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Currently, as the largest family of E3 ubiquitin ligases, Skp1-Cullin 1-F-box (SCF) E3 ligase complexes have attracted extensive attention. Among SCF complexes, Skp2, β-TrCP, and FBXW7 have undergone extensive research on their structures and functions. Previous studies suggest Skp2, β-TrCP, and FBXW7 are overexpressed in numerous cancers. Thus, the SCF E3 ligase complex has become a significant target for the development of anti-cancer drugs. Over the past few decades, a variety of anti-tumor inhibitors targeting the SCF E3 ligase complex have been attempted. However, since almost none of the SCF E3 ligase inhibitors passed clinical trials, the design and synthesis of the new inhibitors are needed. Here, we will introduce the structure and function of Skp2, β-TrCP, and FBXW7, their connections with cancer development, the relevant in vitro and in vivo activities, selectivity, structure-activity relationships, and the therapeutic or preventive application of small molecule inhibitors targeting these three F-box proteins reported in the patent (2010-present). This information will help develop drugs targeting the SCF E3 ubiquitin ligase, providing new strategies for future cancer treatments., Competing Interests: Declaration of competing interest This paper has not been published elsewhere in whole or in part. All authors have read and approved the content, and agree to submit it for consideration for publication in your journal. This paper does not address ethical/legal conflicts., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The proliferative and migratory abilities of vascular smooth muscle cells (VSMCs) play a crucial role in neointima formation following vascular injury. Skp2 facilitates proliferation and migration in cells through cell cycle regulation, presenting an important therapeutic target for atherosclerosis, pulmonary hypertension, and vascular restenosis. This study aimed to identify a natural product capable of inhibiting neointima formation post vascular injury. Here, we demonstrate that troxerutin, a flavonoid, significantly reduced viability and downregulated Skp2 in VSMCs. Moreover, troxerutin exhibited anti-proliferative effects on VSMCs and mitigated neointima formation. These findings collectively elucidate the intrinsic mechanism of troxerutin in treating atherosclerosis, pulmonary hypertension, and vascular restenosis by targeting the E3-linked enzyme Skp2., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Skp1-CUL1-ROC1-F-box E3 ubiquitin ligases' main component S-phase kinase-associated protein 2 (Skp2) is responsible for specifically recognizing ubiquitination-modified substrates to be degraded such as p27 and p21 in the case of binding with adaptor protein Cks1. Pharmacological inhibition of Skp2 has exhibited promising antitumor activity. Herein, we present the design and optimization of a series of [1,2,4]triazolo[1,5- a ]pyrimidine-based small molecules targeting Skp2. Among them, E35 demonstrated excellent inhibitory activities against the binding of Skp2-Cks1. In addition, compound E35 significantly inhibited colony formation and migration, as well as arrested the cell cycle at the S-phase. Mechanistically, compound E35 markedly decreased the expression of Skp2, as well as increased the expression of its substrates p21 and p27. Furthermore, compound E35 showed an obvious inhibitory effect on MGC-803 xenograft mice without obvious toxicity. All of these results suggest that compound E35 might be a valuable lead compound for antitumor agents targeting Skp2.

Introduction: Psoriasis is a chronic immune-mediated disorder affecting over 2-3% of the population worldwide, significantly impacting quality of life. Despite the availability of various therapeutic interventions, concerns persist regarding lesion recurrence and potential alterations in immune surveillance promoting cancer progression. Recent advancements in understanding cellular and molecular pathways have unveiled key factors in psoriasis etiology, including IL-17, 22, 23, TNF-α, PDE-4, JAK-STAT inhibitors, and AhR agonists. This work explores the potential of S-phase kinase-associated protein 2 (Skp2) as a therapeutic target in psoriasis., Area Covered: This review covers the current understanding of psoriasis pathophysiology, including immune dysregulation, and the role of keratinocytes and ubiquitin. It also delves into Skp2 role in cell cycle regulation, and its correlation with angiogenesis and ubiquitin in psoriasis. The evolving therapeutic approaches targeting Skp2, including small molecule inhibitors, are also discussed., Expert Opinion: Targeting Skp2 holds promise for developing novel therapeutic approaches for psoriasis. By modulating Skp2 activity or expression, it may be possible to intervene in inflammatory and proliferative processes underlying the disease. Further research into Skp2 inhibitors and their efficacy in preclinical and clinical settings is warranted to harness the full potential of Skp2 as a therapeutic target in psoriasis management.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 β-galactosidase (SA-β-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., (© 2024 Wiley Periodicals LLC.)

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 (5 mg/kg) produced an antidepressant-like effect in a time-dependent manner in non-stressed male mice, which started 8 days after drug administration. Dose-dependent analysis showed that administration of 5 and 10 mg/kg, but not 1 mg/kg, of NSC689857 produced antidepressant-like effects in both non-stressed male and female mice. Administration of NSC689857 (5 mg/kg) also induced antidepressant-like effects in non-stressed male mice when administered three times within 24 h (24, 5, and 1 h before testing) but not when administered acutely (1 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 (5 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., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 β-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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 SCF SKP2 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., (© 2021. The Author(s).)

