Expression Of S-phase Kinase-associated Protein Research Articles

Abstract P197: Identification and Characterization of Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Proteins in Placenta

Preeclampsia is life threatening condition affecting about 10% of all pregnancies. A potential mechanism is via malfunctioning syncytiotrophoblasts in the placenta. RhoBTB1 is an adapter protein for the CUL3 E3 ubiquitin ligase which delivers protein substrates for proteasomal degradation. An analysis of gene expression databases revealed that the highest level of RhoBTB1 expression is in the placenta. RNAscope in situ hybridization data reveals that RhoBTB1 expression is specifically localized to syncytiotrophoblasts. In blood vessels, RhoBTB1 controls the state of nitric oxide mediated vasodilation because it acts as a substrate adaptor for Phosphodiesterase 5. Thus, we wanted to identify potential protein targets of RhoBTB1 in the placenta to assess if RhoBTB1 plays a role in placental function. To simplify the first identification of RhoBTB1 binding proteins, we employed HTR-8 /SVneo (HTR-8) cells as a model of placental trophoblast cells. We biotinylated the HTR-8 proteome utilizing ascorbate peroxidase (APEX2) proximity labelling system followed by mass spectrometry to identify RhoBTB1 targets. We employed a fusion protein consisting of the C-terminal half of RhoBTB1 (B1B2C), the region required for the binding of other protein substrates, and a control lacking the most C-terminal region (B1B2). We next chose proteins with binding to B1B2C > B1B2 of 2-fold or greater that were expressed in the placenta based on the Human Protein Atlas. Rho GTPase Activating Protein 29 (ARHGAP29), which controls the regulation of small GTP binding proteins, and S-phase Kinase associated Protein 2 (SKP2), a regulator of cell cycle progression was studied further. SKP2 is interesting because it exhibits a tissue specific tropism for placental expression with its highest expression in extravillous trophoblasts, cytotrophoblasts, and syncytiotrophoblasts. Expression of ARHGAP29 and SKP2 in HTR-8 cells were markedly upregulated by MLN4924 suggesting they are regulated by the CUL pathway. They were also modestly upregulated after partial inhibition of RhoBTB1 by siRNA suggesting they may be RhoBTB1-CUL3 targets. Studies are ongoing to validate the physical interaction of ARHGAP29 and SKP2 with RhoBTB1 by co-immunoprecipitation. We are also using RNAscope to assess the co-expression of RhoBTB1, ARHGAP29 and SKP2 in the human placenta from normal and preeclamptic pregnancies.

Suppression of Skp2 contributes to sepsis-induced acute lung injury by enhancing ferroptosis through the ubiquitination of SLC3A2

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The inflammatory cytokine storm causes systemic organ damage, especially acute lung injury in sepsis. In this study, we found that the expression of S-phase kinase-associated protein 2 (Skp2) was significantly decreased in sepsis-induced acute lung injury (ALI). Sepsis activated the MEK/ERK pathway and inhibited Skp2 expression in the pulmonary epithelium, resulting in a reduction of K48 ubiquitination of solute carrier family 3 member 2 (SLC3A2), thereby impairing its membrane localization and cystine/glutamate exchange function. Consequently, the dysregulated intracellular redox reactions induced ferroptosis in pulmonary epithelial cells, leading to lung injury. Finally, we demonstrated that intravenous administration of Skp2 mRNA-encapsulating lipid nanoparticles (LNPs) inhibited ferroptosis in the pulmonary epithelium and alleviated lung injury in septic mice. Taken together, these data provide an innovative understanding of the underlying mechanisms of sepsis-induced ALI and a promising therapeutic strategy for sepsis.

Upregulated SKP2 Empowers Epidermal Proliferation Through Downregulation of P27 Kip1.

