Abstract 3002: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

Abstract

Abstract Autophagy 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 well-known regulator of vesicle trafficking and exosome secretion, also regulates autophagy process. Depletion of Rab27B in colorectal cancer (CRC) cells by CRISPR-Cas9 and siRNA resulted in an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62 indicating a defect in the autophagy flux. To characterize this autophagy defect, we used EGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining and identified that the degradation step of the autophagy flux is inhibited when Rab27B is silenced. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that autophagy defect resulting from Rab27B deletion will cause reduced stress response and tumor growth. Indeed, Rab27B knockout resulted in a 60% reduction in cell viability in response to starvation and a 94% reduction in colony formation in soft agar assay. 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 HCT116 and observed that Rab27B deletion resulted in a significant reduction of tumor size (p<0.0001). Next, to analyze the therapeutic potential of Rab27B inhibition in CRC, we sought to identify small-molecule inhibitors of Rab27B GTPase activity in vitro through high-throughput drug screening from a panel of ∼2,100 compounds contained within the KU-HTSL chemical library. This screening resulted in the identification of three compounds (BTB00809, BTB03584, and KM01532) that demonstrated Rab27B GTPase activity inhibition. Interestingly, inhibition of Rab27B in CRC cells with the compound km01532 resulted in a similar increase of LC3-II in CRC cells HCT116 and SW480 as seen in the Rab27B knockout. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on investigating the mechanism of how Rab27B functions in the autophagy process, and whether Rab27B can be targeted as a potential therapeutic strategy for CRC using the small molecule compound km01532. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan Dixon. Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3002.