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 TNNI1 EmGFP and TNNI3 mCherry 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.

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.

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.

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., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

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 G 2 /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.

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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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 G 1 -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., (©2020 American Association for Cancer Research.)

Timed degradation of the cyclin-dependent kinase inhibitor p27 Kip1 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/p27 Kip1 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).

Cell cycle slowdown or arrest is a prominent feature of cellular senescence. S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCF Skp2 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 β-galactosidase (SA-βGal) 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-βGal 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.

Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest.

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.

Background: Mantle cell lymphoma (MCL) is associated with poor patient prognosis mainly due to incomplete response to chemotherapy. S-phase kinase-related protein 2 (SKP2) is an oncoprotein that promotes cell cycle progression and proliferation. A recent study revealed that SKP2 is also involved in DNA damage response mechanisms. SKP2 induces activation of the Ataxia-telangiectasia-mutated (ATM) protein kinase by regulating NBS1 ubiquitination. The authors thus hypothesized that SKP2-mediated ATM activation is associated with MCL resistance to cisplatin (DDP). Materials and Methods: DDP-resistant MCL cell lines JeKo-1/DDP and Mino/DDP were established by culturing JeKo-1 and Mino cells, respectively, with increasing concentrations of DDP. Protein expression levels of SKP2, ATM, and phosphorylated ATM (p-ATM) in the cell lines were assessed using western blotting. The extent of NBS1 ubiquitination was determined with immunoprecipitation assays. Cell viability, apoptosis, and DNA damage were analyzed using specific detection kits. Results: JeKo-1/DDP and Mino/DDP cells showed higher levels of SKP2 and p-ATM proteins than JeKo-1 and Mino cells, respectively. SKP2 knockdown resulted in a reduced NBS1 ubiquitination and p-ATM protein level in JeKo-1/DDP cells. Both SKP2 knockdown and treatment with an ATM inhibitor enhanced DDP-induced DNA damage in JeKo-1/DDP cells by decreasing amounts of RAD51 and FANCD2, which are factors responsible for DNA repair. Consequently, both SKP2 knockdown and ATM inhibition increased the sensitivity of JeKo-1/DDP cells to DDP treatment, with a more pronounced effect observed by SKP2 depletion. Conclusion: These results suggest that SKP2 is likely to be a more promising target than ATM in the treatment of DDP-resistant MCL.

CD74, a non-polymorphic type II transmembrane glycoprotein and MHC class II chaperone, is the cell surface receptor for the inflammatory cytokine macrophage migration inhibitory factor (MIF) and participates in inflammatory signaling regulation. This study examined the potential role of CD74 in binge drinking-induced cardiac contractile dysfunction. WT and CD74 knockout mice were exposed to ethanol (3 g/kg/d, i.p., for 3 days). Echocardiography, cardiomyocyte function, histological staining and autophagy signaling including AMPK, mTOR, and AMPK downstream signals Skp2 and Sirt1 were evaluated. Our results revealed that ethanol challenge overtly compromised echocardiographic, cardiomyocyte contractile, intracellular Ca 2+ and ultrastructural properties along with overt apoptosis, inflammation (elevated MIF, IL-1β and IL-6) and mitochondrial O 2 - production (p < 0.01), the effect of which was reconciled by CD74 ablation (p < 0.01 vs. ethanol group) with the exception of MIF expression. Ethanol challenge upregulated autophagy (p < 0.001), promoted AMPK phosphorylation and Sirt1 levels (p < 0.003) while suppressing mTOR phosphorylation and Skp2 levels (p < 0.02). These effects were reversed by CD74 ablation. In vitro studies demonstrated that short-term ethanol challenge compromised cardiomyocyte contractile function and facilitated GFP-Puncta formation, which were mitigated by CD74 knockout (p < 0.0001). Moreover, the CD74 ablation-offered beneficial effects against ethanol-induced cardiomyocyte dysfunction, and GFP-Puncta formation were nullified by the AMPK activator AICAR, the Skp2 inhibitor C1 or the Sirt1 activator SRT1720 (p < 0.0001). Taken together, our data revealed that CD74 ablation counteracts acute ethanol challenge-induced myocardial dysfunction, inflammation and apoptosis possibly through an AMPK-mTOR-Skp2-mediated regulation of autophagy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Menin is a nuclear epigenetic regulator that can both promote and suppress tumor growth in a highly tissue-specific manner. The role of menin in colorectal cancer, however, remains unclear. Here, we demonstrate that menin was overexpressed in colorectal cancer and that inhibition of menin synergized with small-molecule inhibitors of EGFR (iEGFR) to suppress colorectal cancer cells and tumor xenografts in vivo in an EGFR-independent manner. Mechanistically, menin bound the promoter of SKP2, a pro-oncogenic gene crucial for colorectal cancer growth, and promoted its expression. Moreover, the iEGFR gefitinib activated endoplasmic reticulum calcium channel inositol trisphosphate receptor 3 (IP3R3)-mediated release of calcium, which directly bound menin. Combined inhibition of menin and iEGFR-induced calcium release synergistically suppressed menin-mediated expression of SKP2 and growth of colorectal cancer. Together, these findings uncover a molecular convergence of menin and the iEGFR-induced, IP3R3-mediated calcium release on SKP2 transcription and reveal opportunities to enhance iEGFR efficacy to improve treatments for colorectal cancer. SIGNIFICANCE: Menin acts as a calcium-responsive regulator of SKP2 expression, and small molecule EGFR inhibitors, which induce calcium release, synergize with Menin inhibition to reduce SKP2 expression and suppress colorectal cancer., (©2019 American Association for Cancer Research.)