Excessive growth of keratinocytes is the critical event in the etiology of psoriasis. However, the underlying molecular mechanism of psoriatic keratinocyte hyperproliferation is still unclear. This study aimed to figure out the potential contributory role of S-phase kinase-associated protein 2 (SKP2) in promoting the hyperproliferation of keratinocytes in psoriasis. We analyzed microarray data (GSE41662) to investigate the gene expression of SKP2 in psoriatic lesion skins compared with their adjacent non-lesional skin. Then, we further confirmed the mRNA and protein expression of SKP2 in human psoriatic skin tissues, imiquimod (IMQ)-induced psoriatic mice back skins and tumor necrosis factor α (TNF-α), interleukin (IL)-17A and IL-6-stimulated keratinocytes by using real-time quantitative polymerase chain reaction and western blot (WB). Furthermore, we explored the potential pathogenic role and its underlying cellular mechanism of SKP2 in promoting keratinocytes hyperproliferation through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle detection, 5-ethynyl-2'-deoxyuridine staining and WB. Finally, we determined whether inhibition of SKP2 can effectively alleviate the keratinocytes hyperproliferation in vivo. We identified that SKP2 is aberrantly upregulated in the psoriatic lesion skin and cytokines-stimulated keratinocytes. Moreover, upregulated SKP2 augments cytokines-induced keratinocytes hyperproliferation. Mechanistically, enhanced SKP2 increased the S phase ratio through inhibiting Cyclin-Dependent Kinase Inhibitor p27 (P27 Kip1) expression. Correspondingly, suppression of SKP2 with SMIP004 can significantly ease the epidermis hyperplasia in vivo. Our results suggest that elevated SKP2 can empower keratinocytes proliferation and psoriasis-like epidermis hyperplasia via downregulation of P27 Kip1. Therefore, targeting SKP2-P27 Kip1 axis might be a promising therapeutic strategy for the treatment of psoriasis in future.

Carbamazepine regulates USP10 through miR-20a-5p to affect the deubiquitination of SKP2 and inhibit osteogenic differentiation.

Antiepileptic drugs (AEDs) harm bone health and are significantly associated with osteoporosis development. In this study, we aimed to explore the mechanisms involved in carbamazepine (CBZ) and microRNA (miR)-20a-5p/ubiquitin-specific peptidase 10 (USP10)/S-phase kinase-associated protein 2 (SKP2) axis in osteoporosis. Human bone marrow mesenchymal stem cells (BMSCs) were treated with different concentrations of CBZ. Knocking down or overexpressing miR-20a-5p, USP10, and SKP2 cell lines were constructed. The expressions of miR-20a-5p, USP10, SKP2, runt-related transcription factor 2 (Runx2), Alkaline phosphatase (ALP), Osterix (Osx), osteocalcin (OCN) and Collagen I were detected with western blot (WB) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Alizarin Red S (ARS) staining was performed to measure calcium deposition. Dual-luciferase assay and RNA immunoprecipitation (RIP) were applied to verify the binding relationship between miR-20a-5p and USP10. USP10 and SKP2 combination was verified by Co-Immunopurification (Co-IP). The stability of the SKP2 protein was verified by Cycloheximide chase assay. CBZ could reduce cell activity. ALP activity and ARS staining were enhanced in the osteogenic induction (OM) group. The expressions of Runx2, ALP, Osx, OCN and Collagen I were increased. CBZ reduced miR-20a-5p expressions. Verification experiments showed miR-20a-5p could target USP10. USP10 increased SKP2 stability and promoted SKP2 expression. CBZ regulated miR-20a-5p/USP10/SPK2 and inhibited BMSCs osteogenic differentiation. CBZ regulated USP10 through miR-20a-5p to affect the deubiquitination of SKP2 and inhibit osteogenic differentiation, which provided a new idea for osteoporosis treatment.

SP1-Induced Upregulation of LncRNA AFAP1-AS1 Promotes Tumor Progression in Triple-Negative Breast Cancer by Regulating mTOR Pathway.

