Abstract B05: The RNA-binding protein HuR enhances exosome secretion in colorectal cancer

Abstract

Abstract Enhanced secretion of exosomes by cancer cells is recognized as a means of transferring oncogenic information within the tumor microenvironment. Through their ability to carry specific RNA and protein cargo, tumor-derived exosomes are now being recognized for their ability to impact the tumor microenvironment and as promising cancer biomarkers. However, our knowledge of the cellular factors that promote increased exosome production from tumor cells and how they impact the loading of specific tumor-promoting RNA cargo is limited. Our prior work has established that colorectal cancer (CRC) cells and tumors overexpress the key RNA-binding protein HuR (ELAVL1) early in GI tumor development. When overexpressed and present in the cytoplasm, HuR can promote mRNA stabilization of tumor-promoting genes through binding of 3'UTR AU-rich elements (ARE). These same mRNAs are within tumor-derived exosomes, suggesting a role for HuR. To test this, Tet-regulated HuR-inducible HeLa cells were used to demonstrate that cytoplasmic HuR overexpression promoted a 4-fold increase in exosome production. Furthermore, HuR was detected in exosomes produced only from HuR-overexpressing cells. The presence of HuR in exosomes directly impacted mRNA cargo, as selective uptake of ARE-containing mRNAs were observed in exosomes derived from HuR overexpressing cells. To assess if these effects were seen in CRC cells that endogenously overexpress HuR, exosome levels from CRC cells were compared to normal human intestinal epithelial and myofibroblast cells. CRC cells secrete ~3-fold greater exosome levels than normal cells and enhanced exosome production was dependent upon HuR. siRNA knockdown and CRISPER/Cas9 knockout of HuR in CRC cells showed a >2-fold decrease in secreted exosomes, similar to the levels observed in normal cells. Furthermore, HuR knockout CRC cells showed a ~3-fold reduction in xenograft tumor growth compared to parental CRC cells. These findings were reflected in vivo where GI-tumor bearing APCMin/+ mice produced ~3-fold more serum exosomes, with HuR as exosomal cargo in APCMin/+ mice, whereas limited expression of exosomal HuR was detected in wild-type mice. This work has identified a novel connection between HuR-mediated post-transcriptional regulation and tumor-derived exosome production, along with providing the first preclinical evidence indicating the presence of exosomal HuR as a serum-based CRC biomarker. Citation Format: Ranjan Preet, Wei-Ting Hung, Shufei Zhuang, Lane K. Christenson, Dan A. Dixon. The RNA-binding protein HuR enhances exosome secretion in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B05.