CircRNA-CREIT inhibits stress granule assembly and overcomes doxorubicin resistance in TNBC by destabilizing PKR

Background Circular RNAs (circRNAs) represent a novel type of regulatory RNA characterized by high evolutionary conservation and stability. CircRNAs are expected to be potential diagnostic biomarkers and therapeutic targets for a variety of malignancies. However, the regulatory functions and underlying mechanisms of circRNAs in triple-negative breast cancer (TNBC) are largely unknown. Methods By using RNA high-throughput sequencing technology, qRT-PCR and in situ hybridization assays, we screened dysregulated circRNAs in breast cancer and TNBC tissues. Then in vitro assays, animal models and patient-derived organoids (PDOs) were utilized to explore the roles of the candidate circRNA in TNBC. To investigate the underlying mechanisms, RNA pull-down, RNA immunoprecipitation (RIP), co immunoprecipitation (co-IP) and Western blotting assays were carried out. Results In this study, we demonstrated that circRNA-CREIT was aberrantly downregulated in doxorubicin resistant triple-negative breast cancer (TNBC) cells and associated with a poor prognosis. The RNA binding protein DHX9 was responsible for the reduction in circRNA-CREIT by interacting with the flanking inverted repeat Alu (IRAlu) sequences and inhibiting back-splicing. By utilizing in vitro assays, animal models and patient-derived organoids, we revealed that circRNA-CREIT overexpression significantly enhanced the doxorubicin sensitivity of TNBC cells. Mechanistically, circRNA-CREIT acted as a scaffold to facilitate the interaction between PKR and the E3 ligase HACE1 and promoted proteasomal degradation of PKR protein via K48-linked polyubiquitylation. A reduced PKR/eIF2 alpha signaling axis was identified as a critical downstream effector of circRNA-CREIT, which attenuated the assembly of stress granules (SGs) to activate the RACK1/MTK1 apoptosis signaling pathway. Further investigations revealed that a combination of the SG inhibitor ISRIB and doxorubicin synergistically inhibited TNBC tumor growth. Besides, circRNA-CREIT could be packaged into exosomes and disseminate doxorubicin sensitivity among TNBC cells. Conclusions Our study demonstrated that targeting circRNA-CREIT and SGs could serve as promising therapeutic strategies against TNBC chemoresistance.

Automated summary

Our study revealed the downregulation of circRNA-CREIT in doxorubicin resistant TNBC cells and its association with poor prognosis. We identified the RNA binding protein DHX9 as responsible for the reduction in circRNA-CREIT by interacting with the flanking inverted repeat Alu (IRAlu) sequences and inhibiting back-splicing. Mechanistically, circRNA-CREIT acted as a scaffold to facilitate the interaction between PKR and the E3 ligase HACE1, leading to proteasomal degradation of PKR protein via K48-linked polyubiquitylation and attenuation of the assembly of stress granules (SGs) to activate the RACK1/MTK1 apoptosis signaling pathway. Moreover, circRNA-CREIT could be packaged into exosomes and disseminate doxorubicin sensitivity among TNBC cells.