Original article
Downregulation of the CXCR4/CXCL12 axis blocks the activation of the Wnt/β-catenin pathway in human colon cancer cells

https://doi.org/10.1016/j.biopha.2015.01.020Get rights and content

Abstract

Chemokine CXCL12 is an extracellular chemokine, which binds to its cell surface receptor CXCR4. High expressions of CXCR4 and CXCL12 are associated with biological malignant potential in colon cancers. We aimed to investigate the roles of the CXCR4/CXCL12 axis in activation of the Wnt/β-catenin pathway in the development of colon cancers. Using colon cancer cell line, we performed the RNA interference assay to downregulate the expression of CXCR4. Cells were exposed to CXCL12 and their growth and metastatic activity were examined. Matrix metalloproteinases (MMPs) activity were analyzed by the gelatin zymography assay. Cell migration ability was estimated by assays of scratch wound and transwell chamber. The expression of CXCR4 and molecules relevant to the Wnt/β-catenin pathway were analyzed by the western blotting and real-time PCR assays. Human colon cancer HT-29 cells identified high expression of CXCR4. HT-29 cells highly responded to CXCL12 stimulation, showing the increase of cell proliferation, invasion and migration through the Matrigeal. The secretion and activity of MMP-2 and MMP-9 were also stimulated in HT-29 cells exposure to CXCL12. However, the CXCR4 knockdown HT-29 cells did not response to CXCL12 stimulation. We suggested that the activation of the CXCR4/CXCL12 axis be blocked in the CXCR4 knockdown cells. This study indicated that one key to the role of the CXCR4/CXCL12 axis is activation of the Wnt/β-catenin pathway. Downregulation of the CXCR4/CXCL12 axis thus reduces cancer growth and metastasis. Targeted therapy utilizing the CXCR4/CXCL12 axis could be an effective strategy for treatment of colon cancers.

Introduction

CXCL12, also as stromal cell-derived factor 1 (SDF-1), is known to function as a chemotactic factor for lymphocytes, dendritic cells and monocytes [1], [2]. Since Kin et al. [3] reported an increased level of chemokine CXCL12 and its receptor in patients with colon cancers, CXCL12/CXCR4 axis has been considered as a valuable marker of cancer metastasis [4], [5], [6]. In human body, CXCL12 is expressed by stroma cells in different tissues, such as liver, lung and heart [7]. High levels of CXCL12 were found in colon carcinoma-associated fibroblasts (CAF) cells in 36.8% of patients [8], [9]. It is hypothesized that elevated CXCL12 secrete from stroma cells, where it work as a telecrine or paracrine interaction factor between stroma cells and cancer cells [10]. CXCR4 is a 352-amino acid seven-span trans-membrane G protein coupled receptor that selectively binds ligand CXCL12. Overexpression of CXCR4 has been considered to associate with cancer invasion and metastasis [11]. In colorectal cancer patients with stage IV, the overall median survival with CXCR4 expressing tumors was 9 months compared to 23 months for CXCR4 negative cases [3]. CXCR4 expression was also found to associate with larger tumor size, advanced tumor-node-metastasis (TNM) stage and poor histological grade [12], [13], [14].

Although the CXCR4/CXCL12 axis was found aberrant expression in cancers, its roles in the development of cancers has not been completely defined and the mechanism underlying the processes of activation has largely remained unknown. We hypothesized that activation of the CXCR4/CXCL12 axis might play crucial roles in cancer invasion and metastasis. High expression of the CXCR4/CXCL12 axis might induce cancer growth and metastasis through activation of the Wnt/β-catenin pathways.

Section snippets

Cell line and cell culture

Human colon cancer cell lines HT-29, LOVO, HCT-116, SW620 and SW480 were purchased from American Type Culture Collection (ATCC; Manassas, USA). All cell lines were maintained in RPMI-1640 medium, supplemented with 10% (v/v) heat-inactivated newborn calf serum, penicillin-streptomycin (100 IU/mL–100 μg/mL), 10 mM HEPES buffer and 2 mM glutamine, at 37 °C in a humid atmosphere containing 5% CO2 and 95% air.

