ABSTRACT
Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.
Subject(s)
S-Phase Kinase-Associated Proteins , Small Molecule Libraries , Humans , S-Phase Kinase-Associated Proteins/metabolism , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemical synthesis , Proteolysis/drug effects , Phenylpropionates/chemistry , Phenylpropionates/pharmacology , Structure-Activity Relationship , Proteasome Endopeptidase Complex/metabolism , Molecular Structure , Ligands , HEK293 Cells , DegronsABSTRACT
BACKGROUND: Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. METHODS: Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. RESULTS: SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated ß-galactosidase (SA-ß-Gal) staining. CONCLUSIONS: Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.
Subject(s)
Apoptosis , Cellular Senescence , Jumonji Domain-Containing Histone Demethylases , Prostatic Neoplasms, Castration-Resistant , Proto-Oncogene Proteins c-akt , S-Phase Kinase-Associated Proteins , Signal Transduction , Humans , S-Phase Kinase-Associated Proteins/metabolism , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/genetics , Male , Apoptosis/drug effects , Cell Line, Tumor , Proto-Oncogene Proteins c-akt/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/drug therapy , Jumonji Domain-Containing Histone Demethylases/metabolism , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/genetics , Cellular Senescence/drug effects , Cellular Senescence/physiology , Signal Transduction/drug effects , Cell Proliferation/drug effects , Disease Progression , Prostatic Neoplasms/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/drug therapy , Cell Movement/drug effects , PC-3 Cells , Nuclear Proteins , Repressor ProteinsABSTRACT
We have previously reported that two inhibitors of an E3 ligase S-phase kinase-associated protein 2 (Skp2), SMIP004 and C1, have an antidepressant-like effect in non-stressed and chronically stressed mice. This prompted us to ask whether other Skp2 inhibitors could also have an antidepressant effect. Here, we used NSC689857, another Skp2 inhibitor, to investigate this hypothesis. The results showed that administration of NSC689857 (5â mg/kg) produced an antidepressant-like effect in a time-dependent manner in non-stressed male mice, which started 8â days after drug administration. Dose-dependent analysis showed that administration of 5 and 10â mg/kg, but not 1â mg/kg, of NSC689857 produced antidepressant-like effects in both non-stressed male and female mice. Administration of NSC689857 (5â mg/kg) also induced antidepressant-like effects in non-stressed male mice when administered three times within 24â h (24, 5, and 1â h before testing) but not when administered acutely (1â h before testing). In addition, NSC689857 and fluoxetine coadministration produced additive antidepressant-like effects in non-stressed male mice. These effects of NSC689857 were not associated with the changes in locomotor activity. Administration of NSC689857 (5â mg/kg) also attenuated depression-like behaviors in male mice induced by chronic social defeat stress, suggesting therapeutic potential of NSC689857 in depression. Overall, these results suggest that NSC689857 is capable of exerting antidepressant-like effects in both non-stressed and chronically stressed mice.
Subject(s)
Antidepressive Agents , Benzothiepins , Dose-Response Relationship, Drug , S-Phase Kinase-Associated Proteins , Animals , Female , Male , Mice , Antidepressive Agents/pharmacology , Behavior, Animal/drug effects , Depression/drug therapy , Disease Models, Animal , Fluoxetine/pharmacology , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Stress, Psychological/drug therapyABSTRACT
Renal cell carcinoma (RCC) is a common malignant tumor in the world. Histologically, most of RCC is classified as clear cell renal cell carcinoma (ccRCC), which is the most prevalent subtype. The overall survival of patients with ccRCC is poor, thus it is urgent to further explore its mechanism and target. S-phase kinase-associated protein 2 (SKP2) is overexpressed in a variety of human cancers and is associated with poor prognosis by enhancing tumor progression. However, it is unclear whether or how SKP2 is involved in ccRCC progression. Here, we reported that overexpression of SKP2 enhanced cell proliferation of ccRCC, while SKP2 depletion exhibited the opposite effect. Bioinformatic analyses found that SKP2 was positively correlated with Aurora-A (Aur-A) in ccRCC. The protein and mRNA levels of SKP2 were elevated or reduced by Aur-A overexpression or silencing, respectively. It was further found that Aur-A caused an increase phosphorylation of FOXO3A, which is a negatively transcription factor for SKP2. Interestingly, SKP2 mediated ubiquitylation and degradation of FOXO3A depend on the kinase activity of Aur-A. The combination of Aur-A inhibitor MLN8237 and SKP2 inhibitor SZL P1-41 showed a synergistic tumor growth inhibition in vivo and in vitro of ccRCC models. Thus, our data reveal that Aurora-A/FOXO3A/SKP2 axis promotes tumor progression in ccRCC, and the double inhibition of SKP2 and Aur-A shows significant synergistic effect, which indicates a potential new therapeutic strategy for ccRCC.
