Study on the Expression and Regulatory Mechanism of Tumor Metastasis Suppressor Nm23-H1: Establishing Platforms for Studying and Screening Chemotherapeutic Agents Using Genomic Promoter Approaches

Carcinoma of the esophagus is one of the most lethal malignant tumors worldwide. Despite recent improvement in the diagnosis and multimodal treatment, the prognosis has not improved substantially in the past decades. The overall 5-year survival rate remains below 15%. Many researches had showed that early lymphatic and distant metastasis and chemoradioresistance are the major causes of treatment failure. Nm23-H1 has been regarded as a metastasis-suppressor factor in various tumors. Our previous studies had demonstrated that overexpression of Nm23-H1 was associated with suppression of lymphatic metastasis and chemosensitivity in oral squamous cell carcinoma (Wang et al., Br J Cancer 2004). In esophageal cancers, negative Nm23-H1 expression was correlated to poor prognosis (Wang et al., Eur J Cardiothorac Surg, 2004). These clinical studies indicated that Nm23-H1 involve some common molecular mechanisms shared by the two phenotypes (metastatic suppressor and chemosensitivity). Cell lines experiments validated that Nm23-H1 knockdown increases migratory/invasive ability and chemoresistance in ESCC cells in our current study, interestingly; it was also found that Nm23-H1 expression is regulated by the chemotherapy treatment and promoter methylation. However, few data is available about the regulation of Nm23-H1 expression related with chemotherapy treatment or epigenetic modification. Based on our observations, it is therefore important to study the expression regulatory mechanism of metastasis-suppressor factor contributing to early invasion/metastasis and chemoresistance in esophageal cancer. We propose that the regulatory mechanism of Nm23-H1 expression in ESCC cells may play a crucial role in Nm23-H1 abundance and thus in modulating the metastatic behavior and chemosensitivity. This basic research project will be designed to establish an analysis platform of Nm23-H1 promoter activity toward linking our clinical outcomes. We expect to identify the cis-acting regulatory elements, methylation profile for Nm23-H1 promoter and define the mechanisms of Nm23-H1 transcriptional regulation in ESCC. This analysis platform of promoter activity will be applied for chemoteraputic agents screening and then provide a novel strategy for clinical practice to overcome common mechanisms shared by these two phenotypes. The specific aims of this project therefore will be: 1) Analyze the DNA methylation status, methylated region and transcriptional activity of Nm23-H1 promoter in ESCC cell lines (i.e. different metastasis/invasion and chemoresistance phenotypes). 2) Establish ESCC cell models carrying a reporter gene under the control of different 5’-flanking regulatory Nm23-H1 promoter region. Identify the minimal promoter region and characterize the cis-acting regulatory elements as well as corresponding transcription factors in ESCC cell models. 3) Using transfected ESCC cell models to define the functional cis-acting regulatory elements during the treatment of chemotherapeutic agents, and screen the chemotherapeutic agents appropriate for highly metastatic and resistant phenotype.