Role of endocytosis in regulating cancer cell migration

Abstract

Cell migration is significant in many physiological and pathophysiological processes, including wound healing, angiogenesis, embryonic development, vascular remodelling and inflammation. Migration is a necessary component of the metastatic cascade as cancer cells have the ability to detach from the primary site, migrate into the blood or lymphatic system and travel to a distant location. Emerging evidence suggests that endocytosis may regulate the migration of tumour cells, but the contribution of different endocytotic pathways have not been addressed in detail. Thus, our aim was to determine the link between endocytosis pathways with cell migration. Focal adhesions are necessary in many physiological and pathophysiological processes including cell survival, morphology, proliferation and cell migration. They are not only structural elements that provide the stability of the cells through its connection to ECM, they also can regulate internal and external signalling to trigger various cellular responses. Several studies have hypothesised that vesicles such as endosomes might interact with focal adhesions or be implicated in their turnover. Therefore, our aim was to examine the possible link between focal adhesions and the early endosomal compartment, including the influence of clathrin and dynamin pathways on focal adhesion dynamics as well as identifying signalling pathways affecting endocytotic regulation of cell migration and focal adhesion turnover. Cell migration using wound-healing assay and time-lapse microscopy on a variety of surfaces demonstrated that both micropinocytosis and caveolae pathways had no effect on cell migration. However, dynamin and clathrin dependent endocytosis pathways significantly decreased cell migration and impaired early endosome trafficking. Inhibition of dynamin or clathrin pathways, decreased the expression of EEA1 and numbers of endosomes containing EEA1, while leading to the enlargement of endosomes. Additionally, dynamin and clatherin dependent pathways significantly increase the number of paxillin and vinculin containing focal adhesions. In addition, both pathways significantly decreased focal adhesion turnover time. Another main finding is that endosomes associate with focal adhesion internalization. Immunocytochemical analysis and live cell imaging revealed that both early endosome markers Rab5 and EEA1 colocalized with focal adhesion proteins. This data was further supported by cell fractionation, and showed that Vinculin, Paxillin, Zyxin, FAK and Talin were found in the same fractions that endosomes were found in. Co-immunoprecipitation was performed to 7 confirm the association between endosomes and focal adhesions, and the results showed that vinculin was found to coimmunoprecipitate with Rab5 and EEA1. Finally, we identified that nitric oxide may be a positive regulator of cell migration and focal adhesion turnover and that relies on early endosome trafficking. Our results show that a significant decrease in migration speed in MDA-MB-231 cells when the total NO synthesis or inducible NO synthase (iNOS) were inhabited. In addition, these treatment cause a significant decrease in focal adhesion turnover time. Importantly, these treatments resulted in a significant increase in endosomes containing EEA1 size and simultaneously decreased its number. This observation was further confirmed by immunocytochemistry results where EEA1 was found to moderately co-localize with eNOS or iNOS, and S-nitrosylated compared to non-modified control proteins like H2B or ubiquitin. This result was further confirmed by bioinformatics analysis using the GPS-SNO algorithm where EEA1 was predicted to be S-nitrosylated at four cysteine residues - Cys-46, Cys-255, Cys-894 and Cys-1102, with a high confidence score. Taken together, our present study provides supporting evidence for the claim that endocytosis plays a crucial role in the migration of cancer cells. Moreover, it is suggested that there is a link between focal adhesions and early endosome markers, which indicates that the early-endosome compartment is involved in focal adhesion turnover.