Subject(s)
Aurora Kinase A , Carcinoma, Renal Cell , Kidney Neoplasms , Aurora Kinase A/antagonists & inhibitors , Aurora Kinase A/metabolism , Azepines/pharmacology , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Forkhead Box Protein O3/metabolism , Humans , Kidney Neoplasms/drug therapy , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Molecular Targeted Therapy , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Signal TransductionABSTRACT
Lung cancer is by far the leading cause of cancer death worldwide, and 85% of patients are diagnosed with non-small cell lung cancer (NSCLC), which is still very difficult to treat. Skp2 functions as an oncogene that participates in processes of many cancers. Here, we report a novel Skp2 inhibitor AAA-237 that binds to Skp2 protein and inhibits the proliferation of the NSCLC cells. We further investigated the anti-NSCLC mechanism of AAA-237 and found that it arrested the cell cycle at the G0/G1 phase by targeting Skp2 to reduce the degradation of p21Cip1 and p27Kip1 or by transcriptionally activating FOXO1 to increase the mRNA expression of p21Cip1 and p27Kip1. More importantly, we found that treatment of a high concentration AAA-237 could induce apoptosis of NSCLC cells and treatment of a low AAA-237 concentration for a longer time could induce senescence of NSCLC cells. Similar results were found in nude mice xenografted with A549 cells. AAA-237 inhibited tumor growth by inducing apoptosis and senescence in a dose-dependent manner. Considering these results, we propose that AAA-237 could be a promising therapeutic drug for treating patients with NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Cell Cycle Checkpoints , Lung Neoplasms , S-Phase Kinase-Associated Proteins , A549 Cells , Animals , Apoptosis , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p27/metabolism , G1 Phase , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Mice , Mice, Nude , S-Phase Kinase-Associated Proteins/antagonists & inhibitorsABSTRACT
Skp1-Cul1-F-box protein (SCF) ubiquitin E3 ligases play important roles in cancer development and serve as a promising therapeutic target in cancer therapy. Brusatol (Bru), a known Nrf2 inhibitor, holds promise for treating a wide range of tumors; however, the direct targets of Bru and its anticancer mode of action remain unclear. In our study, 793 Bru-binding candidate proteins were identified by using a biotin-brusatol conjugate (Bio-Bru) followed by streptavidin-affinity pull down-based mass spectrometry. We found that Bru can directly bind to Skp1 and disrupt the interactions of Skp1 with the F-box protein Skp2, leading to the inhibition of the Skp2-SCF E3 ligase. Bru inhibited both proliferation and migration via promoting the accumulation of the substrates p27 and E-cadherin; Skp1 overexpression attenuated while Skp1 knockdown enhanced these effects of Bru in non-small cell lung cancer (NSCLC) cells. Moreover, Bru binding to Skp1 also inhibited the ß-TRCP-SCF E3 ligase. In both subcutaneous and orthotopic NSCLC xenografts, Bru significantly inhibited the growth and metastasis of NSCLC through targeting SCF complex and upregulating p27 and E-cadherin protein levels. These data demonstrate that Bru is a Skp1-targeting agent that may have therapeutic potentials in lung cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Biotin/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Quassins/therapeutic use , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Biotin/pharmacology , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice, Inbred BALB C , Mice, Nude , Quassins/pharmacology , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolismABSTRACT
Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homoeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCFSKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. The Cancer Genome Atlas (TCGA) data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation.