The long non-coding RNA (lncRNA) actin fiber-associated protein-1 antisense RNA 1 (AFAP1-AS1) exerted oncogenic activity in triple-negative breast cancer (TNBC). We designed this study and conducted it to investigate the upstream regulation mechanism of AFAP1-AS1 in TNBC tumorigenesis. In this work, we proved the localization of AFAP1-AS1 in the cytoplasm. We elucidated the mechanism by which the transcription factor specificity protein 1 (SP1) modulated AFAP1-AS1 in TNBC progression, which has yet to be thoroughly studied. Dual luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay revealed a strong affinity of SP1 toward the promoter regions P3 of AFAP1-AS1, proving the gene expression regulation of AFAP1-AS1 via SP1 in TNBC. Additionally, SP1 could facilitate the tumorigenesis of TNBC cells in vitro and in vivo by regulating the AFAP1-AS1 expression. Furthermore, silenced AFAP1-AS1 suppressed the expression of genes in the mTOR pathway, such as eukaryotic translation initiation factor 4B (EIF4B), mitogen-activated protein kinase-associated protein 1 (MAPKAP1), SEH1-like nucleoporin (SEH1L), serum/glucocorticoid regulated kinase 1 (SGK1), and its target NEDD4-like E3 ubiquitin protein ligase (NEDD4L), and promoted the gene expression of s-phase kinase-associated protein 2 (SKP2). Overall, this study emphasized the oncogenic role of SP1 and AFAP1-AS1 in TNBC and illustrated the AFAP1-AS1 upstream interaction with SP1 and the downstream modulatory of mTOR signaling, thus offering insights into the tumorigenesis mechanism in TNBC.

Gene S-phase kinase associated protein 2 is a novel prognostic marker in human neoplasms

BackgroundNeoplasms are a series of diseases affecting human health. Prognostic and tumor status–related markers for various tumors should be identified.MethodsBased on 19,515 samples from multiple sources, for the first time, this study provided an overview of gene S-phase kinase associated protein 2 (SKP2) in pan-cancer. Differential SKP2 expression in multiple comparison groups was identified by the Kruskal–Wallis test and Wilcoxon rank-sum test. The prognosis significance of SKP2 in individuals with neoplasm was evaluated through univariate Cox regression analysis and Kaplan-Meier curves. The area under the curve was utilized to detect the accuracy of SKP2 in predicting cancer status. Spearman’s rank correlation coefficients were calculated in all correlation analyses. Gene set enrichment analysis was used to identify essential signaling pathways of SKP2 in human neoplasms.ResultsThe study disclosed the upregulated SKP2 expression in 15 neoplasms and decreased SKP2 expression in three cancers (p < 0.05). The transcription factor Forkhead Box M1 may contribute to the increased expression levels of SKP2 in certain tumors. Over-expressed SKP2 represented a risk factor for the prognosis of most cancer patients (hazard ratio > 1, p < 0.05). SKP2 expression made it feasible to distinguish neoplasm and control tissues of 21 neoplasms (sensitivity = 0.79, specificity = 0.87, area under the curve = 0.90), implying its potential in screening a series of neoplasms. Further, the research revealed the close association of SKP2 expression with DNA methyltransferases, mismatch repair genes, microsatellite instability, tumor mutational burden, neoantigen count, and immunity.ConclusionsSKP2 plays an essential role in multiple neoplasms and may serve as a marker for treating and identifying these neoplasms.

Punicalagin alleviates the hyperproliferation of keratinocytes in psoriasis through inhibiting SKP2 expression.

Psoriasis is a chronic inflammatory skin disorder characterized by abnormal keratinocytes proliferation and multiple immune cells infiltration in the dermis and epidermis. Although most psoriasis-related researches have been concentrated on the interleukin-23 (IL-23)/interleukin-17 (IL-17) axis, new data suggest that keratinocytes also play a pivotal role in psoriasis. Previously, we found that punicalagin (PUN), a bioactive ellagitannin extracted from Pericarpium Granati (the pericarpium of Punica granatum L.), exerts a therapeutic effect on psoriasis. However, the underlying mechanism, especially its potential modulatory effect on keratinocytes, remains obscure. Our study aims to reveal the potential regulatory effect and its underlying cellular mechanism of PUN on the hyperproliferation of keratinocytes. We used tumor necrosis factor α (TNF-α), IL-17A and interleukin-6 (IL-6) to induce abnormal proliferation of HaCaT cells (Human Keratinocytes Cells) in vitro. Then, we evaluated the effects of PUN through MTT assay, EdU staining and cell cycle detection. Finally, we explored the underlying cellular mechanisms of PUN via RNA-sequencing, WB in vitro and in vivo. Here, we found that PUN can directly and dose-dependently decrease TNF-α, IL-17A and IL-6-induced abnormal proliferation of HaCaT cells in vitro. Mechanically, PUN suppresses the hyperproliferation of keratinocytes through repressing S-phase kinase-associated protein 2 (SKP2) expression in vitro and in vivo. Moreover, overexpression of SKP2 can partly abolish PUN-mediated inhibition of aberrantly proliferative keratinocytes. These results illustrate that PUN can reduce the severity of psoriasis through directly repressing SKP2-mediated abnormal proliferation of keratinocytes, which gives new insight into the therapeutic mechanism of PUN on psoriasis. Moreover, these findings imply that PUN might be a promising drug candidate for the treatment of psoriasis.

