Exosome-derived circUPF2 enhances resistance to targeted therapy by redeploying ferroptosis sensitivity in hepatocellular carcinoma.

Advanced hepatocellular carcinoma (HCC) can be treated with sorafenib, which is the primary choice for targeted therapy. Nevertheless, the effectiveness of sorafenib is greatly restricted due to resistance. Research has shown that exosomes and circular RNAs play a vital role in the cancer's malignant advancement. However, the significance of exosomal circular RNAs in the development of resistance to sorafenib in HCC remains uncertain. Ultracentrifugation was utilized to isolate exosomes (Exo-SR) from the sorafenib-resistant HCC cells' culture medium. Transcriptome sequencing and differential expression gene analysis were used to identify the targets of Exo-SR action in HCC cells. To identify the targets of Exo-SR action in HCC cells, transcriptome sequencing and analysis of differential expression genes were employed. To evaluate the impact of exosomal circUPF2 on resistance to sorafenib in HCC, experiments involving gain-of-function and loss-of-function were conducted. RNA pull-down assays and mass spectrometry analysis were performed to identify the RNA-binding proteins interacting with circUPF2. RNA immunoprecipitation (RIP), RNA pull-down, electrophoretic mobility shift assay (EMSA), immunofluorescence (IF) -fluorescence in situ hybridization (FISH), and rescue assays were used to validate the interactions among circUPF2, IGF2BP2 and SLC7A11. Finally, a tumor xenograft assay was used to examine the biological functions and underlying mechanisms of Exo-SR and circUPF2 in vivo. A novel exosomal circRNA, circUPF2, was identified and revealed to be significantly enriched in Exo-SR. Exosomes with enriched circUPF2 enhanced sorafenib resistance by promoting SLC7A11 expression and suppressing ferroptosis in HCC cells. Mechanistically, circUPF2 acts as a framework to enhance the creation of the circUPF2-IGF2BP2-SLC7A11 ternary complex contributing to the stabilization of SLC7A11 mRNA. Consequently, exosomal circUPF2 promotes SLC7A11 expression and enhances the function of system Xc- in HCC cells, leading to decreased sensitivity to ferroptosis and resistance to sorafenib. The resistance to sorafenib in HCC is facilitated by the exosomal circUPF2, which promotes the formation of the circUPF2-IGF2BP2-SLC7A11 ternary complex and increases the stability of SLC7A11 mRNA. Focusing on exosomal circUPF2 could potentially be an innovative approach for HCC treatment.

Dong FL ，Xu ZZ ，Wang YQ ，Li T ，Wang X ，Li J ... -  《JOURNAL OF NANOBIOTECHNOLOGY》

Loss of LncRNA DUXAP8 synergistically enhanced sorafenib induced ferroptosis in hepatocellular carcinoma via SLC7A11 de-palmitoylation.

Ferroptosis is an important iron-dependent form of cell death in hepatocellular carcinoma (HCC). Sorafenib, a potent ferroptosis inducer, is used to treat advanced HCC but its efficacy is limited by the development of drug resistance. The effects of DUXAP8 expression on HCC progression were evaluated by TCGA database, Kaplan-Meier analysis, and in situ hybridization analysis. Sorafenib resistant HCC cell lines were modeled in vitro to study the regulation of DUXAP8 on ferroptosis in HCC induced by sorafenib. We used RNA pull-down, immunofluorescence assays, acyl-biotinyl exchange assay and mass spectrometry analysis to assess the molecular mechanism of ferroptosis regulation by DUXAP8. Syngeneic subcutaneous and orthotopic CDX models were used to assess whether DUXAP8 inhibition improves HCC in vivo. LncRNA DUXAP8, which is highly expressed in liver cancer and associated with poor prognosis, contributes to sorafenib resistance through suppression of ferroptosis. In vitro tests revealed that DUXAP8 reduced the sensitivity of HCC to sorafenib-induced ferroptosis by acting on SLC7A11, a subunit of the amino acid antiporter system xc-. DUXAP8 facilitates SLC7A11 palmitoylation and impedes its lysosomal degradation, thereby enhancing SLC7A11 action and suppressing ferroptosis. RNA pull-down and immunofluorescence assays confirmed that DUXAP8 decreased membrane translocation and promoted sorting of de-palmitoylated SLC7A11 to lysosomes by binding of DUXAP8 to SLC7A11. In addition, mass spectrometric analysis found that the Cys414 residue of SLC7A11 might be the predominant mutant site responsible for molecular masking of SLC7A11 lysosomal sorting. Further, the antitumor effect of DUXAP8 knockdown was verified in orthotopic and subcutaneous CDX models. Our findings suggest that a novel translational strategy combining sorafenib with DUXAP8 silencing to overcome drug resistance may improve treatment efficacy in patients with advanced HCC.