Subject(s)
Cell Transformation, Neoplastic/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , Nuclear Proteins/genetics , Phosphoproteins/genetics , S Phase/genetics , S-Phase Kinase-Associated Proteins/genetics , A549 Cells , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Cyclin-Dependent Kinase Inhibitor p21/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p27/metabolism , DNA Damage , Gene Expression Regulation, Neoplastic , HCT116 Cells , HT29 Cells , Humans , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Osteoblasts/metabolism , Osteoblasts/pathology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/metabolism , Sf9 Cells , Signal Transduction , Spodoptera , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/geneticsABSTRACT
One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Receptors, Estrogen/physiology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Myocytes, Cardiac/metabolism , Receptors, Estrogen/chemistry , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Sarcolemma/drug effects , Sarcolemma/metabolism , Sarcomeres/drug effects , Sarcomeres/metabolism , Transcriptome/drug effects , Troponin I/genetics , Troponin I/metabolismABSTRACT
Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in Western countries, and there is still an urgent need for a better understanding of PCa progression to inspire new treatment strategies. Skp2 is a substrate-recruiting component of the E3 ubiquitin ligase complex, whose activity is regulated through neddylation. Slug is a transcriptional repressor involved in the epithelial-to-mesenchymal transition, which may contribute to therapy resistance. Although Skp2 has previously been associated with a mesenchymal phenotype and prostate cancer progression, the relationship with Slug deserves further elucidation. We have previously shown that a high Gleason score (≥8) is associated with higher Skp2 and lower E-cadherin expression. In this study, significantly increased expression of Skp2, AR, and Slug, along with E-cadherin downregulation, was observed in primary prostate cancer in patients who already had lymph node metastases. Skp2 was slightly correlated with Slug and AR in the whole cohort (Rs 0.32 and 0.37, respectively), which was enhanced for both proteins in patients with high Gleason scores (Rs 0.56 and 0.53, respectively) and, in the case of Slug, also in patients with metastasis to lymph nodes (Rs 0.56). Coexpression of Skp2 and Slug was confirmed in prostate cancer tissues by multiplex immunohistochemistry and confocal microscopy. The same relationship between these two proteins was observed in three sets of prostate epithelial cell lines (PC3, DU145, and E2) and their mesenchymal counterparts. Chemical inhibition of Skp2, but not RNA interference, modestly decreased Slug protein in PC3 and its docetaxel-resistant subline PC3 DR12. Importantly, chemical inhibition of Skp2 by MLN4924 upregulated p27 and decreased Slug expression in PC3, PC3 DR12, and LAPC4 cells. Novel treatment strategies targeting Skp2 and Slug by the neddylation blockade may be promising in advanced prostate cancer, as recently documented for other aggressive solid tumors.
Subject(s)
NEDD8 Protein/genetics , Prostatic Neoplasms/genetics , Protein Processing, Post-Translational , S-Phase Kinase-Associated Proteins/genetics , Snail Family Transcription Factors/genetics , Antigens, CD/genetics , Antigens, CD/metabolism , Antineoplastic Agents/pharmacology , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cyclopentanes/pharmacology , Docetaxel/pharmacology , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Humans , Lymphatic Metastasis , Male , NEDD8 Protein/metabolism , Neoplasm Grading , PC-3 Cells , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Pyrimidines/pharmacology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/metabolism , Snail Family Transcription Factors/metabolismABSTRACT
S-phase kinase-associated protein 2 (Skp2) performs oncogenic functions in cancers; however, how Skp2 is regulated post-transcriptionally is elusive in osteosarcoma. Therefore, we determined whether miR-506 could directly target Skp2 in osteosarcoma to perform its tumor suppressive functions. Here, we found that miR-506 mimics suppressed cell viability, induced apoptosis, and attenuated migration and invasion in osteosarcoma cells. Moreover, upregulation of Skp2 accelerated cell viability and motility and rescued the tumor suppressive effect of miR-506 in osteosarcoma cells. Moreover, downregulation of Skp2 inhibited cell viability and decreased cell motility, which enhanced the antitumor activity induced by miR-506 mimic transfection in osteosarcoma cells. Our western blotting results implied that miR-506 inhibited Skp2 expression and subsequently upregulated Foxo1 and p57 in OS cells. In summary, miR-506 performs an anticancer activity via directly targeting Skp2 in osteosarcoma cells, indicating that inactivation of Skp2 by miR-506 might be an alternative strategy for treating osteosarcoma.
