Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer
IntroductionAutophagy is a dynamic, multi-step process that cells use to degrade damaged, abnormal, and potentially harmful cellular substances. While autophagy is maintained at a basal level in all cells, it is activated at a higher level in many cancer cells and promotes tumor growth, anti-tumor immune response, and resistance to cancer therapy. As a result, autophagy is increasingly being recognized to have an important role in cancer progression and emerging as a potential target for cancer therapy. We recently discovered that small GTPase Rab27B, a known regulator of vesicle trafficking and exosome secretion, is also involved in the autophagy process. MethodsRab27B was knocked out using CRISPR/Cas9 in CRC cell line HCT116. Western blotting, Immunofluorescence, MTT assay, spheroid formation assay, soft agar assay and xenograft studies were performed to analyze the effects of Rab27B deletion on CRC cells. ResultsCRISPR/Cas9 deletion or siRNA knockdown of Rab27B in colorectal cancer cells (CRC) showed an abnormal accumulation of autophagy vesicles. Additionally, we observed a significant increase in the autophagy markers LC3-II and p62 by immunocytochemistry and western blot analysis, suggesting a defect in the autophagy flux process. Lysotracker and mCherry-EGFP-LC3 fusion construct indicate an impairment in autophagosome and lysosome fusion when Rab27B is silenced. This defect was rescued by full-length and constitutively active GTP mutant of Rab27B. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that the observed defects in autophagy flux resulting from Rab27B loss would cause reduced stress response and tumor growth. Indeed, Rab27B knockout reduced cell viability in response to starvation and a 94% reduction in soft agar colony formation. Rab27B deletion also prevented spheroid formation in vitro. Finally, to analyze the effect of Rab27B deletion in tumor formation in vivo, we performed a xenograft study with wildtype and Rab27B knockout CRC cells, resulting in a dramatic loss of tumor growth (p<0.0001) in the KO cells. ConclusionsTogether, our results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Future studies will focus on investigating the mechanism of how Rab27B functions in the autophagy pathway and whether Rab27B can be targeted as a potential therapeutic strategy for CRC.