Skp1 Research Articles

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.

Forkhead Box Protein M1 Promotes Nasopharyngeal Carcinoma Cell Tumorigenesis Possibly via the Wnt/β-Catenin Signaling Pathway.

BackgroundForkhead box protein M1 (FoxM1) is an important transcription factor involved in the development and progression of various malignancies. However, its role in nasopharyngeal carcinoma (NPC) remains largely unknown. This study aimed to assess the effect of FoxM1 on NPC cell tumorigenesis as well as the underlying mechanism.Material/MethodsNPC cell lines CNE-1 and CNE-2 were treated with vehicle and FoxM1 inhibitor thiostrepton or transfected with small interfering RNA. CCK-8 assay, flow cytometric assay, and Hoechst 33258 staining were performed to assess the viability, apoptosis and nuclear morphological impairment, and cell cycle, respectively. The expression of apoptosis-related caspase-3 and caspase-9 was detected by western blot analysis The tumor growth in the mouse xenograft model of NPC treated with thiostrepton or control was assessed. The expression of Wnt/β-catenin signaling proteins p27, FoxM1, S phase kinase-associated protein 2 (SKP2), and Cyclin D1 were determined both in cells and xenograft tissues by western blot analysis.ResultsInhibition of FoxM1 by thiostrepton significantly suppressed NPC cell viability, induced apoptosis, increased cell cycle arrest, impaired nuclear morphology, and reduced NPC cell-derived tumor xenograft growth. Mechanistically, inhibition or knockdown of FoxM1 inactivated the Wnt/β-catenin signaling pathway, as demonstrated by altered expression of Wnt/β-catenin signaling-related genes, including p27, SKP2, and cyclin D1, in both NPC cells and xenograft tissues.ConclusionsWe identified FoxM1 as a novel regulator of NPC cell tumorigenesis in vitro and in vivo. Targeting FoxM1 could be a promising therapeutic strategy against NPC.

MicroRNA‐205‐5p targets E2F1 to promote autophagy and inhibit pulmonary fibrosis in silicosis through impairing SKP2‐mediated Beclin1 ubiquitination

Silicosis is an occupational disease characterized by extensive pulmonary fibrosis, and the underlying pathological process remains uncertain. Herein, we explored the molecular mechanism by which microRNA‐205‐5p (miR‐205‐5p) affects the autophagy of alveolar macrophages (AMs) and pulmonary fibrosis in mice with silicosis through the E2F transcription factor 1 (E2F1)/S‐phase kinase‐associated protein 2 (SKP2)/Beclin1 axis. Alveolar macrophages (MH‐S cells) were exposed to crystalline silica (CS) to develop an in vitro model, and mice were treated with CS to establish an in vivo model. Decreased Beclin1 and increased SKP2 and E2F1 were identified in mice with silicosis. We silenced or overexpressed miR‐205‐5p, E2F1, SKP2 and Beclin1 to investigate their potential roles in pulmonary fibrosis in vivo and autophagy in vitro. Recombinant adenovirus mRFP‐GFP‐LC3 was transduced into the MH‐S cells to assay autophagic flow. Knocking down Beclin1 promoted pulmonary fibrosis and suppressed the autophagy. Co‐immunoprecipitation and ubiquitination assays suggested that SKP2 induced K48‐linked ubiquitination of Beclin1. Furthermore, chromatin immunoprecipitation‐PCR revealed the site where E2F1 bound to the SKP2 promoter between 1638 bp and 1645 bp. As shown by dual‐luciferase reporter gene assay, the transfection with miR‐205‐5p mimic inhibited the luciferase activity of the wild‐type E2F1 3′untranslated region, suggesting that miR‐205‐5p targeted E2F1. Additionally, miR‐205‐5p overexpression increased autophagy and reduced the pulmonary fibrosis, while overexpression of E2F1 or SKP2 or inhibition of Beclin1 could annul this effect. The current study elucidated that miR‐205‐5p targeted E2F1, thereby inhibiting SKP2‐mediated Beclin1 ubiquitination to promote macrophage autophagy and inhibit pulmonary fibrosis in mice with silicosis.

