Quantitative proteome analysis of overexpressed Cripto-1 tumor cell reveals 14-3-3γ as a novel biomarker in nasopharyngeal carcinoma
Graphical abstract
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).
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Contributed equally to this work.