This study was designed to examine the potency of SMIP004, an inhibitor of S-phase kinase-associated protein 2 (Skp2), to exert antidepressant-like properties in mouse models following acute or chronic administration to C57BL6/J mice. To this end, the tail suspension test (TST), forced swim test (FST), and sucrose preference test (SPT) were utilized and the antidepressant-like activity of SMIP004 at different concentrations or time points in mice with or without chronic unpredictable stress (CUS) treatment were evaluated. Results showed that in the time- and dose-dependent experiment the antidepressant-like activity of SMIP004 in naïve mice occurred at day 11 and at the dosage of 2 and 4 mg/kg. SMIP004 (2 mg/kg) also produces antidepressant-like activities in naïve mice after three times in a 24-h administration scheme (24, 5, and 1 h before the test) but not after acute treatment (1 h before the test). Combined SMIP004-fluoxetine administration was found to induce coordinated antidepressant-like effects in naïve mice in the TST and FST. These results seem to be specific because the mice in different experimental groups showed no increased locomotor activity in the open field test. Further, long-term SMIP004 treatment at the dosage of 2 but not 1 mg/kg reversed CUS-induced increase in immobility time in the TST and FST as well as CUS-induced decrease in sucrose preference in the SPT, suggesting that SMIP004 can produce antidepressant-like activities at stressed conditions. These results show that SMIP004 displays obvious antidepressant-like activities in all conducted tests, and suggest that SMIP004 might be a novel antidepressant., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Deregulated expression of the transcriptional coactivator with PDZ-binding motif (WWTR1/TAZ) is a common feature of basal-like breast cancer (BLBC). Yet, how oncogenic TAZ regulates cell-cycle progression and proliferation in breast cancer remains poorly understood, and whether TAZ is required for tumor maintenance has not been established. Here, using an integrative oncogenomic approach, TAZ-dependent cellular programs essential for tumor growth and progression were identified. Significantly, TAZ-driven tumor cells required sustained TAZ expression, given that its withdrawal impaired both genesis and maintenance of solid tumors. Moreover, temporal inhibition of TAZ diminished the metastatic burden in established macroscopic pulmonary metastases. Mechanistic investigation revealed that TAZ controls distinct gene profiles that determine cancer cell fate through cell-cycle networks, including a specific, causal role for S-phase kinase-associated protein 2 (SKP2) in mediating the neoplastic state. Together, this study elucidates the molecular events that underpin the role of TAZ in BLBC and link to SKP2, a convergent communication node for multiple cancer signaling pathways, as a key downstream effector molecule. IMPLICATIONS: Understanding the molecular role of TAZ and its link to SKP2, a signaling convergent point and key regulator in BLBC, represents an important step toward the identification of novel therapeutic targets for TAZ-dependent breast cancer., (©2018 American Association for Cancer Research.)

Multiple myeloma (MM) is a malignant neoplasm of plasma, and exhibits several harmful effects including osteolytic injuries, hypercalcemia, and immune dysfunction. Many patients with MM succumb to the underlying malignancy. An S-phase kinase-related protein 2 (Skp2) inhibitor, designated SKPin C1, has been developed and confirmed to have an inhibitory effect on metastatic melanoma cells. This study aimed to determine the effect of SKPin C1 on MM. Normal B lymphocytes, THP-1 cells, and MM U266 and RPMI 8226 cells were exposed to various dosages of SKPin C1 for 48 h. Cell proliferation was determined by MTT, EdU staining, and cell cycle assays. Western blot assays were performed to assess intracellular protein levels of Skp2, p27, and cleaved caspase-3. The amount of ubiquitin attached to p27 was determined using an immunoprecipitation assay. The viability of U266 and RPMI 8226 cells was significantly inhibited by 10 μM SKPin C1 and the inhibitory effect was enhanced with increasing doses of SKPin C1. In contrast, 50 μM SKPin C1 only marginally decreased viability of normal B lymphocytes in 12 h. Skp2 and p27 expression in U266 and RPMI 8226 cells was higher and lower, respectively, than that in the normal B lymphocytes. Treatment with SKPin C1 or Skp2 knockdown increased p27 protein levels in U266 and RPMI 8226 cells by preventing p27 from being ubiquitinated, which slowed the cell cycle, inhibited cell proliferation, and triggered apoptosis. Therefore, this study suggested SKPin C1 as a potent inhibitor against aberrant proliferation and immortalization of MM.