USP2 is an SKP2 deubiquitylase that stabilizes both SKP2 and its substrates

The stability of a protein is regulated by a balance between its ubiquitylation and deubiquitylation. S-phase kinase-associated protein 2 (SKP2) is an oncogenic F-box protein that recognizes tumor suppressor substrates for targeted ubiquitylation by the E3 ligase SKP1-Cullin1-F-box and degradation by proteasome. SKP2 is itself ubiquitylated by the E3 ligases APC/CCDH1 and SCFFBXW2, and deubiquitylated by deubiquitylases (DUBs) USP10 and USP13. Given the biological significance of SKP2, it is likely that the other E3s or DUBs may also regulate its stability. Here, we report the identification and characterization of USP2 as a new DUB. We first screened a panel of DUBs and found that both USP2 and USP21 bound to endogenous SKP2, but only USP2 deubiquitylated and stabilized SKP2 protein. USP2 inactivation via siRNA knockdown or small-molecule inhibitor treatment remarkably shortened SKP2 protein half-life by enhancing its ubiquitylation and subsequent degradation. Unexpectedly, USP2-stabilized SKP2 did not destabilize its substrates p21 and p27. Mechanistically, USP2 bound to SKP2 via the leucine-rich repeat substrate-binding domain on SKP2 to disrupt the SKP2-substrate binding, leading to stabilization of both SKP2 and these substrates. Biologically, growth suppression induced by USP2 knockdown or USP2 inhibitor is partially mediated via modulation of SKP2 and its substrates. Our study revealed a new mechanism of the cross-talk among the E3–DUB substrates and its potential implication in targeting the USP2–SKP2 axis for cancer therapy.

The antioncogenic effect of Beclin-1 and FOXP3 is associated with SKP2 expression in gastric adenocarcinoma.

An overexpression of S-phase kinase-associated protein 2 (SKP2) is frequently observed in human cancer progression and metastasis, and evidence suggests that SKP2 plays a proto-oncogenic role both in vitro and in vivo. However, the function of SKP2 in gastric adenocarcinoma remains largely obscure. We investigated SKP2 expression in human gastric carcinomas.Tissue samples were acquired from 182 cases of gastric adenocarcinoma that were surgically resected from 2006 to 2012. Immunohistochemical staining for SKP2, Beclin-1, and forkhead box protein P3 (FOXP3) was performed. Pearson chi-square test was used to evaluate the associations among clinicopathological variables. The Kaplan–Meier method, the log-rank test, and the Cox proportional-hazards model were used in the analysis of the overall survival (OS) and disease-free survival (DFS).As a result, SKP2 overexpression in gastric adenocarcinomas showed a significant correlation with several favorable clinical factors, including the tumor size, T category, N category, lymphatic invasion, vascular invasion, OS, and DFS. SKP2 expression was positively correlated with the tumoral FOXP3, Beclin-1 expression, and regulatory T cell (Treg) infiltration. The difference in DFS between the SKP2 positive and negative group was attenuated by FOXP3 high expression, Beclin-1 high expression, and Tregs infiltration. Attenuation of the difference in OS by FOXP3 high expression, Beclin-1 high expression, and Tregs infiltration was not significant. In multivariable analysis, SKP2 expression was not correlated with OS and DFS.Our study showed a complex interrelationship between SKP2 and Beclin-1 and FOXP3 expression in gastric adenocarcinoma. The antioncogenic effect of Beclin-1 and FOXP3 expression in gastric adenocarcinoma is related to SKP2 expression.

Overexpression of HOXA10 promotes the growth and metastasis of nasopharyngeal carcinoma.

