Human Cripto-1 is increased in several types of cancer and can function as an oncogene in vitro
and in vivo
]. However, there has been no report about the relations between Cripto-1 and NPC. In this study, we presented the first evidence that Cripto-1 was overexpressed at both transcriptional and translational levels in NPC cell lines. Using immunochemistry and semi-quantitative RT-PCR analysis, we found that Cripto-1 protein was expressed in 54.1% (20 of 37 cases) and 76.5% (13 of 17 cases) human NPC tissues, respectively. However, only 28.6% (2 of 7 cases) nasopharygitis showed Cripto-1 positive. We also found that Cripto-1 overexpression was significantly associated with N classification, distant metastasis, and clinical stage. Our results highlight the clinical significance of Cripto-1 in NPC. Expression of Cripto-1 shows 50-80% variance, as detected by immunohistochemistry, in different types of tumour tissues. More recently, Cripto-1 was detected in almost half of the breast cancer tissue samples analyzed and found to correlate with advanced stage disease[19
]. Our results are consistent with these reports and thus, NPC joins the list of the tumors that express Cripto-1.
Until now, numerous in vitro
and in vivo
studies have shown that cripto-1 act as an important factor during tumorigenesis by promoting cell proliferation, survival, migration and invasion. Overexpression of Cripto-1 cDNA in normal mouse fibroblasts induce these cells to grow in soft agar and increase growth rates in several human breast cancer cell lines[20
]. Human MCF-7 breast cancer cells that overexpress Cripto-1 proliferate at higher rates in serum-free medium, form increased numbers of colonies in soft agar, are more resistant to apoptosis when grown under anchorage independent conditions, and show increased propensity to invade and migrate in vitro
]. MMTV-Cripto-1 transgenic mice and WAP-Cripto-1 transgenic mice that overexpress the human Cripto-1 transgene showed increased incidence of mammary gland tumors[9
]. To explore the possible role of Cripto-1 in promoting NPC cell proliferation and invasion, we knocked down the expression of Cripto-1 in CNE-2 and C666-1 by lentivirus-mediated RNAi silencing. Then through a continuous MTT assay, we found that the cell growth was suppressed after the inhibition of endogenous Cripto-1 protein, suggesting a role of Cripto-1 in promoting tumor cell growth. However, flow cytometry analysis showed no significant change on the cell cycle of CNE-2 cells after Cripto-1 silence. This result suggests that Cripto-1 might promote cell growth in other pathway rather than impact on cell cycle. However, due to high proliferative rate of CNE-2 cells and incomplete RNAi suppressive effect (knock-down rather than knock-out), the negative results of cell cycle analysis need to be further verified.
To investigate the proliferation of the tumor cells in vivo after Cripto -1 gene silencing, we employed the xenotransplant nude mice model with whole-body visualizing instrument. Data showed that both the average volume and weight of tumor in CNE-2/GFP+/Cripto-1- xenotransplant mice group were significantly lower than those in the control group, indicating that the Cripto-1 gene silencing could partially inhibit the growth of CNE-2 cells in vivo. Ki67, a nuclear protein regulating cell cycle, is a biomarker of cell proliferation. Our results showed that the number of cells immunolabeled for Ki67 in the CNE-2/GFP+/Cripto-1- xenotransplant tumors was significantly less than that in the control group, suggesting that a decreased proliferation of CNE-2 cells could be achieved by Cripto-1 knock-down.
Association of Cripto-1 with metastatic potential has been found in a few human cancers. Ciardiello et al. have reported that Cripto-1 mRNA expression is found in 68.2% (30/44) of primary colonic cancers and 61.8% (21/34) of liver metastases, but in only 1.5% (1/65) of normal tissues[22
]. Furthermore, the Cripto-1 expression level was increased in lymph node metastases compared with their primary tumours[23
]. In our study, Cripto-1 mRNA was highly expressed in CNE-2 and C666-1 cell lines which are highly metastatic and proliferative. In the contrary, in CNE-1 cell line with no metastatic ability, Cripto-1 was expressed lowly. We also found Cripto-1 expression correlates with metastasis in NPC patients. In addition, by in vitro
invasion assay, substantial suppression of cell invasion was observed after endogenous Cripto-1 interference. All of these data suggest an association between Cripto-1 and tumour cell motility, invasion, and metastasis. Thus, our studies have shown that Cripto-1, as a potentially oncogenic protein, might play an important role in the tumorigenesis and progression of NPC.
However, the possible mechanism and the exact mode of Cripto-1 action during tumor metastasis and progression are still largely unknown. Although, we have not demonstrated clearly the mechanisms underlined the inhibition of Cripto-1 gene silencing, it has been evidenced that in addition to functioning as a Nodal co-receptor, Cripto-1 has been shown to mediate signaling of other TGF-β
ligands, such as Activin and Xenopus Vg1 and its ortholog in mouse GDF1[24
]. In contrast, binding of Cripto-1 to Activin and TGF-β
1 can inhibit Activin and TGFβ
-1 signaling in mammalian cells[25
]. Moreover, Cripto-1 can also activate the ras/raf/MAPK and PI3-K/AKT/GSK-3β
intracellular signaling pathways independently of Nodal and ALK4[26
]. Some data suggest that Cripto-1 may be involved in regulating integrin signaling either directly by binding to integrins and subsequently activating integrin signaling or indirectly by regulating the expression of extracellular matrix proteins which are also capable of binding integrins and activating integrin signaling[5
]. Several studies have also suggested that the Wnt/b-catenin/Lef-1 signalling pathway may cross-talk with the Cripto-1 signaling pathway, regulating cell adhesion and migration[17
]. Strizzi et al. have reported that Cripto-1 may promote the increased expression of markers and signaling molecules associated with EMT[17
]. Also, in the Cripto-1 transgenic mammary gland tumors, the zinc-finger repressor transcription factor, Snail, known to down-regulate or interfere with the normal expression of E-cadherin, was detected at significantly higher levels as compared to normal control mammary tissue, thus suggesting a novel link between Cripto-1 expression and Snail activity[17
Since high expression of Cripto-1 can be detected in human cancers, as compared to normal tissues, this signaling pathway might represent a target for cancer therapy. This is supported by findings describing the use of antisense oligonucleotides that reduce Cripto-1 expression and cause significant reduction of cell proliferation in vitro
]. In addition, neutralizing antibodies against Cripto-1 were able to significantly inhibit tumor cell growth in two xenograft models with testicular and colon cancer cells that express very high levels of Cripto-1[25
]. Moreover, rat monoclonal antibodies directed against the EGF-like domain of the Cripto-1 peptide also produced a significant inhibition of in vitro
and in vivo
growth of colon cancer and leukemia cells[30
]. Our data also demonstrated that lentivector-mediated RNAi was feasible for the inhibition of the growth and invasion of NPC cells in vitro and in vivo
, indicating the siRNA sequences targeting Cripto-1 could be a potential target for gene therapy of NPC.