] Pils D, Pinter A, Reibenwein J, et al. In ovarian cancer the prognostic influence of HER2/neu is not dependent on the CXCR4/SDF-1 signalling pathway [J]. Br J Cancer, 2007,96(3): [18] Helbig G, Christopherson KW 2nd,Bhat-Nakshatri P, et al. NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4 [J]. J Biol Chem, 2003,278(24): [19] Schioppa T, Uranchimeg B, Saccani A, et al. Regulation of the chemokine receptor CXCR4 by hypoxia [J]. J Exp Med, 2003,198(9): Chinese Journal of Cancer 2009; Vol. 28 Issue 2