RESUMO
Ferroptosis is a new type of programmed cell death, which has been involved in the progression of tumours. However, the regulatory network of ferroptosis in pancreatic cancer is still largely unknown. Here, using datasets from GEO and TCGA, we screened HSPB1, related to the P450 monooxygenase signalling, a fuel of ferroptosis, to be a candidate gene for regulating pancreatic cancer cell ferroptosis. We found that HSPB1 was enriched in the exosomes derived from human pancreatic cancer cell lines SW1990 and Panc-1. Then, hypoxic SW1990 cells were incubated with exosomes alone or together with HSPB1 siRNA (si-HSPB1), and we observed that exosomes promoted cell proliferation and invasion and suppressed ferroptosis, which was reversed by si-HSPB1. Moreover, we found a potential binding affinity between HSPB1 and FUS, verified their protein interaction by using dual-colour fluorescence colocalization and co-IP assays, and demonstrated the promoting effect of FUS on oxidative stress and ferroptosis in hypoxic SW1990 cells. Subsequently, FUS was demonstrated to bind with and stabilize the mRNA of Nrf2, a famous anti-ferroptosis gene that negatively regulates the level of P450. Furthermore, overexpressing FUS and activating the Nrf2/HO-1 pathway (using NK-252) both reversed the inhibitory effect of si-HSPB1 on exosome functions. Finally, our in vivo studies showed that exosome administration promote tumour growth in nude mice of xenotransplantation, which was able to be eliminated by knockdown of HSPB1. In conclusion, exosomal HSPB1 interacts with the RNA binding protein FUS and decreases FUS-mediated stability of Nrf2 mRNA, thus suppressing hypoxia-induced ferroptosis in pancreatic cancer.
Assuntos
Ferroptose , Proteínas de Choque Térmico HSP27 , Fator 2 Relacionado a NF-E2 , Neoplasias Pancreáticas , Proteína FUS de Ligação a RNA , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células , Exossomos/metabolismo , Ferroptose/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/genética , Camundongos Nus , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genéticaRESUMO
Ferroptosis is expected to be a therapeutic target for cancers including pancreatic cancer. We aimed to screen genes that regulate ferroptosis and doxycycline resistance in pancreatic cancer and to explore the underlying mechanisms. Bioinformatics analysis was performed to identify genes that respond to ferroptosis in two human pancreatic cancer cells with GOT1 knocked down or not. 325 and 842 genes were upregulated in MiaPaCa and Tu8902 cells in response to GOT1 knockdown, with 43 genes shared. Among the 43 genes, 14 genes were identified to interact with ferroptosis key genes. MB and HMOX1 were the genes most sensitive to Erastin and doxycycline. Moreover, MB and HMOX1 expression was higher in human normal pancreatic duct epithelial cells than in pancreatic cancer cells. MB and HMOX1 proteins physically bound and promoted each other's expression. By interacting with HMOX1, MB suppressed pancreatic cancer cell proliferation, colony formation and invasion, and promoted cell ferroptosis and sensitivity to erastin and doxycycline. Silencing HMOX1 reversed the promoting effect of MB on cell ferroptosis and sensitivity to doxycycline. A pancreatic cancer xenograft model was established by subcutaneous injection of Panc-1 cells transfected with or without Ad-MB, and doxycycline was administered intraperitoneally. Overexpression of MB enhanced the inhibitory effect of doxycycline on xenograft growth. In conclusion, MB facilitated doxycycline sensitivity in pancreatic cancer cells through promoting HMOX1-mediated ferroptosis.