In the study proposed here, the cDNA microarray technique in combination with statistical and computational methods for whole-genome functional analysis will be used to identify metastasis-related genes in oral squamous cell carcinoma (SCC) in a novel lymph nodal metastatic mouse model. As a result of a unique feature of the mouse model, the abundance of testing samples, and the powerful statistical methods that will be used for data analysis, this study promises to generate significant data and lead to further studies that will increase understanding of the expression of metastatic genes. It is an appropriate application for an Exploratory/Developmental Research Grant as defined in the Purpose section of the program announcement. To study the metastasis of oral SCC, we recently established highly metastatic oral SCC cell lines from a population of poorly metastatic oral SCC cells through in vivo selection using a modified floor-of-mouth (FOM) human tumor animal model. We compared the gene expression in 12 highly metastatic cell lines with their poorly metastatic parental cells using cDNA microarray analysis, rapid analysis of gene expression (RAGE), and northern and western blot analyses. The preliminary experiments revealed the existence of several genes and expressed sequence tags whose expressions were significantly altered in the selected metastatic cell lines. Based on our preliminary findings, we posit that the nodal metastatic mouse model in combination with cDNA microarray analysis can be used to identify alterations of gene expressions that are responsible for enhancing the metastatic potential of oral SCC and for facilitating the migration of SCC cells to and the growth in cervical lymph nodes. There are two Specific Aims in the study. The first is to identify the genes that are up- and down-regulated in highly metastatic oral SCC. DNA microarray and data mining methods such as cluster analysis and feature selection will be used to compare gene expression in highly metastatic primary tumor cells with that in poorly metastatic primary tumor cells and in lymph node metastases. RAGE and northern blot analysis will be done in both oral SCC cell lines and clinical specimens to further confirm the result from DNA microarray analysis. The second is to confirm the effect of differentially expressed genes on the metastatic phenotype of oral SCC cells. The cDNAs of the genes of interest will be inserted into a eukaryotic expression vector in either a sense or antisense format and transfected into either poorly metastatic parental cells or their highly metastatic derivatives. The transfectant will be examined using in vitro and in vivo assays to determine the roles of these genes in metastasis. This study will help to identify genes whose expression levels are crucial for metastatic behaviors. The findings from this project will serve as the basis for an NIH R01 grant application.
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