CLTRN, Regulated by NRF1/RAN/DLD Protein Complex, Enhances Radiation Sensitivity of Hepatocellular Carcinoma Cells Through Ferroptosis Pathway

Abstract

Background Radiotherapy is a viable treatment option for patients with unresectable hepatocellular carcinoma (HCC). However, radiation resistance is an issue that needs to be addressed. In this context, cumulative evidence supports the functional roles of a variety of RNA or proteins in radioresistance, and suggests that the modulation of their expression may constitute a novel radiosensitization approach. Here, we investigated the ability of collectrin (CLTRN) to enhance the radiosensitivity in HCC patients for the rst time. Methods Transcriptome sequencing technology (RNA-seq technology) was used to analyze the transcription-level changes in the genes from the HepG2 cells before and after X-ray irradiation. Combining the results with the HCC tissue RNA-seq data, we determined the ultimate target gene through bioinformatics analysis and cellular veri cation. A series of cellular and molecular biology techniques were applied in vitro and in vivo to con rm whether CLTRN can enhance radiosensitivity in HCC cells. Subsequently, the downstream action mechanism, the upstream transcription factor, and the interaction proteins of CLTRN were determined. Results First, we con rmed the association between CLTRN and radiosensitivity. We observed that CLTRN overexpression led to a signi cant reduction in the proliferation, migration, and invasion potential of Xray-irradiated HCC cells, whereas no observable effect was exerted on cell cycle and apoptosis. The same results were observed in nude mice in vivo. Investigation of the gene regulatory mechanism revealed that the genes analyzed at transcriptome level after CLTRN overexpression were mostly enriched in the glutathione metabolic pathway. As glutathione metabolism forms a vital link in ferroptosis, we surmised that CLTRN is associated with ferroptosis. This was con rmed through the detection of cellular iron, ROS level determination, transmission electron microscopy, and monitoring of ferroptosis-related protein indicators. Lastly, we investigated whether nuclear respiratory factor 1 (NRF1) is the upstream transcription factor of CLTRN, and whether dihydrolipoamide dehydrogenase (DLD) and members of the RAS oncogene family (RAN) are its interacting proteins. Conclusion Our results indicate that CLTRN is a vital regulator of radiation sensitivity and could serve as a novel therapeutic target or prognostic marker in HCC treatment.

Contribution of Lipid Oxidation and Ferroptosis to Radiotherapy Efficacy

“…Depletion of mitochondrial antioxidants including Coenzyme Q with FIN56, a known ferroptosis inducer, diminishes cancer cell survival following radiotherapy. The glycoprotein collectrin improves radiotherapy sensitivity through ferroptosis induction in the setting of hepatocellular carcinoma [54].…”

Immunogenic Cell Death Induction by Ionizing Radiation

“…The connection between IR-induced ferroptosis and antitumor immunity might be linked to many factors such as radiation dose and type. The previous studies have indicated that IR induces tumor cell ferroptosis and ferroptosis improves the radiosensitivity of tumor cells (10,63,64). Based on this, RT combined with ferroptosis inducers may improve the therapeutic effect of RT by increasing lipid peroxidation and promoting immune response rather than DNA damage or caspase activation (65).…”

The Role of Ferroptosis in the Treatment and Drug Resistance of Hepatocellular Carcinoma

“…Radiotherapy induces ferroptosis by down-regulation of SLC7A11 and up-regulation of ACSL4, resulting in GSH production, increasing lipid synthesis, and subsequent oxidative damage ( Lang et al, 2019 ; Lei et al, 2020 ). Studies have found that collectrin (CLTRN), as a target of radiation, is regulated by NRF1 (nuclear respiratory factor 1)/RAN (RAS oncogene family)/DLD (dihydrolipoamide dehydrogenase) protein complex and enhances the radiosensitivity of HCC cells through ferroptosis ( Yuan et al, 2021 ). A combination of gene therapy and radiation therapy is one way forward, allowing the radiation doses to be reduced and the side effects to be reduced.…”