RNA interference assay

Small interfering RNA (siRNA) against CXCR4 was designed by GenePharma (Shanghai, China). The

CXCR4 expression in human colon cancer cell lines

To select CXCR4 with highest expression, we examined five colon cancer cell lines (LOVO, HT-29, HCT-116, SW620, SW480) by the RT–PCR and western blotting assays. CXCR4 at basic levels were significant different in these cell lines. HT-29 was found highest expression of CXCR4 as determined both in the levels of mRNA and protein (Fig. 1A and B). We thus selected HT-29 cells for the subsequent experiments.

We performed the RNA interference assay to knockdown CXCR4 in HT-29 cells. The efficacy of

Discussion

Colon cancer is a multi-factorial disease characterized by excessive adenoma growth, invasion and metastasis. Although the morbidity is decreasing, it is still the second leading cause of deaths due to cancers worldwide [20]. Since most patients are diagnosed at advanced stage, they generally have a poor prognosis with median survival times of less than 1 year. Moreover, the recurrence rate is high even successful initial treatment [21]. Molecular mechanisms of how this cancer develop and spread

Acknowledgment

This project was supported by the grants from Natural Science Foundation of China (grant nos. 81173090, 91229113, 81373435) and from Beijing Science Foundation (7142017). We behold genuine thankfulness to Research Institute McGill University Health Centre for financial support.

References (31)

  • M.C. Ghosh et al.

    CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2

    Am J Physiol Lung Cell Mol Physiol

    (2012)
  • E. Pauls et al.

    Cell cycle control and HIV-1 susceptibility are linked by CDK6-dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells

    J Immunol

    (2014)
  • M.Z. Ratajczak et al.

    The pleiotropic effects of the SDF-1–CXCR4 axis in organogenesis, regeneration and tumorigenesis

    Leukemia

    (2006)
  • S. Saigusa et al.

    Cancer-associated fibroblasts correlate with poor prognosis in rectal cancer after chemoradiotherapy

    Int J Oncol

    (2011)
  • M.D. Shi et al.

    CXCL12-G801A polymorphism modulates risk of colorectal cancer in Taiwan

    Arch Med Sci

    (2013)
  • Cited by (26)

    • Two distinct CXC chemokine receptors (CXCR3 and CXCR4) from the big-belly seahorse Hippocampus abdominalis: Molecular perspectives and immune defensive role upon pathogenic stress

      2017, Fish and Shellfish Immunology
      Citation Excerpt :

      CXCR4 has been widely detected in cells of the central nervous and immune systems [2,3]. The biological functions of CXCR4 and its corresponding ligand (CXCL12) have been extensively studied in mammals, especially with regard to their behavior in pathological conditions such as cancer [17–20]. Most of the studies on fish CXCR4-CXCL12 system have been conducted using the zebrafish model and focused on its involvement in the development of sensory system [21], formation of muscles [22], retinal growth [23], heart regeneration [24] and regulation of cell movement during development [25].

    • Cytokine Regulation of Metastasis and Tumorigenicity

      2016, Advances in Cancer Research
      Citation Excerpt :

      These pathways in turn activate SRF, NF-κB, and Elk-mediated transcription (Begley, MacDonald, Day, & Macoska, 2007; Chinni et al., 2006; Singh et al., 2012). These signaling pathways regulate cell growth, migration, and invasion, and in cooperation with WNT/beta-catenin and Her2 (Chinni et al., 2006, 2008; Garcia-Irigoyen et al., 2015; Song, Gao, Chu, Han, & Qu, 2015). Through a transgenic mammary tumor model, CXCL12 has also been shown to regulate in vivo invasion of carcinoma cells through EGF- and CSF-1-dependent mechanisms (Hernandez et al., 2009).

    • CXCL12-CXCR4/CXCR7 Axis in Cancer: from Mechanisms to Clinical Applications

      2023, International Journal of Biological Sciences
    View all citing articles on Scopus
    1

    Zu-Hua Gao and Zhi-Yu Song contributed equally to this work.

    View full text