Dysregulation of Ral GTPase-activating protein complex activates RalA signaling and supports metastasis of hepatocellular carcinoma through intrinsic and extracellular vesicle-dependent manners

Abstract

Accumulation of oncogenic mutations is a key pathogenetic cause leading to cancer. Although Ras or Ras-like (Ral) mutations are rare in hepatocellular carcinoma (HCC), aberrant RalA activation has been reported in HCC. In this study, our analysis of TCGA HCC cohorts revealed a stepwise increase of RalA along tumour progression and its upregulation was associated with poor prognosis. We further observed a 2-fold upregulation of RalA in 34.4% of our patients’ HCCs (N=90) and its upregulation was correlated with aggressive tumour behavior. Functionally, we observed a reduction of cell proliferation, migration and invasion upon RalA depletion. Furthermore, the cell stemness properties were impeded, as indicated by attenuated sphere forming ability. Consistent observations were obtained using in vivo limiting dilution and orthotopic liver injection models. Reciprocally, overexpression of RalA promoted tumour initiation, growth and metastasis both in vitro and in vivo. Besides, RalA upregulation was associated with copy number gain and transcriptional regulation by SP1 and ETS2. Importantly, double knockdown of SP1 and ETS2, as compared to either one alone, displayed the most prominent RalA protein decrease. Targeting RalA using RBC8 combined with sorafenib synergistically suppressed tumour growth underlying inhibition of p-S6. On the other hand, loss of Ral GTPase Activating Protein subunit A2 (RalGAPA2), the negative regulator of RalA activity, upregulated RalA signaling and further promoted intrahepatic and extrahepatic metastasis in vivo. As the partner of RalGAPA2, the regulatory subunit RalGAPB stabilized the complex and maintained its function as a tumour suppressor through inactivating RalA. Clinically, through targeted DNA-sequencing (N=95), mutations of RalGAP were identified in 12.7% of patients’ HBV-associated HCC tumours, indicating the dysregulation of RalGAP in HCC. Moreover, downregulation of RalGAPA2 was consistently found in our HCC cohorts and associated with poor prognosis. Interestingly, a subgroup with upregulation of RalA and concomitant downregulation of RalGAPA2 showed stronger association with venous invasion and TNM staging, further strengthening the negative relationship between RalGAP and RalA in clinical settings. Of note, we uncovered that RalA was able to regulate the secretion of small extracellular vesicles (sEVs) in HCC. Depletion of RalA decreased the protein density of sEVs while exogenous RalA had the reverse effect. We showed that this pro-migratory effect was abolished when RalA was ablated, and in contrast, exogenous RalA was able to enhance this promoting effect, supporting the critical role of RalA in sEV-mediated pro-migratory function. Upon further dissection of the protein profile of sEVs using mass spectrometry analysis, 8 sEV-associated proteins showed consistent alteration with RalA expression. Importantly, fibronectin (FN1) was downregulated and upregulated in sEVs derived from RalA depleted and overexpressing HCC cells, respectively, as validated by Western Blotting. Taken together, our study revealed RalA upregulation driven by copy number gain and transcriptional promotion contributing to HCC progression. Loss of RalGAPA2 activated RalA signaling to facilitate metastasis. Of significance, RalA also modulated sEV-associated protein FN1 to enhance cell migration in HCC. To conclude, loss of RalGAP-mediated RalA activation favor HCC progression through intrinsic and sEV-dependent manners, targeting RalA combined with sorafenib may serve as alternative therapeutic approach.