Previously, we analyzed expression microarray data before and after treatment with demethylating agent in 2 NPC cell lines and speculated that the TFPI-2 gene may be downregulated by promoter hypermethylation in NPC cells. TFPI-2, also known as placental protein 5 and matrix-associated serine protease inhibitor [7, 23], is a novel serine protease inhibitor[7, 24]. It was reported to be involved in tumorigenesis and metastasis in several types of cancers. Suppression of TFPI-2 gene expression is frequently found in melanoma, liver and pancreatic cancer[9, 25, 26]. Downregulation of TFPI-2 mRNA and protein by promoter hypermethylation has been confirmed by RT-PCR, immunostaining, methylation specific PCR and bisulfate genomic sequencing [9, 27]. In this study, hypermethylation of the TFPI-2 promoter was detected in 4 of 6 (66.7%) NPC cell lines. We also showed a high frequency (88.6%) of TFPI-2 promoter hypermethylation in NPC primary tumor biopsies but not in the NNE tissues, which implied that transcriptional silencing of the TFPI-2 pathway might be involved in NPC tumorigenesis. As well, we found a high frequency of promoter hypermethylation of TFPI-2 in early stage (I and II) NPC, which indicates that this might be an early event in NPC carcinogenesis. In contrast to the high frequency of methylation of TFPI-2 in NPC cell lines and primary tumors, the absence of promoter hypermethylation in 12 histological NNE tissues showed that hypermethylation of TFPI-2 promoter is not necessary to maintain the normal phenotype in the nasopharyngeal epithelium. Thus, hypermethylation of TFPI-2 is a frequent and highly tumor-specific event in NPC. Such characteristics may suggest TFPI-2 methylation as a molecular marker for NPC.
By bisulfite genomic sequencing assay, we found methylation degrees ranging from 60% to 100% in most of the CpG dinucleotides in the TFPI-2-silenced NPC cell lines CNE2 and C666-1. A heavy degree of methylation was also observed in NPC primary tumors, despite the inevitable normal tissue contamination in the biopsy samples without micro-dissection. Moreover, TFPI-2 was completely restored by demethylation treatment in all 3 TFPI-2-silenced NPC cell lines. In this context, epigenetic inactivation of TFPI-2 by promoter hypermethylation is a major, if not the only, mechanism responsible for the loss of TFPI-2 expression in NPC.
Although promoter hypermethylation of TFPI-2 is frequently found in NPC, it is not associated with sex, age, nodal metastasis or cancer stage in our series. This finding is inconsistent with observations in pancreatic and non-small-cell lung cancer; a high methylation rate of TFPI-2 was reported in pancreatic cancer patients with liver metastasis. In non-small-cell lung cancer, TFPI-2 promoter hypermethylation was frequently found in patients with late-stage cancer (stages III and IV) and with lymph node metastases. These findings suggest that the prognostic value of promoter hypermethylation of TFPI-2 is tissue-specific.
The tumor-suppressive functions of TFPI-2 have been demonstrated in several human malignancies such as lung cancer, prostate cancer, glioma, melanoma and esophageal carcinoma. Ectopic expression of TFPI-2 protein in these cancer cell lines negatively regulates colony formation and inhibits cell proliferation[12, 27–30]. Additional studies have shown that TFPI-2 inhibits tumor-related angiogenesis and some members of the extracellular matrix (ECM) therefore implicates tumor invasion and progression[8, 12, 17, 29]. Degradation of the ECM is an essential process for tumor invasion and metastasis and involves various matrix-degrading proteinases. The most important proteolytic ECM enzymes are matrix metalloproteinases (MMPs), and upregulated MMP expression has been demonstrated to be strongly associated with the progression of malignancy in several types of cancer, including NPC[31, 32]. By inhibiting plasmin, TFPI-2 effectively decreases the activation of MMP-1, MMP-3 and MMP-9 and reduces the invasive potential of several cancer cell lines[9, 12, 33, 34]. In accordance with previous studies, we demonstrated that ectopic expression of TFPI-2 significantly inhibited cell proliferation, colony formation and migration in NPC cells. We also demonstrated that restoration of TFPI-2 induces apoptosis in NPC cells. All these evidence strongly implies that TFPI-2 is a putative TSG and plays a role in nasopharyngeal carcinogenesis.
Methylation-mediated inactivation is reversible, up-regulating TFPI-2 by demethylating agent may reverse the malignant phenotype of tumor cells. Therefore TFPI-2 can serve as a novel target for gene therapy in NPC treatment. On the other hand, TFPI-2 functions extracellularly as a secreted protein. Treatment with recombinant TFPI-2 protein inhibited tumor growth and metastasis of esophageal cancer. Thus, TFPI-2 protein might be used directly as an anticancer drug. Further studies will be necessary to explore the great therapeutic potential of TFPI-2 in NPC and other cancer.