Investigation of the transcriptional and epigenetic mechanisms for the Nm23-H1 expression: An nm23-H1-promoter screening platform for novel metastasis-suppressing anticancer drugs

Nm23-H1 (also known as NME1 or Nm23A) was the first tumor metastasis-suppressor gene identified, and has the most data with regard to clinical prognosis. High expressions of Nm23-H1 are generally associated with improved prognosis for various carcinomas (breast, gastric, head/neck, hepatic cancer, melanoma, and NSCLC). 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 clearly indicated that Nm23-H1 expression level in the tumors have a prognostic impact. 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 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 design a sensitive and high-throughput approach for assessing the relationship between the methylation profile and transcriptional activity for of Nm23-H1 promoter, to analysis the cis-acting regulatory elements, and define the mechanisms of Nm23-H1 transcriptional regulation. This analysis platform of promoter activity will be applied for anticancer drugs screening and then provide a novel strategy for clinical practice to overcome metastasis and chemoresistance in the tumors. The specific aims of this project will be: 1) Design a sensitive and high-throughput approach (methylation-sensitive high resolution melting curve analyze) to assess the DNA methylation status for Nm23-H1 promoter region in different metastasis/invasion and chemoresistance phenotypes of ESCC cell lines. 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 cancer cell models to define the functional cis-acting regulatory elements during the treatment of anticancer drugs, and screen those drugs appropriate for highly metastatic and resistant phenotype.