Cancer progression can be described as a continuum ranging from normal, healthy tissue to advanced metastatic disease. In epithelial tissues, glandular disruption and loss of epithelial integrity are salient features of early tumorigenesis. Continued advancement results in cells penetrating the basement membrane, invading neighboring tissue and finally forming distant metastases. This study examines the role of the ARF6 GTPase during tumor progression and describes cellular events that promote the loss of glandular architecture and the regulation of cell invasion.
We begin by quantifying the impact of unregulated ARF6 signaling on epithelial glandular morphology. Constitutive activation of ARF6 in renal epithelial cells (MDCK), or chronic CSF-1R stimulation in mammary epithelial cells (MCF-10A), grown in 3-dimensional reconstituted basement membrane cultures leads to the disruption of normal glandular architectures. These changes are dependent on ARF6-regulated endocytosis and accompanied by internalization of surface receptors and activation of the MAP kinase pathway. Inhibiting endocytosis or ERK signaling results in the reversion of abnormal epithelial cysts/acini to normal glandular morphologies. We go on to demonstrate that the multilumen phenotype in ARF6-GTP epithelial cysts results from a spindle orientation defect. This defect is caused by the formation of signaling endosomes, containing the receptor tyrosine kinase c-Met, which are formed as a result of elevated levels of ARF6-GTP.
In addition to glandular disruption, ARF6 activation has profound effects on cell invasion, in particular the shedding of invasive microvesicles. This dissertation outlines a mechanism through which the protease MT1-MMP is trafficked into nascent microvesicles via association with the SNARE protein VAMP3. VAMP3 knockdown results in the release of microvesicles lacking endogenous MT1-MMP that are incapable of matrix degradation. Additionally, VAMP3 depletion diminishes the ability of melanoma cells to invade through layers of extracellular matrix in an amoeboid manner by disrupting a CD9/BAP31/VAMP3/MT1-MMP trafficking complex. Finally, upon release, ARF6, MT1-MMP and VAMP3 positive microvesicles can be isolated from ascites and serum of ovarian cancer patients. Along with markers being investigated for use in ovarian cancer diagnostics, we?ve confirmed the inclusion of nucleic acids among the repertoire of microvesicle cargo. Together these studies identify the impact of membrane traffic and its regulation on tumor formation via glandular disruption and cell invasion.