Subject(s)
Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Osteosarcoma/genetics , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/genetics , Apoptosis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Survival/genetics , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Forkhead Box Protein O1/metabolism , Humans , Neoplasm Invasiveness/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , S-Phase Kinase-Associated Proteins/metabolism , Transfection , Up-Regulation/geneticsABSTRACT
The objective was to investigate the anti-cancer effects and underlying molecular mechanisms of cytostasis which were activated by an anti-microtubule drug, ABT-751, in two urinary bladder urothelial carcinoma (UBUC)-derived cell lines, BFTC905 and J82, with distinct genetic backgrounds. A series of in vitro assays demonstrated that ABT-751 induced G2/M cell cycle arrest, decreased cell number in the S phase of the cell cycle and suppressed colony formation/independent cell growth, accompanied with alterations of the protein levels of several cell cycle regulators. In addition, ABT-751 treatment significantly hurdled cell migration and invasion along with the regulation of epithelial-mesenchymal transition-related proteins. ABT-751 triggered autophagy and apoptosis, downregulated the mechanistic target of rapamycin kinase (MTOR) and upregulated several pro-apoptotic proteins that are involved in extrinsic and intrinsic apoptotic pathways. Inhibition of autophagosome and autolysosome enhanced apoptosis was also observed. Through the inhibition of the NFκB signaling pathway, ABT-751 suppressed S-phase kinase associated protein 2 (SKP2) transcription and subsequent translation by downregulation of active/phospho-AKT serine/threonine kinase 1 (AKT1), component of inhibitor of nuclear factor kappa B kinase complex (CHUK), NFKB inhibitor alpha (NFKBIA), nuclear RELA proto-oncogene, NFκB subunit (RELA) and maintained a strong interaction between NFKBIA and RELA to prevent RELA nuclear translocation for SKP2 transcription. ABT-751 downregulated stable/phospho-SKP2 including pSKP2(S64) and pSKP2(S72), which targeted cyclin-dependent kinase inhibitors for degradation through the inactivation of AKT. Our results suggested that ABT-751 may act as an anti-cancer drug by inhibiting cell migration, invasion yet inducing cell cycle arrest, autophagy and apoptosis in distinct UBUC-derived cells. Particularly, the upstream molecular mechanism of its anticancer effects was identified as ABT-751-induced cytostasis through the inhibition of SKP2 at both transcriptional and post-translational levels to stabilize cyclin dependent kinase inhibitor 1A (CDKN1A) and CDKN1B proteins.
Subject(s)
Carcinoma/drug therapy , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , S-Phase Kinase-Associated Proteins/genetics , Transcription, Genetic/drug effects , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Carcinoma/genetics , Carcinoma/pathology , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Phosphatidylinositol 3-Kinase/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Mas , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Sulfonamides/pharmacology , Urinary Bladder/drug effects , Urinary Bladder/pathology , Urothelium/drug effects , Urothelium/pathologyABSTRACT
We have reported that SMIP004, an inhibitor of S-phase kinase-associated protein 2 (Skp2), displays antidepressant-like activities in stress-naïve and chronically stressed mice. Here, we investigated the antidepressant-like effect of C1, another inhibitor of Skp2, in mouse models following acute or chronic drug administration at different doses and treatment times by using the tail suspension test (TST), forced swimming test (FST), and social interaction test (SIT). The time- and dose-dependent results showed that the antidepressant-like effect of C1 occurred 8 days after the drug treatment, and C1 produced antidepressant-like activities at the dose of 5 and 10 but not 1 mg/kg in male or female mice. C1 administration (5 mg/kg) also induced antidepressant-like effects in stress-naïve mice in a three-times administration mode within 24 h (24, 5, and 1 h before the test) but not in an acute administration mode (1 h before the test). The C1 and fluoxetine co-administration produced additive effect on depression-like behaviors in stress-naïve mice. The antidepressant-like effect of C1 was not associated with the change in locomotor activity, as no increased locomotor activity was observed in different treatment modes. Furthermore, the long-term C1 treatment (5 mg/kg) was found to ameliorate the depression-like behaviors in chronic social defeat stress-exposed mice, suggesting that C1 can produce antidepressant-like actions in stress conditions. Since C1 is a specific inhibitor of Skp2, our results demonstrate that inhibition of Skp2 might be a potential strategy for the treatment of depression, and Skp2 may be potential target for the development of novel antidepressants.
