44 High Efficacy of Gene Expression Profiling and Regularized Discriminant Analysis in the Prediction of Neuroendocrine Tumor Subtypes and Their Metastases

Abstract

Introduction: Invasion and metastasis are the key characteristics of tumor progression. Epithelial-mesenchymal-transition (EMT) is a developmental program that is often activated during cancer invasion and metastasis, especially at the invasive front. 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 (OKF6D1/d.n.p53) led to immortalization, additional EGFR overexpression (OKF6D1/d.n.p53/EGFR) induced an In Vitro transformed phenotype, whereas additional c-myc overexpression (OKF6D1/ d.n.p53/EGFR/c-myc) resulted in invasive cancer cells. To investigate the role of a master regulator of EMT and metastasis we overexpressed the transcriptional repressor ZEB1 in the different cell types. Additionally, to study the ability of these different cells to migrate we analyzed the expression and localization of different candidates involved in tumor invasion and metastasis: 1. collagen XVII a hemidesmosomal epithelial adhesion protein and 2. cathepsin B a lysosomal cystein protease. Methods: The expression of collagen XVII, cathepsin B and E-cadherin as a ZEB1 target gene was assayed in the distinct cell types by RT-PCR and western blot analysis. The cellular distribution was studied by immunofluorescence. The proteolytic activity of cathepsin B on the cell surface was determined by DQ-Collagen IV. Functional relevance of ZEB1 overexpression was analyzed in several cell types using a matrigel assay. Results: RT-PCR revealed a downregulation of collagen XVII expression in In Vitro transformed cells followed by an upregulation in cancer cells. Immunofluorescence demonstrated that the cellular localization of collagen XVII and cathepsin B is influenced by malignant transformation, i.e. collagen XVII is first translocated to the cytoplasm and relocated to the cell membrane in cancer cells. Cathepsin B is recruited from the lysosomes to the membrane during malignant transformation. Initial results showed a downregulation of the epithelial marker E-cadherin and an upregulation of the mesenchymal marker vimentin in ZEB1 overexpressing cells. Conclusions: In our human cellular model of oral-esophageal carcinogenesis we demonstrated that collagen XVII is differentially expressed during carcinogenesis. Cellular localization of collagen XVII and cathepsin B is defined by the distinct steps of tumor development. The recruitment to the membrane and secretion of cathepsin B leads to an increased degradation of the ECM. In the next step ZEB1 overexpression or at least E-cadherin downregultaion seems to be necessary for a invasive and metastatic phenotype.