Elsevier

Life Sciences

Volume 267, 15 February 2021, 118983
Life Sciences

CD166 promotes cancer stem cell-like phenotype via the EGFR/ERK1/2 pathway in the nasopharyngeal carcinoma cell line CNE-2R

https://doi.org/10.1016/j.lfs.2020.118983Get rights and content

Abstract

Aims

The present study aimed to investigate the role and underlying mechanisms of CD166 in cancer stem cell-like (CSCs) phenotype of the radioresistant nasopharyngeal carcinoma cell CNE-2R.

Main methods

Established CD166-shRNA- CNE-2R cell line by lentivirus-mediated silencing CD166. Then, CSC-related genes mRNAs and proteins, and EGFR/ERK1/2 signaling pathway were detected using RT-PCR and western blot. Sphere formation assay was performed to evaluate the sphere formation capacity in CD166-shRNA- CNE-2R cells. The tumorigenesis ability in vivo was examined in nude mice mode.

Key findings

Downregulation of CD166 inhibited the expression of the CSC-related genes, pEGFR and pERK in vitro and vivo. The capacity to form spheres and tumorigenesis was significantly decreased in CD166-shRNA cells. Furthermore, EGF-stimulated CD166-shRNA cells exhibited an increase in CSC-like traits by activating EGFR/ERK1/2 signaling.

Significance

CD166 induced CSCs formation by activating the EGFR/ERK1/2 signaling pathway in nasopharyngeal carcinoma, which may serve as a critical molecular target for NPC therapeutic strategies.

Introduction

Nasopharyngeal carcinoma (NPC) has a distinct geographical distribution, and is most prevalent in east and southeast Asia, especially in southern China [1,2]. According to Global Cancer Statistics 2018, approximately 129,079 new NPC cases were diagnosed, and 72,987 new deaths occurred in that year around the world [3]. Important to note is that about 42,100 new NPC cases occur each year in China, accounting for 32.6% of all new cancer cases in the world [4]. Regardless of the high response to intensity-modulated radiotherapy, 10–20% of patients still suffer from relapses and metastases after primary treatment [5]. This may be due to the therapeutic resistance of tumors.

Cancer stem cells (CSCs) have been shown to be responsible for therapeutic resistance [6,7]. Probing the role of CSCs in tumor progression and therapy would provide new insights into developing more effective therapies for tumor. Interestingly, a number of studies have demonstrated that CSCs were isolated from various malignant tumors, such as breast cancer [8], head and neck squamous cancer [9] and others [[10], [11], [12]].

CD166 plays an important role in cancer cell resistance to therapies, as well as in recurrence and metastasis [13,14]. Many studies have shown that CD166 is regarded as a part of a CSC-specific phenotypic profile [[15], [16], [17]]. Muraro et al. analyzed the expression of CD166/CD44 in CRC cell lines and evaluated their correlation with CSC properties [15]. In Yan's study, head and neck squamous cell carcinoma cells with high expression of CD166 exhibited stronger ability of sphere formation in vitro and tumor formation ability in vivo than those with low expression [16]. Moreover, Levin et al. found that high-expression of CD166 maintain the stem cells and the stem cell niche in human gastrointestinal tract [17]. All these data indicated that CD166 could serve as a potential marker for cancer stem cells.

Section snippets

Cell lines and culture

CNE-2R, a radioresistant human nasopharyngeal carcinoma cell line, was established by fractional exposure of CNE-2 to radiation at the Cancer Laboratory of Guangxi Medical University [18]. CNE-2R cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS, Biological Industries, Cromwell, CT, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin (both from Solarbio, Beijing, China) and incubated at 37 °C in 5% CO2.

Lentiviral infection

Lentiviral

Successful construction of the CD166-shRNA-CNE-2R cell line

Lentiviruses were successfully constructed and transfected into CNE-2R cells, which were analyzed under a fluorescence microscope. The transfection efficiency was above 90% in CD166-shRNA and NC-shRNA groups, as shown by the green fluorescence protein (GFP)-positive cells (Fig. 1A). Furthermore, the mRNA and protein levels of CD166 were determined by RT-PCR and western blot, respectively. The results showed the lower expression of CD166 in the CD166-shRNA group than that in the NC-shRNA group (

Discussion

Radiation therapy is an effective and common treatment for a variety of malignant tumors, and it is used as a principal treatment for NPC. When it kills most cancer cells, it also induces EMT and the production of CSCs due to their resistance to therapy, eventually generating tumor relapse and metastasis [20]. Cancer cells undergoing EMT not only exhibit enhanced metastatic ability, but also display increased radioresistance by acquiring stemness [21,22]. Accumulating evidence suggests that the

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was supported by National Natural Science Foundation of China (81760544), the Key Research and Development Program Project of Guangxi Zhuang Autonomous Region (Grant No. GuikeAB18221007), the Independent Project of Key Laboratory of Early Prevention & Treatment for Regional High-Incidence-Tumor (Grant No. GKE2019-17), Guangxi Natural Science Foundation Project (2017GXNSFBA198005), the Scientific Research & Technical Development Project of Wuming District, Nanning city (No. 20200214

References (40)

  • M. Al-Hajj et al.

    Prospective identification of tumorigenic breast cancer cells

    Proc. Natl. Acad. Sci. U. S. A.

    (2003)
  • M.E. Prince et al.

    Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma

    Proc. Natl. Acad. Sci. U. S. A.

    (2007)
  • L. Ricci-Vitiani et al.

    Identification and expansion of human colon-cancer-initiating cells

    Nature

    (2007)
  • X. Zhang et al.

    Treatment of radioresistant stem-like esophageal cancer cells by an apoptotic gene-armed, telomerase-specific oncolytic adenovirus

    Clin. Cancer Res.

    (2008)
  • U.H. Weidle et al.

    ALCAM/CD166: cancer-related issues

    Cancer Genomics Proteomics

    (2010)
  • M.G. Muraro et al.

    CD133+, CD166+CD44+, and CD24+CD44+ phenotypes fail to reliably identify cell populations with cancer stem cell functional features in established human colorectal cancer cell lines

    Stem Cells Transl. Med.

    (2012)
  • T.G. Levin et al.

    Characterization of the intestinal cancer stem cell marker CD166 in the human and mouse gastrointestinal tract

    Gastroenterology

    (2010)
  • Y. Guo et al.

    Identification of genes involved in radioresistance of nasopharyngeal carcinoma by integrating gene ontology and protein-protein interaction networks

    Int. J. Oncol.

    (2012)
  • Y. Sun, H. Lin, S. Qu, L. Li, K. Chen, B. Yu, G. Lin, F. Wan, X. Zhu, Downregulation of CD166 inhibits invasion,...
  • S.Y. Lee et al.

    Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionizing radiation

    Mol. Cancer

    (2017)
  • 1

    These authors contributed equally to this work.

    View full text