Subject(s)
Antidepressive Agents/pharmacology , Depression/drug therapy , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Animals , Antidepressive Agents/administration & dosage , Depression/physiopathology , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Synergism , Female , Fluoxetine/pharmacology , Hindlimb Suspension , Locomotion/drug effects , Male , Mice , Mice, Inbred C57BL , Stress, Psychological/drug therapy , Swimming , Time FactorsABSTRACT
Whether glucose is predominantly metabolized via oxidative phosphorylation or glycolysis differs between quiescent versus proliferating cells, including tumor cells. However, how glucose metabolism is coordinated with cell cycle in mammalian cells remains elusive. Here, we report that mammalian cells predominantly utilize the tricarboxylic acid (TCA) cycle in G1 phase, but prefer glycolysis in S phase. Mechanistically, coupling cell cycle with metabolism is largely achieved by timely destruction of IDH1/2, key TCA cycle enzymes, in a Skp2-dependent manner. As such, depleting SKP2 abolishes cell cycle-dependent fluctuation of IDH1 protein abundance, leading to reduced glycolysis in S phase. Furthermore, elevated Skp2 abundance in prostate cancer cells destabilizes IDH1 to favor glycolysis and subsequent tumorigenesis. Therefore, our study reveals a mechanistic link between two cancer hallmarks, aberrant cell cycle and addiction to glycolysis, and provides the underlying mechanism for the coupling of metabolic fluctuation with periodic cell cycle in mammalian cells.
Subject(s)
Citric Acid Cycle/physiology , Glycolysis/physiology , S-Phase Kinase-Associated Proteins/metabolism , Animals , Cell Line , G1 Phase , Glucose/metabolism , Glycolysis/drug effects , Humans , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Mice , Mutagenesis, Site-Directed , Nocodazole/pharmacology , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Interference , RNA, Small Interfering/metabolism , S Phase , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/genetics , UbiquitinationABSTRACT
Bcr-Abl is the primary cause as well as currently key therapeutic target of chronic myeloid leukemia (CML). SKP2, an E3 ligase, is a downstream factor of Bcr-Abl to motivate the cell cycle transition of CML and also found to bind and activate Bcr-Abl in reverse. Therefore, SKP2/Bcr-Abl pathway is an attractive target for CML treatment. This study aims to identify an inhibitor of the SKP2/Bcr-Abl pathway based on a large screening of the natural products. We demonstrate that Diosmetin, a kind of phytoestrogens, notably downregulates the expression of SKP2, Bcr-Abl phosphorylation, and moderately downregulates the Bcr-Abl level. Furthermore, Diosmetin displays a favorable anti-tumor activity in CML cells and xenograft models. Collectively, our study reveals a natural compound in the treatment of CML on the basis of SKP2/Bcr-Abl signaling pathway.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cell Proliferation/drug effects , Flavonoids/pharmacology , Fusion Proteins, bcr-abl/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Protein Kinase Inhibitors/pharmacology , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Animals , Apoptosis/drug effects , Down-Regulation , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Humans , Imatinib Mesylate/pharmacology , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Mice, Inbred BALB C , Mice, Nude , Phosphorylation , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Signal Transduction , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Cell cycle slowdown or arrest is a prominent feature of cellular senescence. S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, is a key regulator of G1/S transition. We investigated whether Skp2 plays a role in the regulation of endothelial progenitor cell (EPC) senescence, which is closely associated with aging-related vasculopathy. Replication-induced senescent EPCs demonstrated more pronounced senescence markers and lower Skp2 levels in comparison with those of their younger counterparts. Depletion of Skp2 induced increases in senescence-associated ß-galactosidase (SA-ßGal) activity and a reduction of telomere length and generated a senescent bioenergetics profile, whereas adenoviral-mediated Skp2 expression reversed the relevant senescence. EPCs isolated from older rats displayed a reduced proliferation rate and increased SA-ßGal activity, both of which were significantly reversed by Skp2 ectopic expression. In addition to reversing senescence, Skp2 also rescued the angiogenic activity of senescent EPCs in the ischemic hind limbs of nude mice. The results revealed that ectopic expression of Skp2 has the potential to rejuvenate senescent EPCs and rescue their angiogenic activity and thus may be pivotal in the development of novel strategies to manage aging-related vascular disease.
