Elsevier

Journal of Proteomics

Volume 83, 27 May 2013, Pages 26-36
Journal of Proteomics

Quantitative proteome analysis of overexpressed Cripto-1 tumor cell reveals 14-3-3γ as a novel biomarker in nasopharyngeal carcinoma

https://doi.org/10.1016/j.jprot.2013.03.001Get rights and content

Highlights

  • Cripto-1 can promote the proliferation and invasion of NPC.

  • 2D-DIGE analysis identified 14-3-3γ as a candidate NPC related protein.

  • High 14-3-3γ expression is associated with poor survival in NPC patients.

Abstract

We previously found that Cripto-1 is involved in the tumorigenesis of nasopharyngeal carcinoma (NPC). Here, to identify new NPC related proteins and to investigate the clinicopathological correlations of it in NPC, Cripto-1 over-expressed cell (CNE1/CR1+) was established. Two-dimensional difference in gel electrophoresis (2D-DIGE) analysis and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify 23 differential proteins in CNE1/CR1+ and parental cells. Among them, 14-3-3γ showed the potential to be a NPC related protein. 14-3-3γ expression was found in 58.3% (60/103) tumor tissues as detected by IHC, and 69.6% (16/23) NPC fresh tumors expressed higher 14-3-3γ than paired non-cancerous tissues as detected by Western blot. Moreover, 14-3-3γ expression was positively correlated with N classification (p = 0.031), distant metastasis (M classification, p = 0.018) and clinical stage (p = 0.046) of NPC patients. As determined by the Kaplan–Meier method, 14-3-3γ expression in NPC was significantly associated with overall survival (p = 0.015). Multivariate analysis also showed that the expression of 14-3-3γ protein was an independent prognostic factor for outcome of NPC. In this study, we identified upregulated 14-3-3γ by 2D-DIGE in CNE1/CR-1+. We also demonstrated that 14-3-3γ might be a potential biomarker for the prognosis of patients with NPC.

Biological significance

We believe that three aspects of this manuscript will make it interesting to general readers of Journal of Proteomics. Firstly, based on our previous report, we further validated that Cripto-1 can promote the proliferation and invasion of nasopharyngeal carcinoma (NPC). In this context, we used 2D-DIGE to identify new NPC related proteins. As a result, 14-3-3γ showed the potential to be a candidate. Secondly, we reported for the first time that the expression level of 14-3-3γ was significantly increased in human NPC patient tissues, and 14-3-3γ overexpression correlated statistically with N classification, distant metastasis, and clinical stage. Our results highlight the clinical significance of 14-3-3γ in NPC. Finally, we found that high 14-3-3γ expression is associated with poor survival in NPC patients. Thus, this study has identified that the 14-3-3γ involves in the carcinogenesis of NPC. Our findings may also provide new insights into understanding the molecular mechanism involved in NPC carcinogenesis and progression, and may lead to the development of new approaches for effective diagnosis and therapy.

Introduction

Nasopharyngeal carcinoma (NPC), a malignant tumor arising from the epithelium of the nasopharynx, has a special racial and geographic distribution. It is one of the most common cancers in Southeast Asia and Southern China. The incidence rate of NPC in Southern China (20–50/100,000 people per year) is nearly 100-fold higher than that in the western world [1]. NPC carcinogenesis is a multi-step process involving many possible etiological factors. It is widely accepted that Epstein–Barr virus (EBV) infection, environmental factors and genetic susceptibility are the major factors involved [2], [3]. However, the molecular mechanisms underlying NPC pathogenesis remain unclear. Additionally, NPC originates from a hidden anatomical site, and is more closely associated with advanced clinical stage with higher incidence of invasion and metastasis at the time of diagnosis. Hence, the prognosis for NPC is poor with a 5-year survival rate of around 60%. Under such circumstances, it is of great clinical values to further understand the molecular mechanisms of this cancer and find valuable early diagnostic markers as well as novel therapeutic strategies.

Human Cripto-1, a member of the EGF-CFC family, is indispensable for early embryonic development. Cripto-1 plays an important role during tumorigenesis and is overexpressed in a wide range of epithelial carcinomas [4]. Our previous work demonstrated that the level of expression of Cripto-1 is significantly increased in NPC [5]. However, the mechanism and the exact mode of Cripto-1 function during tumor metastasis and progression are still largely unknown. To elucidate the possible mechanism of Cripto-1, in this study, we performed gene transfection-mediated overexpression on tumor cell lines to further examine its biological role, and then compared differential proteins with two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) on two CNE1 cell lines with over and minimal Cripto-1 expressions. As a result, in connection with Cripto-1, 14-3-3γ showed the potential to be a NPC related protein.

