Background Cancer cells with relatively high epithelial-mesenchymal transition (EMT) inclination are reported to be vulnerable to ferroptosis. However, the alternative splicing-mediated reprogramming of EMT has been rarely studied in the context of ferroptosis susceptibility. ESRP2 has been discovered as an epithelial splicing regulatory protein. In this study, we elucidated the role of ESRP2 in regulating metastatic potential and ferroptosis vulnerability in hepatocellular carcinoma (HCC).

Methods HCC tissues and adjacent normal tissues were used to study the ESRP2 expression level and its correlation with HCC progression. The Cancer Cell Line Encyclopedia database was used to investigate the correlation between ESRP2 mRNA expression and DNA methylation in cancer cell lines. In vitro and in vivo experiments using HCC cell lines confirmed the regulatory role of ESRP2 in EMT and ferroptosis. The ESRP2-regulated alternative splicing (AS) events were screened by high-throughput RNA sequencing. In vitro and in vivo functional assays using HCC cell lines confirmed the role of RAC1B in ESRP2-mediated HCC metastasis and ferroptosis.

Results ESRP2 was evidently down-regulated in HCC specimens compared with non-tumor tissues, and the down-regulation of ESRP2 correlated with poor prognosis and metastasis in HCC (IDDF2024-ABS-0403 Figure 1). We found that DNA hypermethylation contributes to ESRP2 loss in HCC (IDDF2024-ABS-0403 Figure 2). In addition, ESRP2 loss promoted EMT and metastasis both in vitro and in vivo (IDDF2024-ABS-0403 Figure 2). Moreover, we found that ESRP2 loss in HCC may sensitize tumor cells to ferroptosis (IDDF2024-ABS-0403 Figure 3). By high-throughput RNA sequencing, we identified a total of 4426 ESRP2-regulated AS events, most of which were skipped exon events (IDDF2024-ABS-0403 Figure 4). Further investigation into its molecular mechanism revealed that ESRP2 loss dramatically promoted the exon3b inclusion of the RAC1 gene, termed RAC1B. Splice-switching of RAC1B contributes to HCC metastasis and lipid peroxidation (IDDF2024-ABS-0403 Figure 5).

Conclusions We investigated the roles of ESRP2 in HCC metastasis and ferroptosis by addressing the effect and molecular mechanism of ESRP2 on reprogramming the splicing events. As tumor cells with ESRP2 silencing are always therapy-resistant but prone to ferroptosis, targeting these metastatic cells with ferroptosis inducers might be an effective approach to enhance therapeutic efficacy in HCC treatment.