Deregulation of HOX transcription factor family has frequently been observed in multiple human cancers; however, their role in nasopharyngeal carcinoma remains largely unclear. In the present study, we found that HOX gene family is consistently upregulated in nasopharyngeal carcinoma and identified HOXA10 as one of the mostly upregulated HOX genes. Importantly, we show that HOXA10 overexpression is associated with transcriptional activation of multiple oncogenes essential for nasopharyngeal carcinoma carcinogenesis, including S-phase kinase-associated protein 2 (SKP2), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2), and matrix metalloproteinase 1 (MMP1). Mechanistically, the overexpression of SKP2 induces the degradation of cell cycle inhibitor p27, leading to rapid cell cycle progression and cell proliferation. The overexpression of CAMKK2 is associated with enhanced mTOR signaling activity to meet the increased demand for proteins synthesis in rapid growing nasopharyngeal carcinoma cells. Moreover, MMP1 overexpression facilitates nasopharyngeal carcinoma cell migration and invasion and contributes to cancer metastasis and progression. We thus concluded that HOXA10 overexpression promotes the growth and metastasis of nasopharyngeal carcinoma by transcriptionally activating various oncogenic pathways.

Effect of Continuous Lenalidomide Treatment on Copy Number Alteration and Cytogenetic Abnormalities in Multiple Myeloma Cells

In the last years the role of clonal selection and evolution has been underlined in the progression of multiple myeloma (MM). The potential role of anti-myeloma drugs on clonal selection is currently under investigation. Continuous Lenalidomide (LEN) treatment is becoming the backbone of MM therapy either in young patients as maintenance after stem cell transplantation or in newly diagnosed elderly patients, however the potential role of Len in the clonal selection and the emerging of resistant clones are unknown and has been explored in this study.The experimental approach used was firstly to analyze the sub-clonal evolution in human myeloma cell lines (HMCLs) (JJN3, KMS-12 and RPMI-8226) after long term treatment with low dose of LEN (2 µM) or vehicle (DMSO), mimic the continuous treatment regimens of MM patients, collecting sample at starting point (T0), 1 month and 2 months for single nucleotide polymorphism (SNP)+Comparative Genomic Hybridization (CGH) arrays and fluorescence in situ hybridization (FISH) analyses. Secondly to confirm data by FISH analysis on purified CD138+ plasma cells (PCs) from MM patients treated continuously with LEN.In all collected samples both copy number alterations (CNAs) and loss of heterozygosity (LOH) regions have been detected, ranging from few Kb to more than 10 Mb. These alterations were not mainly specific for LEN treatment being also detected in DMSO treated samples. Moreover, FISH analysis of IGH locus translocations and of cMYC amplification/translocations revealed that the mutational pattern reported at T0 did not change after LEN treatment in the analyzed HMCLs. In the combined CGH+SNP arrays few regions with CNAs and LOH seemed to be specific only in LEN treated samples. In particular, in the RPMI-8226, we detected the presence of a LOH in the 5p region (chr5:14547524-36790726) at T0 but, in the same region, we reported a mosaic duplication in the Len treated samples at 1 month (log ratio: 0,45) and 2 months (log ratio: 0,46) as compared to the vehicle. In this chromosome (Chr) 5p region different genes are coded, including S-phase kinase-associated protein 2 (SKP2) an oncogene known to be involved in tumor polyploidy in cancer cells and in Bortezomib resistance of MM cells. Interestingly our recent data obtained in the LEN resistant cells JJN3 indicated that SKP2 was down-regulated overcoming LEN resistance by hypoxia inducible factor (HIF)-1α knock-out.Based on these data, we performed FISH with SKP2 probe on 5p13.2 locus (RP11-RP23 Empire Genomics) and a control probe on 5p11 locus in RPMI-8226 and in JJN3 samples. FISH analysis revealed in the LEN resistant cell line JJN3 at T0 the coexistence of multiple clones with different degrees of chr5 polysomy, presumably reflecting different ploidy levels, which are not modified by LEN treatment, in all the time point considered. This justified CGH+SNP array data reporting an amplification of SKP2 locus (JJN3 T0 logratio: 1,295; JJN3 DMSO logratio:1,205; JJN3 LEN logratio:1,12), related to chr5 polysomy. Moreover FISH revealed that RPMI-8226 at T0 showed a single clone with chr 5 tetrasomy with SKP2 deletion on two 5p13.2 locus, presumably deriving from the diploid clone with monoallelic SKP2 deletion, reflecting LOH as reported by the CGH+SNP array. After the long-term treatment with either LEN or DMSO, RPMI-8226 developed different sub-clones with different degrees of Chr5 polysomy, maintaining SKP2 deletion suggesting that LEN had not a significant impact on their development. To confirm these observations we analyzed CD138+ cells from 4 MM patients (2 newly diagnosed and 2 relapsed) treated for almost 1 year with LEN and dexamethasone that developed LEN resistance. FISH analyses on bone marrow PCs revealed that 3 of 4 patients showed Chr5 trisomy, coupled with trisomy of Chr9 and Chr15, defining them in the hyperdiploid group; this cytogenetic architecture was not altered at the moment of relapse during LEN treatment. In conclusion, our data suggest that long-term continuous LEN treatment did not induce a clonal MM cell selection and that a possible relationship between the amount of the SKP2 locus and the polyploid level may exist in MM cells. Indeed the potential relationship between SKP2 status and LEN resistance deserve further investigation. DisclosuresNo relevant conflicts of interest to declare.