Shi Z ，Li Z ，Jin B ，Ye W ，Wang L ，Zhang S ，Zheng J ，Lin Z ，Chen B ，Liu F ，Zhang B ，Ding X ，Yang Z ，Shan Y ，Yu Z ，Wang Y ，Chen J ，Chen Q ，Roberts LR ，Chen G ... -  《Clinical and Translational Medicine》

MCM4 potentiates evasion of hepatocellular carcinoma from sorafenib-induced ferroptosis through Nrf2 signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It poses an enormous socioeconomic burden and is a serious public health threat globally due to its poor prognosis. Ferroptosis is a newly identified non-apoptotic form of cell death characterized by lipid peroxidation, iron accumulation, and reactive oxygen species (ROS) generation. However, tumor cells have evolved diverse mechanisms to evade ferroptosis, conferring resistance to drugs. Sorafenib, a first-line therapy for advanced HCC, triggers ferroptosis by selectively targeting solute carrier family 7 member 11 (SLC7A11) to deplete glutathione and inhibit glutathione peroxidase 4 (GPX4), thereby effectively eliminating tumor cells. However, sorafenib resistance has been widely reported, and the precise mechanisms underlying sorafenib drug resistance remain unclear. The minichromosome maintenance (MCM) protein family contains 10 members with vital roles in DNA replication and cell cycle progression. MCM4, a member of the MCM protein family, might be a potential biomarker in pan-cancer analysis. The present study found that MCM4 was upregulated in liver cancer using bioinformatics analysis and sorafenib-treated HCC cells. Moreover, MCM4 might be regarded as a prognostic biomarker for HCC. Further experiments revealed that MCM4-inhibition enhanced the efficacy of sorafenib through elevation of ferroptosis both in vitro and in vivo. Mechanistically, MCM4 potentiates sorafenib-induced ferroptosis evasion in HCC by promoting nuclear factor erythroid 2-related factor 2 (Nrf2) signaling activation. However, no direct interactions were found between Nrf2 and MCM4. Overall, these findings suggest a potential therapeutic strategy for HCC by targeting MCM4 inhibition.

Liu X ，Zhang F ，Fan Y ，Qiu C ，Wang K ... -  《-》

RNA binding protein DAZAP1 promotes HCC progression and regulates ferroptosis by interacting with SLC7A11 mRNA.

RNA-binding proteins (RBPs) closely regulate the whole lifecycle of most RNA molecules, from the very early stage of transcription to RNA decay. Dysregulation of RBPs significantly affects the fate of cancer-related transcripts. Therefore, it is imperative to fully understand the complicated RBP-RNA regulatory networks in malignant diseases and to explore novel therapeutic targets. The RBP DAZAP1 (deleted in azoospermia-associated protein 1), originally identified as an important protein in spermatogenesis, had rarely been studied in the context of carcinogenesis. The role of DAZAP1 in hepatocellular carcinoma (HCC) was unveiled in this study. The relative expression of DAZAP1 was significantly upregulated in HCC and was positively associated with several key malignant characteristics and poor postoperative survival in patients. DAZAP1 knockdown by small interfering RNA markedly inhibited HCC cell proliferation, migration and invasion. Furthermore, DAZAP1 significantly reduced cellular sensitivity to sorafenib (SF), which had been proven to be an inducer of ferroptosis by targeting the system Xc- (composed of a light chain, xCT/SLC7A11, and a heavy chain, 4F2 heavy chain). At the mechanistic level, DAZAP1 was identified as a potent inhibitor of ferroptosis and an efficient binding partner of SLC7A11 mRNA. Further study revealed that DAZAP1 interacted with the 3'UTR (untranslated region) of SLC7A11 mRNA and positively regulated its stability. In our work, we clarified novel functions of DAZAP1 and preliminarily revealed its underlying mechanism in ferroptosis, which may be conducive to the exploration of biomarkers and therapeutic targets in HCC patients.

Wang Q ，Guo Y ，Wang W ，Liu B ，Yang G ，Xu Z ，Li J ，Liu Z ... -  《-》

Dihydroartemisinin induces ferroptosis of hepatocellular carcinoma via inhibiting ATF4-xCT pathway.

Management of hepatocellular carcinoma (HCC) remains challenging due to population growth, frequent recurrence and drug resistance. Targeting of genes involved with the ferroptosis is a promising alternative treatment strategy for HCC. The present study aimed to investigate the effect of dihydroartemisinin (DHA) against HCC and explore the underlying mechanisms. The effects of DHA on induction of ferroptosis were investigated with the measurement of malondialdehyde concentrations, oxidised C11 BODIPY 581/591 staining, as well as subcutaneous xenograft experiments. Activated transcription factor 4 (ATF4) and solute carrier family 7 member 11 (SLC7A11 or xCT) were overexpressed with lentiviruses to verify the target of DHA. Here, we confirmed the anticancer effect of DHA in inducing ferroptosis is related to ATF4. High expression of ATF4 is related to worse clinicopathological prognosis of HCC. Mechanistically, DHA inhibited the expression of ATF4, thereby promoting lipid peroxidation and ferroptosis of HCC cells. Overexpression of ATF4 rescued DHA-induced ferroptosis. Moreover, ATF4 could directly bound to the SLC7A11 promoter and increase its transcription. In addition, DHA enhances the chemosensitivity of sorafenib on HCC in vivo and in vitro. These findings confirm that DHA induces ferroptosis of HCC via inhibiting ATF4-xCT pathway, thereby providing new drug options for the treatment of HCC.

Ji J ，Cheng Z ，Zhang J ，Wu J ，Xu X ，Guo C ，Feng J ... -  《-》