| Notes: | Advisor: Kay F. Macleod.
Includes supplementary digital materials.
Thesis (Ph.D.)--The University of Chicago, Division of the Biological Sciences, and The Pritzker School of Medicine, Committee on Cancer Biology, 2014.
Dissertation Abstracts International, Volume: 75-07(E), Section: B.
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| Summary: | Macro-autophagy (hereafter autophagy) is a catabolic process important for degradation of damaged organelles and protein aggregates and intracellular recycling of proteins and lipids. Autophagy is thought to suppress tumor initiation by promoting genome stability and limiting necrosis and inflammation, but conversely is utilized by tumor cells to survive nutrient stress, hypoxia, and cytotoxic therapies, promoting tumor growth in established tumors. Consequently, autophagy has emerged as a potential therapeutic target in a number of solid tumor types. However, little is known about the role of autophagy during metastasis. Autophagy promotes tumor cell survival during extracellular matrix detachment, growth factor deprivation, and glucose deprivation in vitro, conditions that disseminated tumor cells must survive in order to form overt metastases. Clinical studies have found increased staining for the autophagy marker LC3B in melanoma metastases compared to matched primary tumors, and associated increased LC3B staining with node positivity and decreased overall survival in breast cancer, while over-expression of the key autophagy regulator Beclin1 was also linked to earlier melanoma metastasis. Based on these observations, I hypothesized that autophagy could play an important role in promoting metastasis in vivo.
I demonstrate here that inhibition of autophagy through stable knockdown of key autophagy proteins or treatment with hydroxychloroquine markedly reduced tumor metastasis in vivo. This effect was not due to decreased proliferation or increased cell death. Instead, inhibition of autophagy prevented RhoA-mediated protrusion formation, cell spreading and migration due to a defect in focal adhesion disassembly arising from accumulation of the focal adhesion protein paxillin, which is degraded by autophagy. I also show that active RhoA is localized at autophagosomes, and that inhibition of RhoA activity inhibits not only cellular protrusion but also autophagic flux in metastatic cells. These results establish a new paradigm wherein RhoA signaling is required for autophagic flux that in turn promotes focal adhesion turnover necessary for cell migration and invasion of metastatic tumor cells, and identify a novel function for autophagy in focal adhesion turnover, tumor cell motility and metastasis.
Included supplementary files: Supplemental videos 1-3 are movies of timelapse differential interference contrast (DIC) imaging of control and autophagy-deficient 4T1 tumor cells demonstrating altered cellular morphology and plasma membrane protrusion in autophagy-deficient cells.
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