Prostate cancer (PC) is the most common disease in men over age 50, and its prevalence rate has been gradually increasing since 1980. Taxane-derived anticancer agents are the primary agents used to treat metastatic prostate cancer patients; however, the side effects and acquired drug resistance limit the success of these therapies. Because there is no specific treatment for paclitaxel-resistant prostate cancer, it is necessary to develop new targets and therapeutic strategies to overcome the acquired resistance. In this study, the antitumor activity of a novel selenonucleoside (4'-selenofuranosyl-2,6-dichloropurine, LJ-2618), a third-generation nucleoside, and its plausible mechanisms of action in paclitaxel-resistant prostate cancer (PC-3-Pa) cells were investigated. The established PC-3-Pa cells exhibited over 100-fold resistance against paclitaxel compared to the paclitaxel-sensitive PC-3 cells. LJ-2618, however, effectively inhibited the proliferation of both cell lines with similar IC 50 values in vitro. In PC-3-Pa cells, the activated PI3K/Akt signaling pathway was suppressed by LJ-2618 treatment. In addition, Skp2 was found to be over-expressed in paclitaxel-resistant cells, and the transfection of Skp2 siRNA recovered the sensitivity of paclitaxel in PC-3-Pa cells. Furthermore, LJ-2618 significantly down-regulated Skp2 expression in PC-3-Pa cells by promoting degradation and inducing destabilization of Skp2, which triggers G 2 /M cell cycle arrest. In a xenograft mouse model implanted with PC-3-Pa cells, LJ-2618 (3 or 10 mg/kg) effectively inhibited tumor growth with the enhancement of Skp2 degradation and induction of p27 expression in tumor tissues. These findings suggest that LJ-2618 may have potential for overcoming paclitaxel resistance via promoting Skp2 degradation and stabilizing p27 expression in PC-3-Pa cells. Therefore, the novel selenonucleoside LJ-2618 may lead to the development of a new treatment strategy for patients with paclitaxel-resistant, castration-resistant prostate cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mitophagy is important for cancer development. Notably, the role of Parkin‑mediated mitophagy in colorectal cancer (CRC) mortality has not been fully determined. Therefore, the present study aimed to investigate the effect of Parkin‑mediated mitophagy on CRC apoptosis. In addition, the present study investigated the therapeutic effects of Tanshinone IIA (Tan IIA) on the regulation of CRC cell death via mitophagy. Cellular apoptosis was measured following Tan IIA treatment. In addition, mitophagy activity was evaluated by immunofluorescence and western blotting. The results of the present study revealed that Tan IIA may enhance CRC cell death. In addition, the results demonstrated that Tan IIA enhanced mitochondrial apoptosis, as demonstrated by reduced mitochondrial membrane potential, elevated mitochondrial permeability transition pore opening, and increased oxidative stress, mitochondrial energy disorder and proapoptotic factor expression. Furthermore, the results of the present study demonstrated that Tan IIA induced mitochondrial apoptosis via inhibition of mitophagy. In addition, it was revealed that mitophagy could suppress mitochondrial apoptosis. Functional assays revealed that Tan IIA suppressed the adenosine monophosphate‑activated protein kinase (AMPK) pathway, resulting in the inactivation of S‑phase kinase associated protein 2 (Skp2). Furthermore, reduced levels of Skp2 failed to activate Parkin, thus resulting in inhibition of mitophagy. Conversely, reactivation of AMPK and overexpression of Skp2 rescued mitophagy activity and thus attenuated the Tan IIA‑induced apoptosis of CRC cells. In conclusion, the results of the present study demonstrated the beneficial role of mitophagy in CRC cell survival and suggested that Tan IIA may be an effective therapeutic agent, which suppresses mitophagy activity and enhances CRC apoptosis.

Cell cycle progression in mammals is modulated by two ubiquitin ligase complexes, CRL4 and SCF, which facilitate degradation of chromatin substrates involved in the regulation of DNA replication. One member of the CRL4 complex, the WD-40 containing protein RepID (DCAF14/PHIP), selectively binds and activates a group of replication origins. Here we show that RepID recruits the CRL4 complex to chromatin prior to DNA synthesis, thus playing a crucial architectural role in the proper licensing of chromosomes for replication. In the absence of RepID, cells rely on the alternative ubiquitin ligase, SKP2-containing SCF, to progress through the cell cycle. RepID depletion markedly increases cellular sensitivity to SKP2 inhibitors, which triggered massive genome re-replication. Both RepID and SKP2 interact with distinct, non-overlapping groups of replication origins, suggesting that selective interactions of replication origins with specific CRL components execute the DNA replication program and maintain genomic stability by preventing re-initiation of DNA replication.

