| This dissertation addresses the topic of epithelial morphogenesis in two parts. In part A, I examined the role of two relatively uncharacterized kinases, NDR1 and NDR2. While orthologs of these kinases have diverse effects on morphogenesis pathways in lower organisms, I was unable to find a striking NDR1/2 phenotype in mammalian cells.;In part B, I examined the effects of the K-Ras oncogene, which is mutated early in colon tumorigenesis, in a three-dimensional model of colon epithelial morphogenesis using the cell line Caco-2. Single cells seeded in extracellular matrix generate hollow, polarized structures after ten days. In contrast, expression of an oncogenic version of K-Ras (K-Ras V12) disrupted apical polarity and tight junctions, and promoted hyperproliferation, resulting in large, filled structures. Cells expressing oncogenic B-Raf (B-Raf V600E) phenocopied K-Ras V12 while inhibition of the downstream kinase, MEK, prevented all the observed effects of oncogene expression. These results suggest that the disruption in morphogenesis caused by K-Ras V12 expression is mediated exclusively by activation of the ERK MAP kinase pathway.;To investigate whether the loss of apical polarity and tight junction assembly seen in mature K-Ras V12 and B-Raf V600E structures was due directly to a defect in the first cell division in 3D, i.e. at the two-cell stage. Apical polarity is already established after the first cell division (two-cell stage) in control Caco-2 3D cultures. K-Ras V12 or B-Raf V600E expression, however, prevented the establishment of apical polarity and tight junctions at the two-cell stage and this was also mediated by ERK activation. Furthermore, expression of either K-Ras V12 or B-Raf V600E dramatically increased levels of c-myc. This upregulation of c-myc was due to phosphorylation of c-myc by ERK, which is known to prevent c-myc degradation. C-myc expression disrupted normal polarity establishment. In addition, RNAi-mediated depletion of c-myc prevented disruption of normal polarity establishment in K-Ras V12 or B-Raf V600E cells. I conclude that ERK-mediated upregulation of c-myc by K-Ras or B-Raf oncogenes disrupts the establishment of apical/basolateral polarity, and consequently the morphogenesis, of colon epithelial cells. |