ERR\u03b3 enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes

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.

MiR-101-3p Contributes to α-Synuclein Aggregation in Neural Cells through the miR-101-3p/SKP1/PLK2 Pathway.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive neuronal loss in different brain regions, including the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc). The aggregation of α-synuclein (α-Syn) plays an essential role in the progression of PD-related neuron toxicity. In this study, bioinformatic analysis was used to confirm differentially expressed genes between patients with PD and healthy donors. Immunofluorescence was used to study the aggregation of α-Syn. Flow cytometry was used to confirm the apoptosis of neurons. Western blot was used to investigate the underlying mechanism. Coimmunoprecipitation (co-IP) was used to verify the interaction between proteins. Luciferase activity assay was used to confirm the target gene of miRNA. In vitro protein ubiquitination assay was used to ascertain the role of S-phase kinase-associated protein 1 (SKP1) on the ubiquitination processes of polo-like kinase 2 (PLK2). The result indicated that miR-101-3p was overexpressed in the substantia nigra of the postmortem brains of patients with PD. The underlying role was investigated in the SH-SY5Y cell line. The overexpression of α-Syn did not result in toxicity or aggregation. However, the co-overexpression of miR-101-3p and α-Syn promoted aggregation and neuron toxicity. Luciferase activity assay indicated that SKP1 is a target gene of miR-101-3p. The co-IP experiment confirmed that SKP1 could directly interact with PLK2. In vitro protein ubiquitination assay confirmed that SKP1 could promote the ubiquitination and subsequent protein degradation of PLK2. We also observed that the cotransfection of short hairpin RNA that targets PLK2 and α-Syn overexpression plasmid results in the endoplasmic reticulum stress of neurons. Our results collectively provide evidence that miR-101-3p contributes to α-Syn aggregation in neurons through the miR-101-3p/SKP1/PLK2 pathway.

Downregulation of decidual SKP2 is associated with human recurrent miscarriage

BackgroundRecurrent miscarriage (RM) is a very frustrating problem for both couples and clinicians. To date, the etiology of RM remains poorly understood. Decidualization plays a critical role in implantation and the maintenance of pregnancy, and its deficiency is closely correlated with RM. The F-box protein S-phase kinase associated protein 2 (SKP2) is a key component of the SCF-type E3 ubiquitin ligase complex, which is critically involved in ErbB family-induced Akt ubiquitination, aerobic glycolysis and tumorigenesis. SKP2 is pivotal for reproduction, and SKP2-deficient mice show impaired ovarian development and reduced fertility.MethodsHere, we investigated the expression and function of SKP2 in human decidualization and its relation with RM. A total of 40 decidual samples were collected. Quantitative PCR analysis, western blot analysis and immunohistochemistry analysis were performed to analyze the differential expression of SKP2 between RM and control cells. For in vitro induction of decidualization, both HESCs (human endometrial stromal cells) cell line and primary ESCs (endometrial stromal cells) were used to analyze the effects of SKP2 on decidualization via siRNA transfection.ResultsCompared to normal pregnant women, the expression of SKP2 was reduced in the decidual tissues from individuals with RM. After in vitro induction of decidualization, knockdown of SKP2 apparently attenuated the decidualization of HESCs and resulted in the downregulation of HOXA10 and FOXM1, which are essential for normal human decidualization. Moreover, our experiments demonstrated that SKP2 silencing reduced the expression of its downstream target GLUT1.ConclusionsOur study indicates a functional role of SKP2 in RM: downregulation of SKP2 in RM leads to impaired decidualization and downregulation of GLUT1 and consequently predisposes individuals to RM.

