ARF6 promotes the nuclear translocation of β-catenin to facilitate AFP deficiency-mediated metastasis in CTNNB1S33Y-mutant hepatocellular carcinoma

The mortality rate associated with hepatocellular carcinoma (HCC) remains alarmingly high, positioning it as a primary lethal cancer worldwide, primarily driven by its high degree of malignancy, recurrence, and metastasis. The high heterogeneity of HCC necessitates the urgent identification of new therapeutic targets and molecular pathways. Our previous research observed that alpha-fetoprotein (AFP) deficiency led to pro-metastatic roles in HCC with specific genotypes. Here, we further investigated the underlying molecular mechanism. Through proteome microarray screening, we identified that ADP-ribosylation factor 6 (ARF6) was a key AFP-interacting protein and AFP deficiency upregulated ARF6 activity, which subsequently promoted the metastatic capacity of HepG2 cells. Mechanistically, the abnormal activation of ARF6 induced the dissociation of β-catenin from its membrane-bound anchors, specifically E-cadherin. The consequent increase in free cytoplasmic β-catenin facilitates its nuclear translocation, which in turn activates Wnt/β-catenin pathway. Crucially, we discovered that the pro-metastatic function of activated ARF6 and the sensitivity to the ARF6 inhibitor were strictly dependent on the CTNNB1 genotype. In highly invasive MHCC-97H cells, which harbor a CTNNB1 nonsense mutation, ARF6 inhibitor SecinH3 failed to inhibit migration and invasion. However, the overexpression of CTNNB1S33Y mutation in MHCC-97H cells further amplified the increasing transcriptional activity of β-catenin triggered by the ARF6 abnormal activation. Importantly, the reintroducion of CTNNB1S33Y mutation restored the inhibitory effect of SecinH3 in vitro and significantly suppressed the hepatic and pulmonary metastasis in a tail vein injection HCC metastasis mouse model. These findings establish a CTNNB1S33Y-dependent mechanism for ARF6-mediated metastasis, indicating that CTNNB1 activating mutations serve as a "permissive signal" for ARF6's pro-metastatic program. Our study underscores the importance of CTNNB1 genotype screening for precisely stratifying patients for ARF6-targeted therapies, offering new medication guidance for highly heterogeneous HCC.