Yongchao Zhao

DEPTOR, an mTOR Inhibitor, Is a Physiological Substrate of SCFβTrCP E3 Ubiquitin Ligase and Regulates Survival and Autophagy

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 SCF(βTrCP) 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.

Cullin-RING Ligases as Attractive Anti-cancer Targets

The ubiquitin-proteasome system (UPS) promotes the timely degradation of short-lived proteins with key regulatory roles in a vast array of biological processes, such as cell cycle progression, oncogenesis and genome integrity. Thus, abnormal regulation of UPS disrupts the protein homeostasis and causes many human diseases, particularly cancer. Indeed, the FDA approval of bortezomib, the first class of general proteasome inhibitor, for the treatment of multiple myeloma, demonstrated that the UPS can be an attractive anti-cancer target. However, normal cell toxicity associated with bortezomib, resulting from global inhibition of protein degradation, promotes the focus of drug discovery efforts on targeting enzymes upstream of the proteasome for better specificity. E3 ubiquitin ligases, particularly those known to be activated in human cancer, become an attractive choice. Cullin-RING Ligases (CRLs) with multiple components are the largest family of E3 ubiquitin ligases and are responsible for ubiquitination of ~20% of cellular proteins degraded through UPS. Activity of CRLs is dynamically regulated and requires the RING component and cullin neddylation. In this review, we will introduce the UPS and CRL E3s and discuss the biological processes regulated by each of eight CRLs through substrate degradation. We will further discuss how cullin neddylation controls CRL activity, and how CRLs are being validated as the attractive cancer targets by abrogating the RING component through genetic means and by inhibiting cullin neddylation via MLN4924, a small molecule indirect inhibitor of CRLs, currently in several Phase I clinical trials. Finally, we will discuss current efforts and future perspectives on the development of additional inhibitors of CRLs by targeting E2 and/or E3 of cullin neddylation and CRL-mediated ubiquitination as potential anti-cancer agents.

Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis

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.

Ribosomal protein S27-like and S27 interplay with p53-MDM2 axis as a target, a substrate and a regulator

Several ribosomal proteins regulate p53 function by 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. Similar to 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 colocalizes with MDM2. Both the cytoplasmic and the nuclear p53, induced by ribosomal stress, were reduced upon RPS27L silencing. Our study reveals a multilevel interplay between RPS27L/S27 and p53-MDM2 axis, with RPS27L functioning as a p53 target, a MDM2 substrate and a p53 regulator.

SAG/RBX2/ROC2 E3 ubiquitin ligase is essential for vascular and neural development by targeting NF1 for degradation.

SAG/RBX2/ROC2 protein is an essential RING component of SCF E3 ubiquitin ligase. The role of SAG during embryogenesis remains unknown. We report a critical role for SAG in controlling vascular and neural development by modulating RAS activity via promoting degradation of neurofibromatosis type 1 (NF1). Mice mutant for Sag died at embryonic day 11.5-12.5 with severe abnormalities in vascular and nervous system. Sag inactivation caused Nf1 accumulation and Ras inhibition, which blocks embryonic stem (ES) cells from undergoing endothelial differentiation and inhibits angiogenesis and proliferation in teratomas. Simultaneous Nf1 deletion fully rescues the differentiation defects in Sag(-/-) ES cells and partially rescues vascular and neural defects in Sag(-/-) embryos, suggesting that the effects of Sag deletion may not be solely explained by Nf1 misregulation. Collectively, our study identifies NF1 as a physiological substrate of SAG-CUL1-FBXW7 E3 ligase and establishes a ubiquitin-dependent regulatory mechanism for the NF1-RAS pathway during embryogenesis.

Targeting Neddylation Pathways to Inactivate Cullin-RING Ligases for Anticancer Therapy

SIGNIFICANCE Protein neddylation is catalyzed by an E1 NEDD8-activating enzyme (NAE), an E2 NEDD8-conjugating enzyme, and an E3 NEDD8 ligase. Known physiological substrates of neddylation are cullin family members. Cullin neddylation leads to activation of cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases responsible for ubiquitylation and degradation of many key signaling/regulatory proteins. Thus, through modulating CRLs, neddylation regulates many biological processes, including cell cycle progression, signal transduction, and tumorigenesis. Given that NEDD8 is overexpressed and CRLs are abnormally activated in many human cancers, targeting protein neddylation, in general, and cullin neddylation, in particular, appears to be an attractive anticancer approach. RECENT ADVANCES MLN4924, a small molecule inhibitor of NAE, was discovered that inactivates CRLs and causes accumulation of CRL substrates to suppress tumor cell growth both in vitro and in vivo. Promising preclinical results advanced MLN4924 to several clinical trials for anticancer therapy. CRITICAL ISSUES In preclinical settings, MLN4924 effectively suppresses tumor cell growth by inducing apoptosis, senescence, and autophagy, and causes sensitization to chemoradiation therapies in a cellular context-dependent manner. Signal molecules that determine the cell fate upon MLN4924 treatment, however, remain elusive. Cancer cells develop MLN4924 resistance by selecting target mutations. FUTURE DIRECTIONS In the clinical side, several Phase 1b trials are under way to determine the safety and efficacy of MLN4924, acting alone or in combination with conventional chemotherapy, against human solid tumors. In the preclinical side, the efforts are being made to develop additional neddylation inhibitors by targeting NEDD8 E2s and E3s.

Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis

Cullin-RING ligases (CRLs) are a family of E3 ubiquitin ligase complexes that rely on either RING-box 1 (RBX1) or sensitive to apoptosis gene (SAG), also known as RBX2, for activity. RBX1 and SAG are both overexpressed in human lung cancer; however, their contribution to patient survival and lung tumorigenesis is unknown. Here, we report that overexpression of SAG, but not RBX1, correlates with poor patient prognosis and more advanced disease. We found that SAG is overexpressed in murine KrasG12D-driven lung tumors and that Sag deletion suppressed lung tumorigenesis and extended murine life span. Using cultured lung cancer cells, we showed that SAG knockdown suppressed growth and survival, inactivated both NF-κB and mTOR pathways, and resulted in accumulation of tumor suppressor substrates, including p21, p27, NOXA, and BIM. Importantly, growth suppression by SAG knockdown was partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR. Treatment with MLN4924, a small molecule inhibitor of CRL E3s, also inhibited the formation of KrasG12D-induced lung tumors through a similar mechanism involving inactivation of NF-κB and mTOR and accumulation of tumor suppressor substrates. Together, our results demonstrate that Sag is a Kras-cooperating oncogene that promotes lung tumorigenesis and suggest that targeting SAG-CRL E3 ligases may be an effective therapeutic approach for Kras-driven lung cancers.

Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-κB activation in mouse embryonic stem cells

SAG (sensitive to apoptosis gene; also known as RBX2 or ROC2) is a dual-function protein with antioxidant activity when acting alone or E3 ligase activity when complexed with other components of SCF (Skp1, cullins, F-box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene-trapping strategy significantly sensitized mouse embryonic stem (ES) cells to radiation, with a sensitizing enhancement rate of 1.5-1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24h after irradiation as well as enhancement of radiation-induced apoptosis. Furthermore, Sag elimination abrogated IkappaBalpha degradation leading to inhibition of NF-kappaB activation. Further detailed analysis revealed that IkappaBalpha is a direct substrate of SAG-SCF(beta-TrCP) E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NF-kappaB.

Ribosomal protein S27-like is a physiological regulator of p53 that suppresses genomic instability and tumorigenesis

Cell-based studies showed that several Mdm2-binding ribosomal proteins, upon overexpression, stabilize and activate p53. In contrast, here we show in a mouse knockout study that Mdm2-binding ribosomal protein S27-like (Rps27l), upon disruption, activates p53. Germline inactivation of Rps27l triggers ribosomal stress to stabilize Mdm2, which degrades Mdm4 to reduce Mdm2-Mdm4 E3 ligase towards p53, leading to p53-dependent apoptotic depletion of hematopoietic stem cells and postnatal death, which is rescued by Trp53 deletion. Paradoxically, while increased p53 is expected to inhibit tumorigenesis, Rps27l−/−;Trp53+/− mice develop lymphomas at higher incidence with p53 loss-of-heterozygosity and severe genome aneuploidy, suggesting that Rps27l disruption impose a selection pressure against p53. Thus, Rps27l has dual functions in p53 regulation: under Trp53+/+ background, Rps27l disruption triggers ribosomal stress to induce p53 and apoptosis, whereas under Trp53+/− background, Rps27l disruption triggers genomic instability and Trp53 deletion to promote tumorigenesis. Our study provides a new paradigm of p53 regulation. DOI: http://dx.doi.org/10.7554/eLife.02236.001

Smac-mimetic compound SM-164 induces radiosensitization in breast cancer cells through activation of caspases and induction of apoptosis

Radiotherapy is a treatment choice for local control of breast cancer, particularly after the removal of tumor tissues by surgery. However, intrinsic radioresistance of cancer cells limits therapeutic efficacy. Here, we determined in breast cancer cells the potential radiosensitizing activity of SM-164, a small molecule compound, that mimics the activity of SMAC, a mitochondrial protein released during apoptosis to activate caspases by inhibiting cellular inhibitor of apoptosis proteins, cIAP-1, and XIAP. We found that SM-164 at nanomolar concentrations promoted degradation of cIAP-1, disrupted the inhibitory binding of XIAP to active caspase-9, and sensitized breast cancer cells to radiation with a sensitization enhancement ratio (SER) of 1.7–1.8. In one line of breast cancer cells resistant to SM-164 as a single agent, SM-164 radiosensitization was mediated by intrinsic apoptosis pathway through activation of caspases-9 and -3. In a line of breast cancer cells sensitive to SM-164 as a single agent, SM-164 radiosensitization was mediated by both extrinsic and intrinsic apoptosis pathways through activation of caspases-9, -8, and -3. Consistently, blockage of caspase activation, through siRNA knockdown or treatment with a pan-caspase inhibitor z-VAD-fmk, inhibited apoptosis and abrogated SM-164 radiosensitization. Our study demonstrates that IAPs are valid radiosensitizing targets in breast cancer cells and SM-164 could be further developed as a novel class of radiosensitizers for the treatment of radioresistant breast cancer.