br Receptor interacting serine threonine protein kinase RIPK
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can propagate ligand-activated necroptosis (Linker-mann et al., 2012). Suggesting 673A-induced necroptosis is RIPK1 independent, RIPK1 downregulation did not rescue gamma-Glu-Cys from 673A-induced necroptosis (Figures 4Ai and S2Ci). Similarly,
although RIPK1 is strongly expressed in most ovarian cancer cell lines (Figure S4Cii), pretreatments with the RIPK1 inhibitor Nec-1 did not rescue cells from 673A-induced cell death (Figures 4Aii–4Aiii).
RIPK1-independent necroptosis can be driven by RIPK3. Inter-estingly RIPK3 KD alone (Figure S4Ciii) resulted in a 70% reduc-tion in cell number (Figure 4Bi). This made it difficult to interpret the effect of RIPK3 downregulation on 673A-mediated CSC death (Figure 4Bi). Similarly, high doses of necrosulfonamide (NSA), an inhibitor of the RIPK3 immediate downstream target MLKL also reduced cell numbers. However, low doses of NSA partly rescued cells from 673A-induced CD133+ cell death in A2780 and OVCAR8 cells, suggesting a potential role for RIPK3/MLKL in 673A-triggered death (Figures 4Bii and S4D). We also evaluated the effect of 673A on necroptosome forma-tion. Numerous proteins have been linked with the formation of a necroptosome, including RIP3K, MLKL, DRP1, FADD, and PGAM5 (Wang et al., 2012). PGAM5 acts as a convergence point in RIPK3-dependent and -independent necroptosome formation, resulting in MLKL translocation to cell membranes (Cai et al., 2014) and DRP1 translocation to the mitochondria to increase membrane permeability (Wang et al., 2012). To determine whether 673A treatment induced the formation of the necropto-some complex, we independently immunoprecipitated FADD-, DRP1-, and RIPK3-associated protein complexes in control and 673A-treated cells and performed western blots for PGAM5. For all three immunoprecipitated proteins, we observed increased association of PGAM5 in 673A-treated cells versus control cells (Figure 4C). Eight and 12 h after 673A treatment, we also observed dephosphorylation of DRP1 and increased PGAM5 expression with the appearance of the PGAM5-S splice variant (Figure 4D). Similarly, we observed a significant increase both in the mitochondrial association of DRP1 (Figure 4Ei) and in the proportion of MLKL protein localized to the cell membrane fraction after 673A treatments (Figure 4Eii). Taken together, these data suggest 673A induces calcium-dependent necroptosis.
ALDH1A Family Inhibition Induces the Expression of the Mitochondrial UCPs
ALDH1A family members regulate the biosynthesis of RA to indirectly regulate RA-mediated gene expression. Indicating gene expression may have a role in 673A-mediated CD133+ cell death, 673A-induced CD133+ cell death is prevented by the transcription inhibitor flavopiridol (Figure 5A). qRT-PCR eval-uation of mRNA expression of known RA target genes (Balmer and Blomhoff, 2002) in bulk cells after 673A treatment showed a significant increase in the expression of the mitochondrial UCPs UCP1 and UCP3 (Figure 5Bi). Importantly and potentially explaining the preferential sensitivity of CD133+ cells to ALDH1A family inhibition, qRT-PCR of FACS-isolated cells demonstrated that UCP1 and UCP3 were preferentially induced by 673A in CD133+ cells versus CD133 cells (Figure 5Bii).
Figure 3. 673A Triggers Necroptosis in Ovarian CSCs
(C) IF of HMBG1 localization 24 h after 673A treatment; DAPI was used for nuclear stain.
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UCP1 protein is aberrantly regulated in ALDH1A1-KO mice (Kiefer et al., 2012), supporting UCP induction as an on-target/ ALDH inhibition-mediated effect of 673A. Further supporting that UCP1/UCP3 induction is a result of ALDH1A family inhibi-tion, siRNA KD of the various ALDH1A family members similarly resulted in the induction of UCP1 and UCP3 (Figure 5C), with KD of ALDH1A1 and ALDH1A2 having the most prominent effects. Forced UCP3 expression reduced cell numbers 2-fold, whereas UCP1 expression reduced cell numbers 30% compared with a control (Figure 5Di). Cells with UCP1 or UCP3 overexpression