Paeoniflorin (PF), a natural compound isolated from Paeoniae radix, has been shown to exert antitumor effects in various types of human cancers including glioma. However, the mechanism of action is not well understood. S-phase kinase-associated protein (Skp)2 functions as an oncogene in many cancers. In the present study, we investigated whether Skp2 mediates the anti-glioma activity of PF. We found that PF inhibited glioma cell proliferation, migration and invasion, and induced G2/M arrest and apoptosis. Skp2 expression was downregulated in glioma cells treated with PF. PF-induced antitumor effects in glioma cells were abolished by Skp2 overexpression but were enhanced by RNA interference of Skp2. Moreover, PF treatment inhibited U87 cell-derived tumor growth in a xenograft mouse model. These results demonstrate that PF exerts its antitumor effects in part by inhibiting Skp2 expression in glioma cells and could be a promising therapeutic agent for glioma therapy.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and no curative therapies. SCF-Skp2 E3 ligase is a target for cancer therapy, but there have been no reports about Skp2 as a target for IPF. Here we demonstrate that Skp2 is a promising therapeutic target for IPF. We examined whether disrupting Skp2 suppressed pulmonary fibrosis in a bleomycin (BLM)-induced mouse model and found that pulmonary fibrosis was significantly suppressed in Skp2 -deficient mice compared with controls. The pulmonary accumulation of fibrotic markers such as collagen type 1 and fibronectin in BLM-infused mice was decreased in Skp2 -deficient mice. Moreover, the number of bronchoalveolar lavage fluid cells accompanied with pulmonary fibrosis was significantly diminished. Levels of the Skp2 target p27 were significantly decreased by BLM-administration in wild-type mice, but recovered in Skp2 -/- mice. In vimentin-positive mesenchymal fibroblasts, the decrease of p27-positive cells and increase of Ki67-positive cells by BLM-administration was suppressed by Skp2 -deficency. As these results suggested that inhibiting Skp2 might be effective for BLM-induced pulmonary fibrosis, we next performed a treatment experiment using the Skp2 inhibitor SZL-P1-41. As expected, BLM-induced pulmonary fibrosis was significantly inhibited by SZL-P1-41. Moreover, p27 levels were increased by the SZL-P1-41 treatment, suggesting p27 may be an important Skp2 target for BLM-induced pulmonary fibrosis. Our study suggests that Skp2 is a potential molecular target for human pulmonary fibrosis including IPF., Competing Interests: The authors declare no conflicts of interest.

We have previously shown that the anti-malarial compound Quinacrine (QC) inhibits ovarian cancer (OC) growth by modulating autophagy. In the present study we extended these studies to identify the molecular pathways regulated by QC to promote apoptosis independent of p53 status in OC. QC exhibited strong anti-cancer properties in OC cell lines in contrast to other anti-malarial autophagy inhibiting drugs. QC treatment selectively upregulated cell cycle inhibitor p21, and downregulated F box protein Skp2 and p62/SQSTM1 expression independent of p53 status. Genetic downregulation of key autophagy protein ATG5 abolished QC-mediated effects on both cell cycle protein p21/Skp2 as well as autophagic cargo protein p62. Furthermore, genetic silencing of p62/SQSTM1 resulted in increased sensitivity to QC-mediated apoptosis, downregulated Skp2 mRNA and increased accumulation of p21 expression. Likewise, genetic knockdown of Skp2 resulted in the upregulation of p21 and p27 and increased sensitivity of OC cells to QC treatment. In contrast, transient overexpression of exogenous p62-HA plasmid rescued the QC-mediated Skp2 downregulation indicating the positive regulation of Skp2 by p62. Collectively, these data indicate that QC-mediated effects on cell cycle proteins p21/Skp2is autophagy-dependent and p53-independent in high grade serious OC cells.

The aim of the present study was to investigate the anti-cancer effects of the natural plant flavonoid, taxifolin, on human osteosarcoma cancer cells. Taxifolin was demonstrated to exhibit anti‑cancer effects on U2OS and Saos‑2 osteosarcoma cell lines. Treatment of cells with taxifolin inhibited proliferation and diminished colony formation in soft agar in a dose‑dependent manner. In vivo, intraperitoneal administration of taxifolin in nude mice bearing U2OS xenograft tumors, significantly inhibited tumor growth. In addition, taxifolin treatment was demonstrated to promote G1 cell cycle arrest and cell apoptosis in U2OS and Saos‑2 cell lines, as demonstrated by flow cytometry analysis. Western blot analysis demonstrated that taxifolin treatment was associated with a reduction in the expression levels of AKT serine/threonine kinase 1 (AKT), phosphorylated (p‑Ser473) AKT, v‑myc avian myelocytomatosis viral oncogene homolog (c‑myc) and S‑phase kinase associated protein 2 (SKP‑2) in U2OS and Saos‑2 cell lines. Overexpression of AKT considerably reversed the taxifolin‑induced decrease in AKT, c‑myc and SKP‑2 protein expression and the decrease in AKT phosphorylation, suggesting that inactivation of AKT was a mediator of taxifolin‑induced inhibition of c‑myc and SKP‑2. Furthermore, overexpression of SKP‑2 in U2OS cells partially reversed the growth inhibition mediated by taxifolin. Finally, taxifolin treatment repressed cell migration and invasion in U2OS cells and this effect was markedly reversed by SKP‑2 overexpression. The results of the present study indicate that taxifolin may present a potential novel therapeutic agent for osteosarcoma treatment.

