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posted on 2023-03-31, 21:29 authored by Yinfei Tan, Eleonora Sementino, Mitchell Cheung, Suraj Peri, Craig W. Menges, Anna-Mariya Kukuyan, Ting Zhang, Vladimir Khazak, Lauren A. Fox, Eric A. Ross, Suresh Ramanathan, Suresh C. Jhanwar, Raja M. Flores, Siddharth Balachandran, Joseph R. Testa <p>Re-expression of RIPK3 expression triggers necroptosis in RIPK3-negative MM cell lines, and RIPK3 inhibition rescues the cell death caused by necroptosis. A, RIPK3-negative MM cell lines M12 and M22 were transduced with WPI lentivirus expressing RIPK3 WT or empty vector and treated with the apoptosis inhibitor zVAD and/or the RIPK3 inhibitor GSK'872 for 1-4 d. Results of immunoblot analysis (24 h) and clonogenic assay (4 d) are shown. B, Cell viability determined with hemocytometer and trypan blue dye exclusion test.</p>
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ARTICLE ABSTRACT
Receptor-interacting protein kinase 3 (RIPK3) phosphorylates effector molecule MLKL to trigger necroptosis. Although RIPK3 loss is seen in several human cancers, its role in malignant mesothelioma is unknown. This study aimed to determine whether RIPK3 functions as a potential tumor suppressor to limit development of malignant mesothelioma.
RIPK3 expression was examined in 66 malignant mesothelioma tumors and cell lines. Promoter methylation and DNMT1 siRNA studies were performed to assess the mode of RIPK3 silencing in RIPK3-deficient malignant mesothelioma cells. Restoration of RIPK3 expression in RIPK3-negative malignant mesothelioma cells, either by treatment with 5-aza-2′-deoxycytidine or lentiviral expression of cDNA, was performed to assess effects on cell viability, necrosis, and chemosensitization.
Loss of RIPK3 expression was observed in 42/66 (63%) primary malignant mesotheliomas and malignant mesothelioma cell lines, and RT-PCR analysis demonstrated that downregulation occurs at the transcriptional level, consistent with epigenetic silencing. RIPK3-negative malignant mesothelioma cells treated with 5-aza-2′-deoxycytidine resulted in reexpression of RIPK3 and chemosensitization. Ectopic expression of RIPK3 also resulted in chemosensitization and led to necroptosis, the latter demonstrated by phosphorylation of downstream target MLKL and confirmed by rescue experiments. Mining of RIPK3 expression and survival outcomes among patients with malignant mesothelioma available from The Cancer Genome Atlas repository revealed that promoter methylation of RIPK3 is associated with reduced RIPK3 expression and poor prognosis.
These data suggest that RIPK3 acts as a tumor suppressor in malignant mesothelioma by triggering necroptosis and that epigenetic silencing of RIPK3 by DNA methylation impairs necroptosis and contributes to chemoresistance and poor survival in this incurable disease.
