Background and Aims Ras‐like (Ral) small guanosine triphosphatases (GTPases), RalA and RalB, are proto‐oncogenes directly downstream of Ras and cycle between the active guanosine triphosphate‐bound and inactive guanosine diphosphate‐bound forms. RalGTPase‐activating protein (RalGAP) complex exerts a negative regulation. Currently, the role of Ral up‐regulation in cancers remains unclear. We aimed to examine the clinical significance, functional implications, and underlying mechanisms of RalA signaling in HCC. Approach and Results Our in‐house and The Cancer Genome Atlas RNA sequencing data and quantitative PCR data revealed significant up‐regulation of RalA in patients’ HCCs. Up‐regulation of RalA was associated with more aggressive tumor behavior and poorer prognosis. Consistently, knockdown of RalA in HCC cells attenuated cell proliferation and migration in vitro and tumorigenicity and metastasis in vivo. We found that RalA up‐regulation was driven by copy number gain and uncovered that SP1 and ETS proto‐oncogene 2 transcription factor cotranscriptionally drove RalA expression. On the other hand, RalGAPA2 knockdown increased the RalA activity and promoted intrahepatic and extrahepatic metastasis in vivo. Consistently, we observed significant RalGAPA2 down‐regulation in patients’ HCCs. Intriguingly, HCC tumors showing simultaneous down‐regulation of RalGAPA2 and up‐regulation of RalA displayed a significant association with more aggressive tumor behavior in terms of more frequent venous invasion, more advanced tumor stage, and poorer overall survival. Of note, Ral inhibition by a Ral‐specific inhibitor RBC8 suppressed the oncogenic functions in a dose‐dependent manner and sensitized HCC cells to sorafenib treatment, with an underlying enhanced inhibition of mammalian target of rapamycin signaling. Conclusions Our results provide biological insight that dysregulation of RalA signaling through dual regulatory mechanisms supports its oncogenic functions in HCC. Targeting RalA may serve as a potential alternative therapeutic approach alone or in combination with currently available therapy.

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