Abstract
Sensitive to Apoptosis Gene (SAG), also known as RBX2 (RING box protein-2), is the RING component of SCF (SKP1, Cullin, and F-box protein) E3 ubiquitin ligase. Our previous studies have demonstrated that SAG is an anti-apoptotic protein and an attractive anti-cancer target. We also found recently that Sag knockout sensitized mouse embryonic stem cells (mES) to radiation and blocked mES cells to undergo endothelial differentiation. Here, we reported that compared to wild-type mES cells, the Sag−/− mES cells were much more sensitive to all-trans retinoic acid (RA)-induced suppression of cell proliferation and survival. While wild-type mES cells underwent differentiation upon exposure to RA, Sag−/− mES cells were induced to death via apoptosis instead. The cell fate change, reflected by cellular stiffness, can be detected as early as 12 hrs post RA exposure by AFM (Atomic Force Microscopy). We then extended this novel finding to RA differentiation therapy of leukemia, in which the resistance often develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE), that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination.
Highlights
Sensitive to Apoptosis Gene (SAG), known as RBX2, ROC2 (Regulator of Cullins) or RNF7 (RING finger protein-7), was originally cloned in our laboratory as a redox-inducible antioxidant protein [1], and later characterized as the second RING family member of the SCF E3 ubiquitin ligase
Our recent work showed that during mouse embryonic stem cells (mES) cell differentiation induced by LIF withdrawal, Sag deletion had no effect on the formation of embryoid bodies, but prevented the endothelial differentiation to form blood island structure [13]
We found that Sag deletion prevented mES cells from undergoing differentiation, rather triggered apoptosis instead
Summary
SAG, known as RBX2, ROC2 (Regulator of Cullins) or RNF7 (RING finger protein-7), was originally cloned in our laboratory as a redox-inducible antioxidant protein [1], and later characterized as the second RING family member of the SCF E3 ubiquitin ligase (for review, see [2]). The Sag knockout in mouse caused embryonic lethality, which is associated with growth retardation and other developmental defects [13], whereas Sag knockout in mES cells induced radiosensitization [14], and blocked their endothelial differentiation [13]. These cellular functions were mediated via its antioxidant activity by scavenging ROS [1,4,6], and via its E3 ubiquitin ligase activity by promoting the degradation of p27, cJun, pro-caspase-3, IkBa, HIF-1a, and NOXA in a cell context dependent manner [9,10,12,15,16,17]. The findings suggest that SAG may play a role in human tumorigenesis and could serve as an anticancer target
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