Regulation of Clathrin Mediated Endocytosis and Its Role in Cancer Progression

Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities in 29 independently isolated non-small cell lung cancer (NSCLC) cell lines to determine whether there were systematic changes in the three different endocytic pathways. However we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. We also used the In-Cell ELISA assay to characterize Ikarugamycin (IKA), a previously discovered antibiotic, which inhibits the uptake of oxidized low-density lipoproteins in macrophages, as well as clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA had yet been performed. Therefore, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We showed that IKA acutely inhibits CME, but not other endocytic pathways with an IC50 of 2.7 μM. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME were reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking. Finally, we investigated possible mechanisms that lead to altered endocytosis in cancer cells. We discovered that dynamin 1 (Dyn1), previously thought to be neuron specific is frequently upregulated and postranslationally regulated in cancer cells. Dyn1 expression alters the proliferation rates, growth in soft agar, and tumor growth of cancer cells. We hypothesize that these changes are due to alteration in cell surface protein expression and downstream signaling pathways and have developed protocols to test these hypothesizes. Taken together, our results suggest that endocytic alterations in cancer cells can significantly influence cancer-relevant phenotypes.