The role of Cripto-1 in the tumorigenesis and progression of oral squamous cell carcinoma
Introduction
Oral cancer is a major health problem worldwide, with 264,000 new cases and 128,000 deaths reported annually.1 Squamous cell carcinoma is the most common malignancy of the oral cavity. Oral squamous cell carcinoma (OSCC) remains a lethal disease in over 50% of the cases diagnosed annually, due mostly to late detection of advanced stage cancer.2 Although there have been advances in the treatment and understanding of the underlying molecular pathogenesis of oral cancer, survival rates over the last several decades have not improved significantly. Therefore, there is great interest in determining novel molecular targets for treatment as well as early detection of OSCC.
Cripto-1, also known as teratocarcinoma-derived growth factor-1(TDGF-1), is a member of the EGF–CFC protein family. These proteins contain an epidermal growth factor (EGF)-like domain and a cysteine-rich region called the Cripto/FRL1/Cryptic (CFC) domain (Cripto in humans, FRL1 in Xenopus, and Cryptic in mice), and are involved in the activation of several different signaling pathways during embryonic development and cellular transformation.3 The major signaling pathways activated by Cripto-1 are the Nodal/ALK4/Smad-2 signaling pathway and the Glypican-1/c-Src/MAPK/AKT signaling pathway.3, 9 During embryogenesis, Cripto-1 functions as a cell-surface co-receptor for Nodal and other TGF-β ligands such as Xenopus Vg1 and its mouse ortholog, GDF-1; upon ligand binding, Cripto-1 binds to the Activin type I (ALK4)/ type II receptor complex, stimulating Smad-2 phosphorylation and activation.3 By participating in this signaling pathway, mouse Cripto/Cryptic regulates the formation of the primitive streak, patterning of the anterior/posterior axis, specification of the mesoderm and endoderm during gastrulation, and establishment of the left/right asymmetry of developing organs.4 Cripto-1 can also bind to Activin B and sequester it from the Activin type II receptor, thereby blocking Activin B’s ability to inhibit the growth of tumor cells.5 Therefore, antagonism of Activin signaling by Cripto-1 may represent one of the mechanisms by which Cripto-1 promotes tumorigenesis. In addition, Cripto-1 binding to Glypican-1 activates the downstream c-Src/MAPK/AKT signaling pathway in a Nodal- and ALK4-independent manner.8 Because MAPK and AKT signal transduction pathways have been shown to be essential for cell proliferation and survival,6, 7 Cripto-1 could also be involved in the pathogenesis of human cancer by inappropriately activating the Glypican-1/c-Src/MAPK/AKT signaling pathway.
Although the Cripto-1 protein has been identified as a marker of embryogenesis and is generally not expressed in normal adult tissues, Cripto-1 has been found to be overexpressed in several human cancers by immunohistochemistry, including breast, colon, lung, cervix, stomach, and pancreatic cancer.9 High expression of Cripto-1 in human cancers, as compared to that in normal tissues, suggests that Cripto-1 might be a target for cancer therapy.10, 11 Furthermore, it has been reported that Cripto-1 expression is significantly increased in premalignant lesions such as colon adenomas, intestinal metaplasia of the gastric mucosa, and ductal carcinoma in situ (DCIS) of the breast.9 These findings suggest that up-regulation of Cripto-1 may be an early event in carcinogenesis. In addition, in vitro and in vivo studies have shown that Cripto-1 plays an important oncogenic role during tumorigenesis by promoting cell proliferation, migration, invasion, and tumor angiogenesis, as well as inducing the epithelial-to-mesenchymal transition (EMT).9, 12, 13
To the best of our knowledge, no previous report has evaluated Cripto-1 expression in OSCC. Therefore, our aims in this study were to determine Cripto-1 expression in tissue samples of normal oral mucosa, epithelial dysplasia, and OSCC by immunohistochemistry, to analyze the correlation between Cripto-1 expression and clinicopathologic parameters, and to identify the oncogenic effects of Cripto-1 on OSCC cells.
Section snippets
Patients and tissue samples
Ten normal oral mucosa samples, 15 epithelial dysplasia samples, and 60 OSCC samples were studied by immunohistochemistry. All tumors were surgically removed at the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, between 1997 and 2002. Of the 60 OSCC patients, 41 were male, and the mean patient age was 59 years (range, 27–93 years). The patients’ clinical information, including age, gender, tumor size, lymph node metastasis, recurrence, and TNM stage, is
Immunohistochemical expression of Cripto-1 in normal oral mucosa, epithelial dysplasia, and OSCC tissue samples
Diffuse Cripto-1 staining was observed in the cytoplasm of mucosal epithelial cells and tumor cells. Cripto-1 expression was absent in eight normal oral mucosa tissue samples, but was weakly detected in the remaining two samples (20%) (Fig. 1A). Cripto-1 expression was detected in 11 (73.3%) of 15 epithelial dysplasia samples (Fig. 1B). Thirty-three (55.5%) of 60 OSCC samples were positive for Cripto-1. Cripto-1 positivity was significantly more frequently detected in both epithelial dysplasia
Discussion
Several studies have reported that Cripto-1 is differentially expressed in normal mucosa and premalignant lesions.16, 17, 18, 19 Furthermore, differential expression of Cripto-1 between noninvolved epithelium adjacent to tumors and the tumors themselves has been detected in stomach, colon, breast, and bladder carcinomas.20 A gradual increase in Cripto-1 expression from normal mucosa to premalignant lesions and from premalignant lesions to carcinoma was demonstrated in colon17 and breast tissue,
Conclusions
We evaluated the expression of Cripto-1 in OSCC tissues and cell lines using immunohistochemistry, RT-PCR, and western blot analysis. The in vitro role of Cripto-1 was also investigated by cell growth assays and migration assays after treatment of cells with rhCritpo-1 protein. Our results suggest that overexpression of Cripto-1 likely plays a role in the malignant transformation of oral mucosa and may be involved in the tumorigenesis and progression of OSCC by enhancing tumor cell growth and
Conflict of interest statement
None declared.
Acknowledgement
We are grateful to Prof. Bo-Hyong Jin for kind consultation and helpful remarks about statistics.
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