S-phase kinase-associated protein 2 (SKP2), a potent oncogene was revealed to be upregulated in gastric cancer (GC) tissue samples, in which SKP2 was inversely correlated with microRNA (miR)‑508‑5p transcripts. In present study, the functional effect of miR‑508‑5p on SKP2 and its metastatic potential were investigated in SGC‑7901 GC cells. Significant downregulation of the miR‑508‑5p transcript was associated with the progression of GC. Furthermore, the overexpression of miR‑508‑5p was demonstrated to inhibit the proliferation, migration and invasion of SGC‑7901 cells, as well as induced cell apoptosis and cell cycle arrest at the G0/G1 phase in vitro. The overexpression of miR‑508‑5p was able to downregulate the expression of the SKP2 oncogene, through a mechanism by which miR‑508‑5p directly targeted the SKP2 gene. Thus, regulating transcriptional and post‑transcriptional SKP2 expression, as demonstrated using luciferase reporter assays, reverse transcription‑quantitative polymerase chain reaction analysis and immunoblotting assays. The results of the present study identified that miR‑508‑5p functionally affects the SKP2 gene and reduces metastatic potential in GC, suggesting a novel role of miR‑508‑5p in the regulation of SKP2 and cell cycle.

Osteosarcoma (OS) is a common bone tumor that mainly affects children and young adults. S-phase kinase‑associated protein 2 (Skp2) has been characterized to play a critical oncogenic role in a variety of human malignancies. However, the biological function of Skp2 in OS remains largely obscure. In the present study, we elucidated the role of Skp2 in cell growth, cell cycle, apoptosis and migration in OS cells. We found that depletion of Skp2 inhibited cell growth in both MG-63 and SW 1353 cells. Moreover, we observed that depletion of Skp2 triggered cell apoptosis in two OS cell lines. Furthermore, downregulation of Skp2 induced cell cycle arrest in the G0/G1 phase in OS cells. Notably, our wound healing assay results revealed that inhibition of Skp2 suppressed cell migration in OS cells. Invariably, our western blot results demonstrated that depletion of Skp2 in OS cells inhibited activation of pAkt and increased p27 expression in OS cells, suggesting that Skp2 exerted its oncogenic function partly through the regulation of Akt and p27. Our findings revealed that targeting Skp2 could be a promising therapeutic strategy for the treatment of OS.

Nitidine chloride (NC) has been reported to exert its anti-tumor activity in various types of human cancers. However, the molecular mechanism of NC-mediated tumor suppressive function is largely unclear. In the current study, we used several approaches such as MTT, FACS, RT-PCR, Western blotting analysis, invasion assay, transfection, to explore the molecular basis of NC-triggered anti-cancer activity. We found that NC inhibited cell growth, induced cell apoptosis, caused cell cycle arrest in ovarian cancer cells. Emerging evidence has demonstrated that Skp2 plays an important oncogenic role in ovarian cancer. Therefore, we also explored whether NC exerts its biologic function via downregulation of Skp2 in ovarian cancer cells. We observed that NC significantly inhibited the expression of Skp2 in ovarian cancer cells. Notably, overexpression of Skp2 abrogated the anti-cancer activity induced by NC in ovarian cancer cells. Consistently, downregulation of Skp2 expression enhanced the sensitivity of ovarian cancer cells to NC treatment. Thus, inactivation of Skp2 by NC could be a novel strategy for the treatment of human ovarian cancer.

Cyclin-dependent kinases (cdks) are central catalytic units of cell division cycle. Among the cdk family members, cdk1 has critical roles in multiple phases of the cell cycle. Aberrant expression or hyper-actions of cdk1 are tumorigenic and yet the complex oncogenic network that regulates its turnover is poorly understood. We found a hitherto unexplored functional connection between skp2 and cdk1 turn over. In vitro knockdown or overexpression of skp2 in cultured cells reduced or induced cdk1 expression indicating skp2 as a positive driver for cdk1. A partial inhibitory role for p27 was identified in this context. Interestingly, concurrent overexpression of skp2 and p27 favored cdk1 upregulation in vitro, which correlated well with similar observations in clinical tumor samples. We found that the transcription factor FOXM1 may play a central role in the skp2-cdk1 loop. Additional molecular involvement in the skp2-cdk1 loop was also explored. In conclusion, our results revealed hitherto unexplored p27 independent molecular mechanisms for skp2 driven tumor progression. Our results support the previous findings that skp2 may be a potential therapeutic target for the management of tumors. J. Cell. Biochem. 118: 797-807, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Purpose: Prolactinoma (prolactin-secreting pituitary adenoma) is one of the most common estrogen-related functional pituitary tumors. As an agonist of the dopamine D2 receptor, bromocriptine is used widely to inhibit prolactinoma progression. On the other hand, it is not always effective in clinical application. Although a dopamine D2 receptor deficiency contributes to the impaired efficiency of bromocriptine therapy to some extent, it is unknown whether there some other underlying mechanisms leading to bromocriptine resistance in prolactinoma treatment. That is the main point addressed in this project., Materials and Methods: Human prolactinoma samples were used to analyze the S-phase kinase associated protein 2 (SKP2) expression level. Nutlin-3/adriamycin/cisplatin-treated GH3 and MMQ cells were used to analyze apoptosis in SKP2 overexpression or knockdown cells. SKP2 expression and the interaction partners of SKP2 were also detected after a bromocriptine treatment in 293T. Apoptosis was analyzed in C25 and bromocriptine-treated GH3 cells., Results: Compared to normal pituitary samples, most prolactinoma samples exhibit higher levels of SKP2 expression, which could inhibit apoptosis in a p53-dependent manner. In addition, the bromocriptine treatment prolonged the half-life of SKP2 and resulted in SKP2 overexpression to a greater extent, which in turn compromised its pro-apoptotic effect. As a result, the bromocriptine treatment combined with C25 (a SKP2 inhibitor) led to the maximal apoptosis of human prolactinoma cells., Conclusion: These findings indicated that SKP2 inhibition sensitized the prolactinoma cells to bromocriptine and helped promote apoptosis. Moreover, a combined treatment of bromocriptine and C25 may contribute to the maximal apoptosis of human prolactinoma cells.

