Summary DEPTOR, an inhibitor of mTORC1 and mTORC2, is degraded via ubiquitin-proteasome pathway by an unknown E3 ubiquitin ligase. Here we report that DEPTOR is a physiological substrate of SCFβTrCP E3 ligase for targeted degradation. Upon growth factor stimulation, RSK1 and S6K1 kinases are activated to phosphorylate DEPTOR, which is then recognized by the F-box protein, βTrCP via its degron sequence for subsequent ubiquitination and degradation by SCF E3. Endogenous DEPTOR levels are negatively regulated by βTrCP. DEPTOR half-life is shortened by βTrCP but extended by a dominant negative mutant of βTrCP, by RSK1/S6K1 inhibition, and by βTrCP degron site mutations. Biologically, DEPTOR accumulation upon βTrCP knockdown inactivates mTORC1 and activates AKT in cancer cells to confer resistance to rapamycin and paclitaxel. Furthermore, DEPTOR accumulates upon glucose deprivation and mTOR inhibition, to induce autophagy. Thus, βTrCP-DEPTOR-mTOR intertwine to regulate cell survival and autophagy.

MLN4924, a newly discovered small molecule inhibitor of NEDD8-activating enzyme (NAE), inactivates Cullin-RING E3 ubiquitin Ligases (CRLs) by blocking cullin neddylation. As a result, MLN4924 causes accumulation of several key substrates of CRLs and effectively suppresses tumor cell growth by inducing apoptosis and senescence. However, the role of MLN4924 in induction of autophagy and its biological significance are totally unknown. Here we showed that MLN4924 effectively induces autophagy in both time- and dose-dependent manners in multiple human cancer lines, indicating a general phenomenon. Mechanistically, by inactivating CRLs, MLN4924 causes accumulation of DEPTOR and HIF1α. The siRNA knockdown and gene KO studies showed that DEPTOR and the HIF1-REDD1-TSC1 axis are responsible for MLN4924-induced autophagy via inhibiting mTORC1. Biologically, autophagy is a survival signal to tumor cells, and blockage of autophagy via siRNA knockdown, gene KO and small molecule inhibitor remarkably enhanced MLN4924-induced apoptosis. Our study reveals an uncharacterized mechanism of MLN4924 action and provides the proof-of-concept evidence for strategic drug combination of MLN4924 with an autophagy inhibitor for maximal killing of tumor cells via enhancing apoptosis.

Several ribosomal proteins regulate p53 function via modulating MDM2. We recently found that RPS27L, a RPS27 like protein, is a direct p53 inducible target. Here we showed that RPS27 itself is a p53 repressible target. Furthermore, the N-terminal region of either RPS27L or RPS27 binds to MDM2 on the central acidic domain of MDM2. RPS27L or RPS27 forms an in vivo triplex with MDM2-p53 and competes with p53 for MDM2 binding. Like p53, RPS27L, but not RPS27, is a short-lived protein and a novel MDM2 substrate. Degradation of RPS27L requires the RING or acidic domain of MDM2. Ectopic expression of RPS27L or RPS27 inhibits MDM-2-mediated p53 ubiquitination and increases p53 levels by extending p53 protein half-life, whereas siRNA silencing of RPS27L decreases p53 levels by shortening p53 half-life with a corresponding reduction in p53 transcription activity. RPS27L is mainly localized in the cytoplasm, but upon p53-activating signals, a portion of RPS27L shuttled to the nucleoplasm where it co-localizes with MDM2. Both cytoplasmic and nuclear p53, induced by ribosomal stress, were reduced upon RPS27L silencing. Our study reveals a multi-level interplay among RPS27L/S27 and p53-MDM2 axis with RPS27L acting as a p53 target, an MDM2 substrate, and a p53 regulator.

Purpose: Sensitive to apoptosis gene (SAG; also known as RBX2 or ROC2) was originally cloned as a redox-inducible antioxidant protein and was later characterized as a RING component of SCF E3 ubiquitin ligases. SAG overexpression inhibits apoptosis induced by many stimuli both in vitro and in vivo. SAG mRNA was overexpressed in human lung tumor tissues with a correlation to poor patient survival. To investigate whether SAG serves as an anticancer target, we determined the effect of SAG silencing on cell proliferation, survival, and radiosensitivity.Experimental Design: SAG protein expression in human tumors was evaluated by immunohistochemical staining using tumor tissue arrays. SAG expression in cancer cells was knocked down by siRNA silencing. The anticancer effects of SAG silencing were evaluated by in vitro assays for cell growth and survival and by an in vivo orthotopic xenograft tumor model. Radiosensitization by SAG silencing of human cancer cells was determined by clonogenic survival assay. Apoptosis induction was evaluated by fluorescence-activated cell sorting analysis, caspase-3 activation assay, and Western blotting of apoptosis-associated proteins.Results: SAG was overexpressed in multiple human tumor tissues compared with their normal counterparts. SAG silencing selectively inhibited cancer cell proliferation, suppressed in vivo tumor growth, and sensitized radiation-resistant cancer cells to radiation. Mechanistically, SAG silencing induced apoptosis with accumulation of NOXA, whereas SAG overexpression reduced NOXA levels and shortened NOXA protein half-life.Conclusions: The findings showed that SAG E3 ubiquitin ligase plays an essential role in cancer cell proliferation and tumor growth and may serve as a promising anticancer and radiosensitizing target.

Proteolysis-targeting chimeric molecules (PROTACs) represent an emerging technique that is receiving much attention for therapeutic intervention. The mechanism is based on the inhibition of protein function by hijacking a ubiquitin E3 ligase for protein degradation. The hetero-bifunctional PROTACs contain a ligand for recruiting an E3 ligase, a linker, and another ligand to bind with the protein targeted for degradation. Thus, PROTACs have profound potential to eliminate "undruggable" protein targets, such as transcription factors and non-enzymatic proteins, which are not limited to physiological substrates of the ubiquitin-proteasome system. These findings indicate great prospects for PROTACs in the development of therapeutics. However, there are several limitations related to poor stability, biodistribution, and penetrability in vivo. This review provides an overview of the main PROTAC-based approaches that have been developed and discusses the promising opportunities and considerations for the application of this technology in therapies and drug discovery.