Small molecule-mediated modulation of ubiquitination and neddylation improves HSC function ex vivo.

Hematopoietic stem cells (HSCs) are particularly characterized by their quiescence and self-renewal. Cell cycle regulators tightly control quiescence and self-renewal capacity. Studies suggest that modulation of ubiquitination and neddylation could contribute to HSC function via cyclin-dependent kinase inhibitors (CDKIs). S-phase kinase-associated protein 2 (SKP2) is responsible for ubiquitin-mediated proteolysis of CDKIs. Here, we modulated overall neddylation and SKP2-associated ubiquitination in HSCs by using SKP2-C25, an SKP2 inhibitor, and MLN4924 (Pevonedistat) as an inhibitor of the NEDD8 system. Treatments of SKP2-C25 and MLN4924 increased both murine and human stem and progenitor cell (HSPC) compartments. This is associated with the improved quiescence of murine HSC by upregulation of p27 and p57 CDKIs. A colony-forming unit assay showed an enhanced in vitro self-renewal potential post inhibition of ubiquitination and neddylation. In addition, MLN4924 triggered the mobilization of bone marrow HSPCs to peripheral blood. Intriguingly, MLN4924 treatment could decrease the proliferation of murine bone marrow mesenchymal stem cells or endothelial cells. These findings shed light on the contribution of SKP2, and associated ubiquitination and neddylation in HSC maintenance, self-renewal, and expansion.

A Machine Learning Approach to Prioritizing Functionally Active F-box Members in Arabidopsis thaliana.

Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating, and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex. The S-phase Kinase-associated Protein 1 (Skp1), CUL1, F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants due to the dramatic number expansion of the FBX genes in plant genomes. Since it is the FBX proteins that recognize and determine the specificity of SCF substrates, much effort has been done to characterize their genomic, physiological, and biochemical roles in the past two decades of functional genomic studies. However, the sheer size and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this work, we first identified 82 known FBX members that have been functionally characterized up to date in Arabidopsis thaliana. Through comparing the genomic structure, evolutionary selection, expression patterns, domain compositions, and functional activities between known and unknown FBX gene members, we developed a neural network machine learning approach to predict whether an unknown FBX member is likely functionally active in Arabidopsis, thereby facilitating its future functional characterization.

SKP2-Promoted Ubiquitination of FOXO3 Promotes the Development of Asthma.

Asthma is a respiratory disease with a dramatically increasing incidence globally. The present study explored the roles of S-phase kinase-associated protein 2 (SKP2) and forkhead box O3 (FOXO3) in asthma and their involvement in the Krüppel-like factor 15-lipoprotein receptor-related protein 5 (KLF15-LRP5) axis. SKP2 expression in patients with asthma and OVA-induced asthmatic Sprague Dawley rats was detected by reverse transcription quantitative PCR and Western blot assays. Alterations in SKP2 and LRP5 expression were evaluated in OVA-induced asthmatic rats, followed by measurement of inflammatory cytokines using ELISA and airway resistance using a methacholine challenge test. We applied TGF-β1 to establish the airway smooth muscle cell (ASMC) proliferation model of asthma. The FOXO3 ubiquitination and changes in cell biological behaviors were detected using immunoprecipitation, MTT, and Annexin V/propidium iodide assays. Flow cytometry was adopted to detect cell cycle, and ELISA was used to measure the concentrations of IL-4, IL-5, IL-13, and IgE in rat bronchoalveolar lavage fluid. SKP2 was highly expressed and FOXO3 was poorly expressed in patients with asthma and in OVA-induced asthmatic rats. SKP2 silencing decreased IL-4, IL-5, IL-13, and IgE expression in rat bronchoalveolar lavage fluid, whereas SKP2 enhanced FOXO3 ubiquitination to upregulate KLF15, which bound to the LRP5 promoter in TGF-β1-induced ASMCs and increased LRP5 expression. SKP2 enhanced airway hyperresponsiveness and inflammation in the OVA-induced rat model and augmented TGF-β1-induced ASMC proliferation by inhibiting the FOXO3/KLF15/LRP5 axis. Additionally, overexpressed SKP2 resulted in reduced numbers of ASMCs in the G1 phase but increased numbers in the G2/M phase. Collectively, we show that SKP2 promotes FOXO3 ubiquitination to suppress the KLF15-LRP5 axis, thereby exacerbating asthma.