Promoter methylation and enhanced SKP2 are associated with the downregulation of CDKN1C in cervical squamous cell carcinoma

PurposeCervical Squamous Cell Carcinoma (CSCC) is one of the significant causes of cancer deaths among women. Distinct genetic and epigenetic-altered loci, including chromosomal 11p15.5–15.4, have been identified. CDKN1C (Cyclin-Dependent Kinase Inhibitor 1C, p57KIP2), a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs), located at 11p15.4, is a putative tumor suppressor. Apart from transcriptional control, S-Phase Kinase Associated Protein 2 (SKP2), an oncogenic E3 ubiquitin ligase, regulates the protein turnover of CDKN1C. But the molecular status of CDKN1C in CSCC and the underlying mechanistic underpinnings have yet to be explored. MethodsTCGA and other publicly available datasets were analyzed to evaluate the expression of CDKN1C and SKP2. The expression (transcript/protein) was validated in independent CSCC tumors (n = 155). Copy number alteration and promoter methylation were correlated with the expression. Finally, in vitro functional validation was performed. ResultsCDKN1C was down-regulated, and SKP2 was up-regulated at the transcript and protein levels in CSCC tumors and the SiHa cell line. Notably, promoter methylation (50%) was associated with the downregulation of the CDKN1C transcript. However, high expression of SKP2 was found to be associated with the decreased expression of CDKN1C protein. Independent treatments with 5-aza-dC, MG132, and SKP2i (SKPin C1) in SiHa cells led to an enhanced expression of CDKN1C protein, validating the mechanism of down-regulation in CSCC. ConclusionCollectively, CDKN1C was down-regulated due to the synergistic effect of promoter hyper-methylation and SKP2 over-expression in CSCC tumors, paving the way for further studies of its role in the pathogenesis of the disease.

SKP2 Contributes to AKT Activation by Ubiquitination Degradation of PHLPP1, Impedes Autophagy, and Facilitates the Survival of Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid carcinoma. Despite a good prognosis, approximately a quarter of PTC patients are likely to relapse. Previous reports suggest an association between S-phase kinase-associated protein 2 (SKP2) and the prognosis of thyroid cancer. SKP1 is related to apoptosis of PTC cells; however, its role in PTC remains largely elusive. This study aimed to understand the expression and molecular mechanism of SKP2 in PTC. SKP2 expression was upregulated in PTC tissues and closely associated with clinical diagnosis. In vitro and in vivo knockdown of SKP2 expression in PTC cells suppressed cell growth and proliferation and induced apoptosis. SKP2 depletion promoted cell autophagy under glucose deprivation. SKP2 interacted with PH domain leucine-rich repeat protein phosphatase-1 (PHLPP1), triggering its degradation by ubiquitination. Furthermore, SKP2 activates the AKT-related pathways via PHLPP1, which leads to the cytoplasmic translocation of SKP2, indicating a reciprocal regulation between SKP2 and AKT. In conclusion, the upregulation of SKP2 leads to PTC proliferation and survival, and the regulatory network among SKP2, PHLPP1, and AKT provides novel insight into the molecular basis of SKP2 in tumor progression.

