Role of the ARF6 GTPase in Epithelial Gland Morphogenesis

Morphogenesis and differentiation of internal epithelial organs is dependent on various processes including cell-cell and cell-matrix interactions, cell polarity and the interplay of cell survival and cell death pathways. Epithelial cancer progression is often accompanied by the disruption of epithelial gland architecture, although little is understood about the signaling pathways and cellular processes involved. However, various oncogenes have been shown to affect internal epithelial organ development and polarization by deregulating one or more of the aforementioned cellular processes described above.

This study describes a novel role for the ARF6 GTPase in the regulation of epithelial architecture and gland development. ARF6 functions downstream of Src, the activation of which leads to the targeting of the cell-cell adhesion molecule, E-cadherin, to the lysosome. By use of three dimensional cell-culture, the role of ARF6 in the development of tubules and cysts, the building blocks of epithelial glands has also been investigated. Tubules form in response to cues derived from morphogens such as hepatocyte growth factor. ARF6 is transiently activated during tubulogenesis and perturbing the ARF6 GTP/GDP cycle by inducible expression of ARF6 mutants defective in GTP binding or hydrolysis inhibits the development of mature tubules. Further, we show that activation of ARF6 is necessary and sufficient to initiate tubule extension. ARF6 regulates the subcellular distribution of the GTPase, Rac1, to tubule extensions.

Furthermore, ARF6-induced ERK activation regulates Rac1 activation during tubule initiation through the expression of the receptor for urokinase plasminogen activator.

Sustained activation of ARF6 also inhibits the normal progression of cyst development. Cysts expressing an ARF6-GTP mutant exhibit a significant pool of internalized E-cadherin thereby disrupting cyst architecture. In addition, the levels of Bcl-2 are higher in ARF6-GTP cysts relative to normal cysts thereby affecting lumen clearance by apoptosis. This latter step is dependent on ARF6 regulated ERK activation, which in turn is dependent on PLD. ARF6-dependent PLD activation promotes receptor endocytosis that ultimately permits ERK signaling from intracellular endosomes. Thus, the studies described in this research dissertation have identified a signaling apparatus downstream of ARF6 activation that regulates epithelial morphogenesis and could potentially contribute to epithelial cancer initiation and progression.