Subject(s)
Cell Cycle/genetics , Endothelial Progenitor Cells/metabolism , Ischemia/therapy , Leukocytes, Mononuclear/metabolism , Neovascularization, Physiologic , S-Phase Kinase-Associated Proteins/genetics , Animals , Cell Proliferation , Cellular Senescence/genetics , Endothelial Progenitor Cells/cytology , Endothelial Progenitor Cells/transplantation , Gene Expression Regulation , Hindlimb/blood supply , Hindlimb/pathology , Humans , Ischemia/genetics , Ischemia/metabolism , Ischemia/pathology , Leukocytes, Mononuclear/cytology , Mice , Mice, Inbred BALB C , Mice, Nude , Mitochondria/metabolism , Primary Cell Culture , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/metabolism , Telomere , Telomere HomeostasisABSTRACT
The RB1 tumor suppressor gene is mutated in highly aggressive tumors including small-cell lung cancer (SCLC), where its loss, along with TP53, is required and sufficient for tumorigenesis. While RB1-mutant cells fail to arrest at G1-S in response to cell-cycle restriction point signals, this information has not led to effective strategies to treat RB1-deficient tumors, as it is challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/Trp53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, which was confirmed as the mechanism of protection by using knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind/inhibit Skp2 have in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using genetic and pharmacologic approaches, antitumor activity was seen with Skp2 loss or inhibition in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. Our data highlight a downstream actionable target in RB1-deficient cancers, for which there are currently no targeted therapies available. SIGNIFICANCE: There are no effective therapies for SCLC. The identification of an actionable target downstream of RB1, inactivated in SCLC and other advanced tumors, could have a broad impact on its treatment.
Subject(s)
CDC2-CDC28 Kinases/antagonists & inhibitors , Lung Neoplasms/drug therapy , Retinoblastoma Protein/deficiency , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Small Cell Lung Carcinoma/drug therapy , Animals , CDC2-CDC28 Kinases/genetics , CDC2-CDC28 Kinases/metabolism , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Mice , Mice, Knockout , Molecular Targeted Therapy , Retinoblastoma Binding Proteins/deficiency , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , S-Phase Kinase-Associated Proteins/metabolism , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/metabolism , Small Molecule Libraries/pharmacology , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Xenograft Model Antitumor AssaysSubject(s)
Diosgenin/analogs & derivatives , Neoplasms/drug therapy , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Animals , Biological Products/therapeutic use , Cell Line, Tumor , Diosgenin/therapeutic use , Humans , Mice , S-Phase Kinase-Associated Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Quadruple negative breast cancer (QNBC), lacking the expression of ER (estrogen receptor), PR (progesterone receptor), HER2 (human epidermal growth factor receptor-2) and AR (androgen receptor), was regarded as one breast cancer subtype with the worst prognosis. Recently, the molecular features of QNBC are not well understood. Different from AR-positive triple-negative breast cancer, QNBC is insensitive to conventional chemotherapeutic agents and has no efficient treatment targets. However, QNBC has been shown to express unique proteins that may be amenable to use in the development of targeted therapies. Here we reviewed the features of QNBC and proteins that may serve as effective targets for QNBC treatment, such as ACSL4, SKP2, immune checkpoint inhibitors, EGFR, MicroRNA signatures and Engrailed 1.
Subject(s)
Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/antagonists & inhibitors , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/metabolism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/metabolism , Humans , MicroRNAs/metabolism , Prognosis , Receptor, ErbB-2/metabolism , Receptors, Androgen/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/metabolismABSTRACT
Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).
Subject(s)
Apoptosis , Cell Proliferation , Gene Expression Regulation, Neoplastic , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Protein Kinase Inhibitors/pharmacology , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , S-Phase Kinase-Associated Proteins/physiology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.