Emerging Roles of SKP2 in Cancer Drug Resistance.

More than half of all cancer patients receive chemotherapy, however, some of them easily acquire drug resistance. Resistance to chemotherapy has become a massive obstacle to achieve high rates of pathological complete response during cancer therapy. S-phase kinase-associated protein 2 (Skp2), as an E3 ligase, was found to be highly correlated with drug resistance and poor prognosis. In this review, we summarize the mechanisms that Skp2 confers to drug resistance, including the Akt-Skp2 feedback loop, Skp2-p27 pathway, cell cycle and mitosis regulation, EMT (epithelial-mesenchymal transition) property, enhanced DNA damage response and repair, etc. We also addressed novel molecules that either inhibit Skp2 expression or target Skp2-centered interactions, which might have vast potential for application in clinics and benefit cancer patients in the future.

LxCxD motif of the APC/C coactivator subunit FZR1 is critical for interaction with the retinoblastoma protein

Retinoblastoma protein (pRB) regulates cell cycle by utilizing different regions of its pocket domain for interacting with E2F family of transcription factors and with cellular and viral proteins containing an LxCxE motif. An LxCxE-like motif, LxCxD, is present in FZR1, an adaptor protein of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C). The APC/CFZR1 complex regulates the timely degradation of multiple cell cycle proteins for mitotic exit and maintains G1 state. We report that FZR1 interacts with pRB via its LxCxD motif. By using point mutations, we found that the cysteine residue in the FZR1 LxCxD motif is critical for direct interaction with pRb. The direct binding of the LxCxD motif of FZR1 to the pRB LxCxE binding pocket is confirmed by using human papillomavirus protein E7 as a competitor, both in vitro and in vivo. While mutation of the cysteine residue significantly disrupts FZR1 interaction with pRB, this motif does not affect FZR1 and core APC/C association. Expression of the FZR1 point mutant results in accumulation of S-phase kinase-associated protein 2 (SKP2) and Polo-like kinase 1 (PLK1), while p27Kip1 and p21Cip1 proteins are downregulated, indicating a G1 cell cycle defect. Consistently, cells containing point mutant FZR1 enter the S phase prematurely. Together our results suggest that the LxCxD motif of FZR1 is a critical determinant for the interaction between FZR1 and pRB and is important for G1 restriction.

FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

Vascular smooth muscle cells (VSMCs) normally exhibit a very low proliferative rate. Vessel injury triggers VSMC proliferation, in part, through focal adhesion kinase (FAK) activation, which increases transcription of cyclin D1, a key activator for cell cycle-dependent kinases (CDKs). At the same time, we also observe that FAK regulates the expression of the CDK inhibitors (CDKIs) p27 and p21. However, the mechanism of how FAK controls CDKIs in cell cycle progression is not fully understood. We found that pharmacological and genetic FAK inhibition increased p27 and p21 by reducing stability of S-phase kinase-associated protein 2 (Skp2), which targets theCDKIs for degradation. FAK N-terminal domain interacts with Skp2 and an APC/C E3 ligase activator fizzy-related 1 (Fzr1) in the nucleus, which promote ubiquitination and degradation of both Skp2 and Fzr1. Notably, overexpression of cyclin D1 alone failed to promote proliferation of genetic FAK kinase-dead (KD) VSMCs, suggesting that the FAK-Skp2-CDKI signalling axis is distinct from the FAK-cyclin D1 pathway. However, overexpression of both cyclin D1 and Skp2 enabled proliferation of FAK-KD VSMCs, implicating that FAK ought to control both activating and inhibitory switches for CDKs. In vivo, wire injury activated FAK in the cytosol, which increased Skp2 and decreased p27 and p21 levels. Both pharmacological FAK and genetic FAK inhibition reduced Skp2 expression in VSMCs upon injury, which significantly reduced intimal hyperplasia through elevated expression of p27 and p21. This study revealed that nuclear FAK-Skp2-CDKI signalling negatively regulates CDK activity in VSMC proliferation.