Pan-Cancer Analysis of the Expression and Prognostic Value of S-Phase Kinase-Associated Protein 2

BACKGROUND: S-Phase Kinase-Associated Protein 2 (SKP2) is essential in modulating metabolism processes, cell proliferation, and carcinogenesis DUE to its capacity to ubiquitinate and degrade various tumor-suppressive substrates. However, the actual biological and mechanism significance of SKP2 in the development of tumors and as a possible therapeutic target remains to be completely understood. AIM: This study aimed to explore the potential roles of the SKP2 gene in the oncologic pathogenesis of various cancers through an in-depth pan-cancer analysis including gene expression assessment, survival analysis, genetic alteration, and enrichment analysis. METHODS: Public databases including the Cancer Genome Atlas database, Genotype-Tissue Expression Project database, cBioPortal database, Gene Expression Profiling Interactive Analysis 2 database, Tumor Immune Estimation Resource version 2.0 database, and STRING database were used to detect the SKP2 expression, molecular mechanism, and its association with the prognosis across pan-cancer. RESULTS: SKP2 was significantly highly expressed in most types of cancers and was substantially correlated to the poor survival of patients with specific cancers based on the log-rank test. SKP2 had the highest frequency of alteration in lung cancer and amplification was the most common genetic alteration type. Finally, SKP2-related genes were identified and enrichment analyses were conducted. CONCLUSION: This study presented the first demonstration of the pan-cancer landscape of abnormal SKP2 expression, it could potentially serve as a predictive indicator and prospective therapeutic target.

232 (PB112) - Low SKP2 expression is predictive of sensitivity to an MDM2 antagonist in p53 wild-type AML

Background: Non-mutational aberrations of p53 function are a common event in AML. Using fragment-based drug discovery we have identified a Murine double minute 2 (MDM2) antagonist that potently activates p53 pathway. Here, we present the discovery of a novel patient selection strategy for MDM2 antagonist-induced apoptosis in wild-type p53 AML. Material and Methods: Cell panel screen and bioinformatics analyses of p53 wild-type AML cell lines that undergo apoptosis following Compound 1 (MDM2 antagonist) treatment versus those that do not were employed to identify novel patient selection strategy. Correlation between low levels of SKP2 (S-Phase Kinase Associated Protein 2) and induction of apoptosis following Compound 1 treatment was tested in vitro using Western Blot and flow cytometry analysis. Genetic manipulation of SKP2 levels using knockdown and over-expression approaches in wild-type p53 AML cells were used to validate the role of SKP2 expression in in vitro and in vivo experiments. Finally, bone marrow-derived primary AML blasts were tested to confirm the novel patient selection strategy. Results: Cell panel screen of p53 wild-type cell lines showed a wide range of sensitivity to Compound 1 suggesting that additional biomarkers are required to ensure patient stratification. Further bioinformatics analysis of apoptotic and non-apoptotic AML cancer cell lines following treatment with Compound 1 demonstrated differential gene expression levels and identified several potential predictive biomarkers. Western blot and flow cytometry analyses demonstrated that basal SKP2 expression level is lower in apoptotic AML cell lines than in non-apoptotic cells. Over-expression of SKP2 levels in MV4-11 cells reduced MDM2 antagonist-dependent cell death in previously apoptotic cell line. Similarly, SKP2 knockdown in OCI-AML3 cells increased MDM2 antagonist-induced cytotoxicity. Studies performed in mouse systemic AML model in vivo also demonstrated that high levels of SKP2 reduced p53 response to Compound 1. Finally, the correlation of SKP2 expression and Compound 1 sensitivity was confirmed in primary AML blasts isolated from patients. Conclusions: This work demonstrates in vitro and in vivo activity of MDM2 antagonist in AML models with low basal SKP2 levels. These pre-clinical data validate low levels of SKP2 as a novel patient selection strategy in AML and support the clinical investigation of MDM2 antagonist as therapeutic strategy for the treatment of SKP2low p53wt AML. No conflict of interest.

The SKP2-p27 axis defines susceptibility to cell death upon CHK1 inhibition.

