Epigenetic Inactivation of RIPK3-Dependent Necroptosis Augments Cisplatin Chemoresistance in Human Osteosarcoma

Abstract

Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents. Unfortunately, drug resistance limits the efficacy of chemotherapeutic treatment and compromises therapeutic outcomes in a substantial proportion of cases. Aberrant CpG island methylation-associated transcriptional silencing contributes to chemoresistance in pediatric solid tumors. Here, using whole-genome DNA methylation screening on 16 human primary OS specimens, we identify receptor interacting protein kinase-3 (RIPK3), a molecular regulator of the necroptosis programmed cell death pathway, as a gene target of aberrant CpG methylation and demonstrate its role in human OS chemoresistance. We validated these findings via enforced expression and DsiRNA silencing, and evaluated the role of RIPK3 in cisplatin chemosensitivity and necroptosis activation through MLKL phosphorylation. We found that CpG island methylation results in RIPK3 silencing in primary human OS samples and cell lines. Enforced RIPK3 expression significantly enhanced cisplatin cytotoxicity in OS cells and DsiRNA knockdown reversed the cisplatin-sensitive phenotype. In cells with enforced RIPK3 expression, cisplatin treatment significantly increased phosphorylation of both RIPK3 and its target, MLKL, indicative of induction of necroptosis. Here, we identify RIPK3 as an important mediator of chemoresistance in OS and a potential pharmacologic target to improve chemotherapy efficacy in drug-resistant tumors.

Keywords: CpG islands; RIPK3; chemoresistance; necroptosis; osteosarcoma.

Figure 1

Hypermethylation of CpG islands is prevalent in human primary osteosarcoma (OS) tumor samples compared to levels detected in normal bone and primary human osteoblasts.

Figure 2

Transcription and translation of RIPK3 is inhibited in OS cell lines. (A) qPCR demonstrates RIPK3 mRNA levels are significantly reduced compared to small intestine positive control. (B) Western blot analysis demonstrates RIPK3 expression is absent in OS cell lines HOS, 143B, MG63, G292, and MNNG/HOS, in hMSCs, and hFOBs. G292 RIPK3 transfected cells were used as a positive control. (C) Levels of MLKL, measured via qPCR, were also significantly reduced in OS cell lines compared to hMSCs and hFOBs. RIPK3; 57 kDa.

Figure 3

Forced expression of RIPK3 augments cisplatin sensitivity in 143B and G292 cell lines. (A) Enforced expression of RIPK3 in 143B and G292 cell lines does not alter overall survival. (B) Western blot confirms forced expression of RIPK3 in stably transduced 143B cells (GFP-tagged RIPK3; 87 kDa). (C) Western blot confirms forced expression of RIPK3 in stably transfected G292 cells (RIPK3; 57 kDa). (D) 143B cells expressing RIPK3 (143B-RIPK3) demonstrate reduced survival following 48 h cisplatin treatment compared to empty vector (EV) and untreated controls. (E) G292 cells expressing RIPK3 (G292-RIPK3) are more susceptible to cisplatin-induced cell death compared to EV and untreated controls. ns = not significant.

Figure 4

DsiRNA silencing of RIPK3 restores cisplatin chemoresistance. (A) DsiRNA treatment successfully reduced RIPK3 mRNA and (B,C) protein levels in 143B cells with stable RIPK3 expression. Similarly, DsiRNA treatment reduced RIPK3 (D) mRNA and protein levels (E,F) in RIPK3 expressing G292 (1: 50 nM DsiRNA, 2: 75 nM DsiRNA). Following 48 h of cisplatin treatment (20 µM), (G) 143B and (H) G292 cells with DsiRNA treatment to reduce RIPK3 expression demonstrated increased chemoresistance compared to untreated and non-targeting (NT) controls.

Figure 5

Cisplatin treatment results in phosphorylation of RIPK3 and activation of MLKL-associated necroptosis. (A) 143B cells stably expressing RIPK3 demonstrate elevated levels of phosphorylated RIPK3 (p-RIPK3; 87 kDa) following cisplatin treatment compared to EV controls. (B) Quantification of 143B p-RIPK3 Western blot densitometry with untreated as control. (C) We compared expression of p-RIPK3 to total protein by Westerbn blot and (D) quantified changes following RIPK3 forced expression. (E) RIPK3 expressing G292 cells also show a significant increase in p-RIPK3 (60 kDa) levels compared to EV and untreated controls. (F) Quantification of G292 p-RIPK3 Western blot densitometry with untreated RIPK3 as control. (G) We compared changes in p-RIPK3 to total protein changes by Western blot and (H) through densitometry analysis. (I) RIPK3 mRNA expression is elevated in RIPK3 expressing 143B and G292 cells. (J) 143B cells stably expressing RIPK3 show significantly elevated levels of phosphorylated MLKL (p-MLKL; 54 kDa) following both 24 and 48 h cisplatin treatment compared to EV and untreated controls. (K) Quantification of 143B p-MLKL Western blot densitometry with untreated EV as control. (L) Examination of total MLKL protein levels by Western blot and (M) densitometry. (N) Finally, we examined expression levels of p-MLKL/total MLKL and demonstrate that forced expression of RIPK3 leads to an activation of necroptosis via p-MLKL.