While clinical benefit of the proteasome inhibitor (PI) bortezomib (BTZ) for multiple myeloma (MM) patients remains unchallenged, dose-limiting toxicities and drug resistance limit the long-term utility. The E3 ubiquitin ligase Skp1-Cullin-1-Skp2 (SCF Skp2 ) promotes proteasomal degradation of the cell cycle inhibitor p27 to enhance tumor growth. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance. SCF Skp2 activity is increased by the Cullin-1-binding protein Commd1 and the Skp2-binding protein Cks1B. Here we observed higher CUL1, COMMD1 and SKP2 mRNA levels in CD138 + cells isolated from BTZ-resistant MM patients. Higher CUL1, COMMD1, SKP2 and CKS1B mRNA levels in patient CD138 + cells correlated with decreased progression-free and overall survival. Genetic knockdown of CUL1, COMMD1 or SKP2 disrupted the SCF Skp2 complex, stabilized p27 and increased the number of annexin-V-positive cells after BTZ treatment. Chemical library screens identified a novel compound, designated DT204, that reduced Skp2 binding to Cullin-1 and Commd1, and synergistically enhanced BTZ-induced apoptosis. DT204 co-treatment with BTZ overcame drug resistance and reduced the in vivo growth of myeloma tumors in murine models with survival benefit. Taken together, the results provide proof of concept for rationally designed drug combinations that incorporate SCF Skp2 inhibitors to treat BTZ resistant disease.

The S phase kinase-associated protein 1 (Skp1), an adaptor protein of the Skp1-Cul1-F-box protein complex, binds the ubiquitin E3 ligase Skp2 and is critical to its biological functions. Targeting of Skp1 by a small compound 6-O-angeloylplenolin (6-OAP) results in dissociation and degradation of Skp2 and mitotic arrest of lung cancer cells. Here, by using a proteome microarray containing 16,368 proteins and a biotinylated 6-OAP, we identified 99 proteins that could bind 6-OAP, with Skp1 and STAT3 sitting at the central position of the 6-OAP interactome. 6-OAP formed hydrogen bonds with Ser611/Ser613/Arg609 at the SH2 domain of STAT3 and inhibited the constitutive and interleukin-6-induced phosphorylated STAT3 (pSTAT3), leading to inhibitory effects on lung cancer cells and suppression of Skp2 transcription. STAT3 was overexpressed in tumor samples compared to counterpart normal lung tissues and was inversely associated with prognosis of the patients. 6-OAP inhibited tumor growth in SCID mice intravenously injected with lung cancer cells, and downregulated both STAT3 and Skp2 in tumor samples. Given that 6-OAP is a Skp1 inhibitor, our data suggest that this compound may target Skp1 and STAT3 to suppress Skp2, augmenting its anti-lung cancer activity.

Gastric cancer is a malignant disease of the digestive system with high rates of incidence and mortality. S‑phase kinase‑associated protein 2 (Skp2) is a novel oncogene, which has been identified to be important in tumor progression and metastasis. In order to clarify the role of Skp2 in human gastric cancer, the present study detected the expression of Skp2 in human gastric cancer tissues, and investigated the molecular mechanism of Skp2 in the progression of gastric carcinoma. The results of the initial bioinformatics analysis showed that Skp2 was significantly upregulated in 31 specimens of primary gastric cancer from a UK patient cohort, and in 10 gastric cancer lines of a side population, compared with normal gastric tissues (P<0.01). Specimens from 47 patients with gastric cancer and 19 normal gastric tissue specimens were obtained and analyzed using western blot analysis. The positive rate of expression of Skp2 was 87.2%, indicating that the expression of Skp2 was observed in 41 specimens of the detected gastric cancer samples, whereas the positive rate of the expression of Skp2 was 5.6% in the normal gastric samples (P<0.01). In the human gastric cancer cell lines, the defective regulation of Skp2 or presence of an Skp2 inhibitor inhibited the proliferation of BGC‑823 and MKN‑45 cells. In addition, the Skp2 inhibitor suppressed the proliferation of gastric cancer cells in a time‑ and dose‑dependent manner. Furthermore, transfection with Skp2 short hairpin (sh)RNA or treatment with SKP inhibitor C1 for 48 and 72 h led to the accumulation of p27kip1 in Hela cells. Tumorigenicity experiments involving nude mice showed that interference of the expression of Skp2 inhibited the growth of the human gastric tumor cells in the nude mice, and the tumor weights and volumes in the Skp2 shRNA group were significantly lower, compared with those in the negative control shRNA group (P<0.01) and untreated group (P<0.01). Taken together, these data suggested that Skp2 acted as an oncogene in human gastric cancer, and that Skp2‑mediated p27kip1 degradation contributed to the progression of gastric cancer. Abrogating the effects of Skp2 may effectively inhibit the growth of gastric cancer cells, which may be useful as a novel target in the clinical treatment of gastric cancer.