14-3-3γ is a member of 14-3-3 proteins, a family of highly conserved phosphoserine/threonine-binding proteins that regulates diverse cellular processes including cell cycle progression, apoptosis, transcriptional regulation, and cell proliferation by functioning as chaperones and adaptors [6], [7], [8]. Seven isoforms (β, γ, ε, η, σ, τ/θ and ξ) have been identified. Although 14-3-3 proteins were initially identified as brain proteins, there are growing evidences that they may have an important role in the development of human cancers, including lung [9], gastric [10], breast [11], hepatocellular carcinoma (HCC) [12] and oral carcinoma [13]. 14-3-3γ, a 28 KDa acidic polypeptide, targets more than 200 proteins in vivo by binding specific phosphoserine/threonine motifs using dimerization properties. It has been reported that the expression was elevated in lung cancer, HCC and vulvar squamous cell carcinoma [14], [15], [16].

However, there is no evidence that has shown the association between 14-3-3γ expression and carcinogenesis of NPC, neither the relation between the 14-3-3γ and Cripto-1. In this study, we examined the 14-3-3γ expression and the clinicopathological factors in human NPC tissues to determine its prognostic significance.

Section snippets

Ethics statement

All the clinical materials used for these research purposes, prior consent of the patients and approval from the Ethics Committee of Southern Medical University were obtained.

Cell line, patients and tissue specimens

Human well differentiated nasopharyngeal squamous carcinoma cell line CNE-1 was kept in our lab [5], and was grown in RPMI-1640 medium (Hyclone, Logan, UT) supplemented with 10% fetal calf serum (ExCell, Shanghai, China) and 1% l-glutamine. A total of 103 primary NPC patients treated at the Nanfang Hospital, Southern

Cripto-1 promoted the proliferation and migration of NPC cells in vitro

In our previous study, we used siRNA to knockdown endogenous Cripto-1 expression, and found that Cripto-1 can inhibit tumor cell growth and invasion. To further validate this gene's function, Human Cripto-1 cDNA was successfully transfected into the CNE1 cell line, in which endogenous Cripto-1 expression is minimal [5], to establish a stable Cripto-1-expressing cell line. As shown in Fig. 1, the results of Western blot (Fig. 1A) and quantitative RT-PCR (Fig. 1B) revealed that the expression of

Discussion

In this study, after performing gene transfection-mediated overexpression of Cripto-1 in CNE1 cells, we presented the evidence that it can promote the growth and invasion of cells in vitro, which is consistent with our previous results after down-regulated the expression of Cripto-1. Similarly, overexpression of Cripto-1 cDNA in normal mouse fibroblasts induces these cells to grow in soft agar and increases growth rates in several human breast cancer cell lines [16]. Human MCF-7 breast cancer

Author contributions

Conceived and designed the experiments: ZW and DW. Performed the experiments: ZW. Analyzed the data: ZW and DW. Contributed reagents/materials/analysis tools: ZW and GL. Wrote the paper: ZW and DW.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81001215).

References (40)

  • S.S. Agarwala et al.

    Current and future adjuvant immunotherapies for melanoma: blockade of cytotoxic T-lymphocyte antigen-4 as a novel approach

    Cancer Treat Rev

    (2011)
  • S. Kannan et al.

    Cripto enhances the tyrosine phosphorylation of Shc and activates mitogen-activated protein kinase (MAPK) in mammary epithelial cells

    J Biol Chem

    (1997)
  • W.H. Jia et al.

    Trends in incidence and mortality of nasopharyngeal carcinoma over a 20–25 year period (1978/1983-2002) in Sihui and Cangwu counties in southern China

    BMC Cancer

    (2006)
  • X. Shao et al.

    Expression of an Epstein–Barr-virus receptor and Epstein–Barr-virus-dependent transformation of human nasopharyngeal epithelial cells

    Int J Cancer

    (1997)
  • L. Strizzi et al.

    Cripto-1: a multifunctional modulator during embryogenesis and oncogenesis

    Oncogene

    (2005)
  • Z. Wu et al.

    Cripto-1 overexpression is involved in the tumorigenesis of nasopharyngeal carcinoma

    BMC Cancer

    (2009)
  • S. Wang et al.

    Gene expression profile changes and possible molecular subtypes in differentiated-type nonkeratinizing nasopharyngeal carcinoma

    Int J Cancer

    (2011)
  • T. Fan et al.

    Up-regulation of 14-3-3zeta in lung cancer and its implication as prognostic and therapeutic target

    Cancer Res

    (2007)
  • J.S. Jang et al.

    The differential proteome profile of stomach cancer: identification of the biomarker candidates

    Oncol Res

    (2004)
  • I.N. Lee et al.

    Identification of human hepatocellular carcinoma-related biomarkers by two-dimensional difference gel electrophoresis and mass spectrometry

    J Proteome Res

    (2005)
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