CCL20 and CXCL8 synergize to promote progression and poor survival outcome in patients with colorectal cancer by collaborative induction of the epithelial–mesenchymal transition
Introduction
Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second leading cause of cancer-related death in the developed countries [1]. Approximately 50% of CRC patients die from distant metastases within 6 years of the initial diagnosis, and the liver is the predominant and often the only organ for distant metastasis [2]. Recent studies in CRC have suggested that the chemotactic responses of tumor cells are necessary for metastatic spread to the liver, and chemokines as well as their receptors, in particular CCL20/CCR6 and CXCL8/CXCR1/2, play an important role in the metastatic process. CCL20 is the only chemokine known to interact with the chemokine receptor CCR6 [3], [4], [5], and expressed mainly in mucosa-associated and lymphoid tissues and the liver [5]. CCL20 and CCR6 are expressed predominantly in the microenvironment of CRC tumors [6], [7], [8], [9], [10]. A strong association has been found between CCL20/CCR6 staining intensity in CRC samples and the development of synchronous liver metastases [6], [7], [8]. Stimulation of colon cancer cells by CCL20 has been shown to result in signaling events associated with cell proliferation, adhesion and migration [11]. In a murine model, stimulation of CCR6-expressing plasmocytoma cells with CCL20 before intravenous application resulted in a marked increase in their homing to the liver [12]. CXCL8, also referred to as interleukin-8 (IL-8), stimulated the migratory capacity of a distinct set of leukocytes [13]. Increased expression of CXCL8 has been detected in the tumor microenvironment and blood of CRC patients [14], [15], [16], [17], [18], [19], [20], [21], [22]. Overexpression of CXCL8 promotes tumor growth, progression, and metastasis [16], [17], [18], [19], and is associated with tumor size, tumor grading, depth of infiltration, liver metastasis and survival in CRC [15], [16], [17], [18], [19], [22].
To date, however, the precise mechanisms by which these chemokines determine tumor cell detachment and migration away from the primary tumor tissues remain to be established. Over the past decade, accumulating evidence has shown that the epithelial–mesenchymal transition (EMT) is a pathological process contributing to cancer progression, particularly to cancer invasion, dissemination, and metastasis [23]. While epithelial cells undergo EMT, loss of the epithelial marker E-cadherin (E-cad) and concomitant expression of distinct mesenchymal markers like vimentin (Vim) and beta-catenin play a vital role in this reversible transdifferentiation. Therefore, the EMT provides a mechanism for carcinoma cells to acquire a more aggressive phenotype [23].
Several chemokines for induction of EMT in epithelial tumor cells has been documented, such as CXCL12 for oral squamous cell carcinoma cells B88 and HNt [24], CXCL16 for CRC cell HT29 [25], and CCL18 or CXCL8 for breast cancer cells [26], [27], and CXCL8 for nasopharyngeal carcinoma cells [28]. However, CCL20 or CXCL8-induced EMT in CRC cells has not been reported. In the present study, we demonstrated for the first time that CCL20 or CXCL8 alone was not sufficient to induce complete EMT in human CRC cells, but both of them could jointly induce EMT-like phenotype that was required to maintain CRC cell proliferation, migration and invasion. We further found that the phosphatidylinositol 3-kinase (PI3K)/AKT-ERK1/2 pathway crosstalk mediated by CCL20 and CXCL8 appeared to be responsible for the EMT in the CRC cells. Furthermore, clinical evidence showed that there was a strong association of the coexpression of CCL20 and CXCL8 with the loss of E-cadherin expression in CRC tissues. Patients with CRC who had coexpression of CCL20 and CXCL8 were more likely to develop liver metastases and had a most poor prognosis. The present data indicate that CCL20 and CXCL8 synergize to promote CRC progression and metastasis by collaborative induction of EMT via PI3K/AKT-ERK1/2 crosstalk, therefore, detection of both coexpression can be used to predict patient outcomes, and targeting both CCL20 and CXCL8 or the PI3K/AKT pathway could be highly valuable for the therapy of recurrent or metastatic CRC.
Section snippets
Patients and sample collection
Two hundred thirteen patients with CRC at the Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University (Kunming, China) between February 2003 and May 2005 were enrolled onto this study. The median age of patients at the time of admission was 62 years, range 21–79 years. All the patients underwent radical resection of the primary tumor with regional lymph node dissection. All diagnoses of adenocarcinoma were confirmed histopathologically, and the paraffin
Both CCL20 and CXCL8 coordinately induced EMT-like phenotype in colon cancer cells
CCL20 or CXCL8 alone at a concentration of 100 ng/mL for a period of 48 h was not enough to induce an EMT-like phenotype in SW480 cells, but after cotreatment with both chemokines at the same concentration, most cells not only changed to a spindle-shaped, elongated morphology, but also exhibited a loose association and occasional misorientation compared to the closely contacted monolayer, polygon cobblestone-like cells (Fig. 1A). Concomitant expression of epithelial and mesenchymal markers is often
Discussion
Chemokines have been demonstrated to be involved in the pathogenesis and metastasis of tumors [36]. In CRC, the tumor cells selectively upregulate expression of some chemokines and chemokine receptors, providing a basis for efficient autocrine and paracrine loops [37], [38]. Multiple clinical studies implicate chemokines in the development, progression, and metastasis of CRC [39]. However, almost all of these studies focused respectively on a single chemokine involvement and did not address the
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by Grants from the National Natural Science Foundation of China, China (30960445), Joint Special Funds for the Department of Science and Technology of Yunnan Province-Kunming Medical University (2012FB071) and Funds for Equipped Research Institutions of Yunnan Provincial Health Department, China (2010NS092; 2011WS077; 2011WS076). The use of human tissue samples and clinical data was approved by the ethics committee of Third Affiliated Hospital, Kunming Medical
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These authors contributed equally to this work.