| The loss of homotypic adhesion is a crucial precursor to metastasis in solid tumors. Homotypic adhesion, mediated by cadherins, claudins, occludins, desmosomes, and a variety of other structural proteins, is especially strong and tightly regulated in healthy epithelium, and the disruption of these bonds is a hallmark of epithelial oncogenic progression. This work correlated gene expression with homotypic adhesion in a panel of human colon cancer cell lines in order to identify novel factors regulating adhesion. This correlative approach identified CD99 and CD99L2 as being functionally involved in homotypic adhesion. When expression of the two genes was silenced in SW480 cells, a reduction in the expression of CDH1 protein and an increase in the expression of vimentin protein was observed. This expression pattern is consistent with the activation of the epithelial-mesenchymal transition (EMT), a genetic program usually active in development but frequently aberrantly activated in cancer cells. Cells undergoing EMT have reduced homotypic adhesion and increased motility and invasion. Moreover, we examined expression in patient samples and found that CD99 is expressed on the cell surface membrane of normal colon tissue, but that expression is aberrant or lost in matched tumor tissue. This evidence suggested that CD99 and CD99L2 contribute to homotypic adhesion by signaling to maintain an epithelial phenotype, and when their expression was lost, the cells undergo EMT. Because the two proteins are structurally similar and have similar effects on homotypic adhesion through EMT, we hypothesized that they would share a common or overlapping genomic signaling pattern. We characterized the shared genomic signaling by observing total gene expression in SW480 cells following siRNA-mediated knockdown of either CD99 or CD99L2. A set of genes that were similarly affected by both knockdowns was identified. Recurrent network analysis of these genes identified several gene networks that promote cancer-related phenotypes such as invasion, anti-apoptosis, and proliferation. Analysis of these networks identified transcription factors, including NF-kappaB, PAX4, and MYC, whose binding sites are overrepresented in the promoter regions of the genes residing in these networks. This suggested that the genomic signaling caused by loss of CD99 or CD99L2 signaling could be mediated by changes in the activity of these transcription factors. We demonstrated that the binding of NF-kappaB, PAX4, and MYC was increased following knockdown of CD99. The increased activity of NF-kappaB was of particular interest because NF-kappaB is a master regulator of EMT. We demonstrated that knockdown of CD99 led to the activation of the canonical pathway of NF-kappaB, mediated by increased phosphorylation of AKT. Further, we demonstrated that inhibition of NF-kappaB activation significantly abrogated the effects of CD99 knockdown. This dissertation identified CD99 and CD99L2 as mediators of homotypic adhesion in colon cancer, demonstrated that these cell surface markers contribute by maintaining the cells' epithelial phenotype, established that in their absence cells undergo EMT, and characterized the genomic and non-genomic signaling caused by the loss of their expression. |
