Abstract RalA is a Ras-related small GTP binding protein A; however, its functional roles and regulatory mechanisms in hepatocellular carcinoma (HCC) are unclear. In this study, using the RNA-Seq data from TCGA database and our in-house HKU database, we observed that RalA expression was significantly up-regulated in human HCCs (P=0.001). This RalA over-expression was validated in a separate cohort of our HCC patients. Upon clinicopathological correlation of RalA in HCC, we found that over-expression of RalA was associated with more aggressive features of HCC patients, with more frequent tumor microsatellite formation (P=0.001), venous invasion (P=0.005) and absence of tumor encapsulation (P=0.005). The over-expression also correlated with poorer overall survival of HCC patients (P<0.001). Functionally, we established RalA stable knockdown (KD) in three HCC cell lines (Hep3B, BEL7402 and MHCC-97L-Luc) and demonstrated that KD of RalA significantly inhibited cell proliferation, colony formation, migration and invasion in vitro. Conversely, ectopic expression of RalA, by transfecting the RalA dominant active form G23V construct into HCC cells, promoted HCC metastasis using transwell invasion assays. Also, with immunofluorescence assay, RalA mainly located in the cytoplasm and cell membrane. Over-expression of RalA changed the HCC cell morphology from polygonal to spindled shape, suggestive of epithelial-mesenchymal transition. We conducted in vivo study using the orthotopic liver injection model of RalA stable-KD MHCC-97L cells in nude mice, and observed that KD of RalA suppressed HCC tumorigenicity and reduced lung metastasis (P<0.001). Moreover, KD of RalA also reduced sphere formation ability of HCC cells, as well as suppressed the expression of stemness markers, including CD24, NANOG, NOTCH1, NESTIN, and EpCAM, using qPCR. For the liver cancer stem cell surface markers, the expression level of CD24 was significantly decreased in RalA KD Hep3B and BEL7402 cells by flow cytometry. Additionally, with RNA-Seq analysis of Hep3B and BEL7402 shRalA clones, 12 genes were found to be significantly altered (among them 5 were up-regulated and 7 were down-regulated) for both cell lines, when compared with in-house cohort as well as TCGA cohort. Further analysis showed that RalA expression level was positively and negatively correlated with a small group of genes. Taken together, our findings have shown that RalA is significantly up-regulated in human HCCs and its over-expression enhances HCC metastasis and cancer stemness. Further investigation of the interplay between RalA and its downstream targets will derive novel mechanistic insight regarding the oncogenic role of RalA in HCC. Citation Format: Luqing Zhao, Lo-Kong Chan, Daniel Wai-Hung Ho, Goofy Yu-Man Tsui, Macrina Wai-Ling Lam, Charles Shing Kam, Karen Man-Fong Sze, Vanilla Xin Zhang, Abdullah Husain, Joyce Man-Fong Lee, Irene Oi-Lin Ng. RalA is frequently up-regulated in human hepatocellular carcinoma and promotes metastasis and cancer stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 91.