CEMIP Promotes Extracellular Matrix-detached Prostate Cancer Cells Survival By Inhibiting Ferroptosis

Objective: Patients with metastatic prostate cancer have a poor prognosis and are the main cause of death of prostate cancer.The survival of tumor cells after detachment from the extracellular matrix(ECM)is a prerequisite for metastasis.Ferroptosis is a new type of regulated cell death,the ferroptosis resistance of cells is closely related to the occurrence,development and treatment response of various tumors.After detachment from the ECM,some prostate cancer cells died due to the altered microenvironment.Whether ferroptosis is involved in this death process and whether the surviving cells acquired ferroptosis resistance has not yet been reported.This study focused on evaluating the differences in ferroptosis resistance between detachment-resistant(DR)prostate cancer cells and their parental cells,and exploring the regulatory mechanism of ferroptosis resistance in DR prostate cancer cells.Methods: A DR prostate cancer cell model was constructed by continuous suspension culture and reattachment culture.The viability of cells was detected by CCK-8 assay.The ferroptosis resistance ability of prostate cancer cells was assessed by detecting intracellular reactive oxygen species levels,malondialdehyde levels,GSH/GSSG ratio,cystine uptake levels,ferrous ion levels and the ultrastructural changes of mitochondria.The expression of cell migration inducing protein(CEMIP)in DR prostate cancer cells was silenced by short hairpin RNA,whereas CEMIP was overexpressed in parental prostate cancer cells,and then the effect of CEMIP expression on ferroptosis resistance were evaluated.The relationship between solute carrier family 7 member 11(SLC7A11)and clinical features and prognosis of patients with prostate cancer were evaluated by means of bioinformatics analysis and immunohistochemistry.Rescue experiments were performed in DR prostate cancer cells by overexpressing SLC7A11 while silencing CEMIP to evaluate the effect of CEMIP-regulated SLC7A11 expression on ferroptosis resistance,anchorage-independent growth,invasion and migration ability of DR prostate cancer cells.The nude mice lung metastasis model was used to evaluate the effects of CEMIP-regulated SLC7A11 expression on metastasis and prognosis.The distribution of nuclear factor erythroid 2-related factor 2(NRF2)in parental prostate cancer cells and DR prostate cancer cells was detected by nucleoplasmic protein separation experiments.The transcriptional regulation of SLC7A11 by NRF2 was verified by dual-luciferase reporter gene experiment.The interaction between CEMIP and ITPR3 in DR prostate cancer cells was verified by co-immunoprecipitation experiments,and the effect of the interaction between CEMIP and inositol 1,4,5-trisphosphate receptor type 3(ITPR3)on cytoplasmic calcium levels was detected by calcium fluorescent probe.Results: Ferroptosis inhibitor and apoptosis inhibitor synergistically increased the cell viability of suspension cultured parental prostate cancer cells.Compared with parental prostate cancer cells,the resistance of DR prostate cancer cells to ferroptosis inducers were significantly enhanced.Silencing CEMIP decreased the antioxidant capacity and resistance to ferroptosis inducers in DR prostate cancer cells.CEMIP enhanced the ferroptosis resistance and malignant biological behavior of DR prostate cancer cells in vivo and in vitro by promoting the expression of SLC7A11.Compared with parental prostate cancer cells,the phosphorylation level of NRF2 in DR prostate cancer cells were significantly increased and aggregated to the nucleus,which promoted the transcription of SLC7A11.The interaction between CEMIP and the endoplasmic reticulum calcium channel ITPR3 in DR prostate cancer cells was enhanced compared to that in parental cells,which increased cytoplasmic calcium levels and the phosphorylation level of Ca MKII in DR prostate cancer cells.Conclusion: Ferroptosis is involved in the death process of prostate cancer cells after detachment from the ECM.The ferroptosis resistance of DR prostate cancer cells was significantly enhanced.The interaction of CEMIP and the endoplasmic reticulum calcium channel ITPR3 up-regulated the calcium level in DR prostate cancer cells,which in turn activated the Ca MKII/NRF2/SLC7A11 signaling pathway and promoted the expression of SLC7A11,thereby enhances the ferroptosis resistance of DR prostate cancer cells and their malignant progression both in vivo and in vitro.