Characterization of candidate genes influencing migration, invasion and metastasis in genetically generated oral-esophageal cancer cells

Introduction: The ability of a tumor cell to migrate across the extracellular matrix (ECM) is a prerequisite for metastasis. In our cellular model of human oral-esophageal carcinogenesis we were able to recapitulate tumor development in a stepwise fashion. Cyclin D1 overexpression and p53 inactivation led to immortalization, additional EGFR overexpression induced an in vitro transformed phenotype, whereas additional c-myc overexpression resulted in invasive cancer cells. To study the ability of these different cells to migrate, we analyzed the expression and localization of different candidates thought to be involved in tumor invasion and metastasis: 1. Collagen XVII, a hemidesmosomal epithelial adhesion protein, 2. E-Cadherin, a cell adhesion molecule of adherens junctions, 3. the cell surface protein CD24 and 4. the lysosomal cysteine protease Cathepsin.

Methods: The expression of these candidate genes was assayed in immortalized oral keratinocytes (OKF6D1/d.n.p53), in in vitro transformed OKF6D1/d.n.p53/EGFR and OKF6D1/d.n.p53/EGFR/c-myc cancer cells by real-time PCR and western blot analysis. The cellular distribution of these proteins was studied by immunofluorescence. Furthermore, we analyzed the migration behavior in our cellular model using a matrigel assay. To eventually increase the metastatic potential we additionally knocked down the E-Cadherin gene via siRNA.

Results: RT-PCR revealed a downregulation of collagen XVII expression in transformed cells followed by an upregulation in cancer cells. Immunofluorescence demonstrated that the cellular localization of collagen XVII, E-Cadherin and Cathepsin B is influenced by malignant transformation. Migration and invasion appears to be increased only in the genetically generated cancer cells overexpressing c-myc.

Conclusions: In our human cellular model of oral-esophageal carcinogenesis we demonstrated that only collagen XVII is differentially expressed during carcinogenesis. Nevertheless, cellular localization of collagen XVII, E-Cadherin and Cathepsin B is defined by the distinct steps of tumor development. Finally, these different findings lead to the conclusion, that in vitro generated cancer cells acquire features, potentially allowing to migrate through the ECM and ultimately to metastasize.