The Hippo Pathway Effector YAP Promotes Ferroptosis via the E3 Ligase SKP2.

Ferroptosis is a new form of regulated cell death resulting from the accumulation of lipid-reactive oxygen species. A growing number of studies indicate ferroptosis as an important tumor suppressor mechanism having therapeutic potential in cancers. Previously, we identified TAZ, a Hippo pathway effector, regulates ferroptosis in renal and ovarian cancer cells. Because YAP (Yes-associated protein 1) is the one and only paralog of TAZ, sharing high sequence similarity and functional redundancy with TAZ, we tested the potential roles of YAP in regulating ferroptosis. Here, we provide experimental evidence that YAP removal confers ferroptosis resistance, whereas overexpression of YAP sensitizes cancer cells to ferroptosis. Furthermore, integrative analysis of transcriptome reveals S-phase kinase-associated protein 2 (SKP2), an E3 ubiquitin ligase, as a YAP direct target gene that regulates ferroptosis. We found that the YAP knockdown represses the expression of SKP2. Importantly, the genetic and chemical inhibitions of SKP2 robustly protect cells from ferroptosis. In addition, knockdown of YAP or SKP2 abolishes the lipid peroxidation during erastin-induced ferroptosis. Collectively, our results indicate that YAP, similar to TAZ, is a determinant of ferroptosis through regulating the expression of SKP2. Therefore, our results support the connection between Hippo pathway effectors and ferroptosis with significant therapeutic implications. IMPLICATIONS: This study reveals that YAP promotes ferroptosis by regulating SKP2, suggesting novel therapeutic options for YAP-driven tumors.

MiR-506 exerts antineoplastic effects on osteosarcoma cells via inhibition of the Skp2 oncoprotein.

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.

Identification of the antidepressive properties of C1, a specific inhibitor of Skp2, in mice.

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.

Pharmacological inhibition of noncanonical EED-EZH2 signaling overcomes chemoresistance in prostate cancer.

Rationale: Chemoresistance is a major obstacle in prostate cancer (PCa) treatment. We sought to understand the underlying mechanism of PCa chemoresistance and discover new treatments to overcome docetaxel resistance.Methods: We developed a novel phenotypic screening platform for the discovery of specific inhibitors of chemoresistant PCa cells. The mechanism of action of the lead compound was investigated using computational, molecular and cellular approaches. The in vivo toxicity and efficacy of the lead compound were evaluated in clinically-relevant animal models.Results: We identified LG1980 as a lead compound that demonstrates high selectivity and potency against chemoresistant PCa cells. Mechanistically, LG1980 binds embryonic ectoderm development (EED), disrupts the interaction between EED and enhancer of zeste homolog 2 (EZH2), thereby inducing the protein degradation of EZH2 and inhibiting the phosphorylation and activity of EZH2. Consequently, LG1980 targets a survival signaling cascade consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B 1 (ABCB1) and survivin. As a lead compound, LG1980 is well tolerated in mice and effectively suppresses the in vivo growth of chemoresistant PCa and synergistically enhances the efficacy of docetaxel in xenograft models.Conclusions: These results indicate that pharmacological inhibition of EED-EZH2 interaction is a novel strategy for the treatment of chemoresistant PCa. LG1980 and its analogues have the potential to be integrated into standard of care to improve clinical outcomes in PCa patients.