Checkpoint kinase 1 (CHK1; encoded by CHEK1) is an essential gene that monitors DNA replication fidelity and prevents mitotic entry in the presence of under‐replicated DNA or exogenous DNA damage. Cancer cells deficient in p53 tumor suppressor function reportedly develop a strong dependency on CHK1 for proper cell cycle progression and maintenance of genome integrity, sparking interest in developing kinase inhibitors. Pharmacological inhibition of CHK1 triggers B‐Cell CLL/Lymphoma 2 (BCL2)‐regulated cell death in malignant cells largely independently of p53, and has been suggested to kill p53‐deficient cancer cells even more effectively. Next to p53 status, our knowledge about factors predicting cancer cell responsiveness to CHK1 inhibitors is limited. Here, we conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen to identify genes defining sensitivity to chemical CHK1 inhibitors. Next to the proapoptotic BCL2 family member, BCL2 Binding Component 3 (BBC3; also known as PUMA), the F‐box protein S‐phase Kinase‐Associated Protein 2 (SKP2) was validated to tune the cellular response to CHK1 inhibition. SKP2 is best known for degradation of the Cyclin‐dependent Kinase Inhibitor 1B (CDKN1B; also known as p27), thereby promoting G1‐S transition and cell cycle progression in response to mitogens. Loss of SKP2 resulted in the predicted increase in p27 protein levels, coinciding with reduced DNA damage upon CHK1‐inhibitor treatment and reduced cell death in S‐phase. Conversely, overexpression of SKP2, which consequently results in reduced p27 protein levels, enhanced cell death susceptibility to CHK1 inhibition. We propose that assessing SKP2 and p27 expression levels in human malignancies will help to predict the responsiveness to CHK1‐inhibitor treatment.

Cardioprotective Effect of Paeonol on Chronic Heart Failure Induced by Doxorubicin via Regulating the miR-21-5p/S-Phase Kinase-Associated Protein 2 Axis.

BackgroundThis study primarily explored the role of paeonol in doxorubicin (DOX)-induced chronic heart failure (CHF), considering the cardioprotective effect of paeonol on an epirubicin-induced cardiac injury.MethodsDOX-induced CHF-modeled rats were treated with paeonol. Cardiac function and myocardial damage in rats were evaluated by using the multifunction instrument, and the histopathology, apoptosis, and the expression of miR-21-5p and S-phase kinase-associated protein 2 (SKP2) in myocardium were detected. The target gene of miR-21-5p was confirmed by a dual-luciferase reporter assay. After the required transfection or paeonol treatment, the viability, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) of the DOX-induced cardiomyocytes were determined. Reverse-transcription quantitative-PCR (RT-qPCR) and Western blot were performed to quantify the expressions of miR-21-5p, SKP2, and apoptosis-related factors.ResultsPaeonol improved cardiac function and also ameliorated the cardiac damage of CHF-modeled rats, where the downregulation of abnormally elevated myocardial damage markers, including brain natriuretic peptide, lactate dehydrogenase, renin, angiotensin II, aldosterone, and endothelin 1, was observed. Paeonol alleviated the histopathological injury and suppressed the apoptosis in CHF-modeled rats, inhibited miR-21-5p expression, and upregulated SKP2 expression in vitro and in vivo. miR-21-5p targeted SKP2. Paeonol and SKP2 increased the viability and MMP, but reduced apoptosis and ROS in the DOX-induced cardiomyocytes. miR-21-5p exerted effects opposite to PAE and SKP2, and it downregulated the expression of Bcl-2 and mitochondrion-Cytochrome c (Cyt c) and upregulated the expression of Bax, C-caspase-3, and cytoplasm-Cyt c. miR-21-5p reversed the effects of paeonol, and its effects were further reversed by SKP2.ConclusionPaeonol shows a cardioprotective effect on DOX-induced CHF via regulating the miR-21-5p/SKP2 axis.

The Impact of SKP2 Gene Expression in Chronic Myeloid Leukemia.