Prostate cancer is the most commonly diagnosed tumor in men in the United States. Patients with hormone-refractory prostate cancer are often treated with paclitaxel, but most of them eventually develop drug resistance. S-phase kinase associated protein 2 (Skp2) is a component of the SCF (Skp1-Cullin1-F-box) type of E3 ubiquitin ligase complexes. In the present study, we investigated the role of Skp2 in paclitaxel-resistant DU145-TxR or PC-3-TxR cells by Skp2 silencing or using Skp2 inhibitors. We first confirmed that Skp2 expression is up-regulated in DU145-TxR or PC-3-TxR cells compared with their parental cells DU145 or PC-3, respectively. Knockdown of Skp2 or Skp2 inhibitor treatment in DU145-TxR or PC-3-TxR cells restored paclitaxel sensitivity. E-cadherin was decreased while Vimentin was increased in PC-3-TxR or DU145-TxR cells. In addition, p27 expression was inversely correlated with Skp2 expression in DU145-TxR or PC-3-TxR cells. Moreover, p27 was found to increase in both Skp2 silencing PC-3-TxR and DU145-TxR cells. These results suggest that Skp2 is associated with prostate cancer cell resistance to paclitaxel. Skp2 may be a potential therapeutic target for drug-resistant prostate cancer.

Autophagy is a highly conserved self-digestion process, which is essential for maintaining homeostasis and viability in response to nutrient starvation. Although the components of autophagy in the cytoplasm have been well studied, the molecular basis for the transcriptional and epigenetic regulation of autophagy is poorly understood. Here we identify co-activator-associated arginine methyltransferase 1 (CARM1) as a crucial component of autophagy in mammals. Notably, CARM1 stability is regulated by the SKP2-containing SCF (SKP1-cullin1-F-box protein) E3 ubiquitin ligase in the nucleus, but not in the cytoplasm, under nutrient-rich conditions. Furthermore, we show that nutrient starvation results in AMP-activated protein kinase (AMPK)-dependent phosphorylation of FOXO3a in the nucleus, which in turn transcriptionally represses SKP2. This repression leads to increased levels of CARM1 protein and subsequent increases in histone H3 Arg17 dimethylation. Genome-wide analyses reveal that CARM1 exerts transcriptional co-activator function on autophagy-related and lysosomal genes through transcription factor EB (TFEB). Our findings demonstrate that CARM1-dependent histone arginine methylation is a crucial nuclear event in autophagy, and identify a new signalling axis of AMPK-SKP2-CARM1 in the regulation of autophagy induction after nutrient starvation.

Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Skp2 is thought to have two critical roles in tumorigenesis. As part of the SCF(Skp2) ubiquitin ligase, Skp2 drives the cell cycle by mediating the degradation of cell cycle proteins. Besides the proteolytic activity, Skp2 also blocks p53-mediated apoptosis by outcompeting p53 for binding p300. Herein, we exploit the Skp2/p300 interaction as a new target for Skp2 inhibition. An affinity-based high-throughput screen of a combinatorial cyclic peptoid library identified an inhibitor that binds to Skp2 and interferes with the Skp2/p300 interaction. We show that antagonism of the Skp2/p300 interaction by the inhibitor leads to p300-mediated p53 acetylation, resulting in p53-mediated apoptosis in cancer cells, without affecting Skp2 proteolytic activity. Our results suggest that inhibition of the Skp2/p300 interaction has a great potential as a new anticancer strategy, and our Skp2 inhibitor can be developed as a chemical probe to delineate Skp2 non-proteolytic function in tumorigenesis., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Skp2 is frequently overexpressed in many human cancers and plays a key role in tumorigenesis. As a component of the SCFSkp2ubiquitin E3 ligase complex, Skp2 is responsible for recruiting substrate proteins for their ubiquitination and subsequent degradation by the 26S proteasome. Thus, Skp2 promotes the cell cycle by down-regulating cell cycle proteins such as the tumor suppressor p27. Alternatively, Skp2 suppresses p53-dependent apoptosis by outcompeting p53 for binding to p300, thereby perturbing p300-mediated p53 acetylation and stabilization. Taken together, inhibition of Skp2 functions (either proteolytic function or non-proteolytic function) is emerging as a promising and novel anti-cancer strategy. In the present review, we highlight the development of Skp2 inhibitors with different mechanisms of action.