The Study of the Mechanisms of Carbonic Anhydrase IX in Oral Cancer Carcinogenesis

• Main Authors: Jia-Sin Yang, 楊嘉欣
• Other Authors: 楊順發
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/15465548379656797566

博士 === 中山醫學大學 === 醫學研究所 === 101 === Oral cancer has the fastest growing incidence and mortality rates among all cancer types in Taiwan. Oral cancer is a solid malignant tumor that is prone to occur following hypoxia. Carbonic anhydrase IX (CAIX), which is a hypoxia-induced transmembrane protein, is highly expressed in various forms of cancer. However, the correlation between CAIX and the incidence of oral cancer in Taiwan has not been clarified. In this study, we examined the expression of CAIX in clinical specimens, and used the results to determine the correlation between CAIX and clinicopathological parameters. In addition, we conducted cell experiments to investigate the role of CAIX in the molecular mechanisms of oral cancer. First, we used an enzyme-linked immunosorbent assay (ELISA) to determine the plasma CAIX concentrations of 100 healthy control participants, 30 patients diagnosed with oral submucosal fibrosis (OSF), and 191 oral cancer patients. The results indicated that the CAIX expression in the oral cancer patients (p<0.001) and OSF patients (p<0.001) were substantially higher than that of the healthy participants, and the plasma CAIX levels of oral cancer patients were positively correlated to patient age (p<0.05), history of chewing areca nuts (p<0.05), tumor size (p<0.05), and tumor stage (p<0.05). We then performed an immunohistochemistry (IHC) test to analyze the CAIX expression in 259 oral cancer tissue specimens. The results indicated that the CAIX expression in oral cancer tissue was positively correlated to distant metastasis (p<0.05) and tumor stage (p<0.05). Subsequently, we conducted a wound healing assay and a Boyden chamber assay to examine an SCC-9 human oral cancer cell line, which consistently overexpresses CAIX. The results indicated that the overexpression of CAIX increases the movement and invasion capacity of SCC-9 cells. In addition, we performed reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, and western blot tests and found that CAIX overexpression increases the mRNA and protein expressions of matrix metalloproteinases-9 (MMP-9), phosphorylation expression in the focal adhesion kinase (FAK), steroid receptor coactivator (Src), and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proteins, and nucleoprotein expression in transcription factors nuclear factor-κB (NF-κB), c-Jun, and c-Fos. We also performed a luciferase reporter gene assay and a chromatin immunoprecipitation (ChIP) assay and found that CAIX overexpression increases the binding capacity of NF-κB, c-Jun, and c-Fos regarding the NF-κB and activator protein-1(AP-1) binding sites on the MMP-9 gene promoter. According to the results obtained from the clinical specimens, we inferred that CAIX expression can be used as a biomarker to predict the course of oral cancer. In addition, the cell experiments confirmed that the overexpression of CAIX increases the phosphorylation of FAK, Src, and ERK1/2, thereby enabling transcription factors NF-κB, c-Jun, and c-Fos to translocate and enter the nucleus, binding to the NF-κB and AP-1 binding sites on the MMP-9 gene promoter. This reaction increases the mRNA and protein expressions of MMP-9, which consequently induces the metastasis of oral cancer cells.