Introduction: The prognosis of chronic myeloid leukemia (CML) patients has been dramatically improved with the introduction of imatinib (IM), the first tyrosine kinase inhibitor (TKI). TKI resistance is a serious problem in IM-based therapy. The human S-phase kinase-associated protein 2 (SKP2) gene may play an essential role in the genesis and progression of CML. Aim of the study: We try to explore the diagnostic/prognostic impact of SKP2 gene expression to predict treatment response in first-line IM-treated CML patients at an early response stage. Patients and methods: The gene expression and protein levels of SKP2 were determined using quantitative RT-PCR and ELISA in 100 newly diagnosed CML patients and 100 healthy subjects. Results: SKP2 gene expression and SKP2 protein levels were significantly upregulated in CML patients compared to the control group. The receiver operating characteristic (ROC) analysis for the SKP2 gene expression level, which that differentiated the CML patients from the healthy subjects, yielded a sensitivity of 86.0% and a specificity of 82.0%, with an area under the curve (AUC) of 0.958 (p < 0.001). The ROC analysis for the SKP2 gene expression level, which differentiated optimally from the warning/failure responses, yielded a sensitivity of 70.59% and a specificity of 71.21%, with an AUC of 0.815 (p < 0.001). Conclusion: The SKP2 gene could be an additional diagnostic and an independent prognostic marker for predicting treatment responses in first-line IM-treated CML patients at an early time point (3 months).

SKP2 regulates ZEB1 expression and stimulates eutopic endometrial stromal cell invasion and proliferation of adenomyosis

Though endometriosis is benign, however, it shares certain characteristics with cancers, such as the ability to invade and metastasize. Previous studies have demonstrated that S-phase kinase associated protein2 (SKP2) promotes invasion, tumorigenesis, and metastasis. However, its correlation with adenomyosis is unclear. Herein, we aimed to look into SKP2 expression patterns and regulatory effects on endometrial stromal cell (ESC) proliferation and invasion, and its internal mechanism in adenomyosis. Western blot, qRT-PCR, and immunochemistry were carried out for detecting SKP2 and ZEB1 expression in ESC of adenomyosis and adenomyosis endometrial tissue. The primary ESCs were identified using immunofluorescence. SKP2 knockdown was accomplished in vitro by transfecting a particular lentivirus vector. The colony formation and CCK-8 assays were carried out for assessing cell proliferation, while cell invasion potential was assessed using the transwell assay. Both SKP2 and ZEB1 were found to be significantly upregulated in adenomyosis endometrial tissue. Knockdown of SKP2 inhibited adenomyotic ESC invasion and proliferation. Further experiments showed that knocking out SKP2 reduced ZEB1 expression in adenomyotic ESCs. Our results showed that SKP2 could regulate ZEB1 expression, and increased SKP2 may play a role in the pathogenesis of adenomyosis and stimulating ESC proliferation and invasion.

CircCRIM1 Promotes Hepatocellular Carcinoma Proliferation and Angiogenesis by Sponging miR-378a-3p and Regulating SKP2 Expression.

Mounting evidence has demonstrated that circular RNAs have an important function in tumorigenesis and cancer evolvement. CircCRIM1 has been shown to be a poor prognostic element in multiple human malignancies. However, the clinical significance and mechanism of circCRIM1 in hepatocellular carcinoma (HCC) is still unclear. The present study confirmed the expression level of circCRIM1 using quantitative real-time PCR. In addition, circCRIM1 siRNA and overexpression vectors were used for transfection into LM3 or Huh7 cells to down- or up-regulate the expression of circCRIM1. In vitro and in vivo experiments were performed to explore the function of circCRIM1 in HCC. RNA pull-down, RNA immunoprecipitation, fluorescent in situ hybridization, and luciferase reporter assays were conducted to confirm the relationship between miR-378a-3p and circCRIM1 or S-phase kinase-associated protein 2 (SKP2) in HCC. Then, circCRIM1 was up-regulated in HCC and its expression level was significantly associated with poor prognosis and clinicopathologic characteristics. CircCRIM1 enhanced the proliferation and angiogenesis of HCC cells in vitro and promoted xenograft growth in vivo. Moreover, circCRIM1 upregulated the expression of SKP2 by functioning as a sponge for miR-378a-3p. These findings suggest that circCRIM1 boosts the HCC progression via the miR-378-3p/SKP2 axis and may act as a crucial epigenetic therapeutic molecule target in HCC.

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.

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.

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.

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.