Ubiquitin-specific-processing Protease 7 Research Articles

USP7 regulates HMOX-1 via deubiquitination to suppress ferroptosis and ameliorate spinal cord injury in rats

Heme oxygenase 1 (HMOX-1) is overexpressed in spinal cord injury (SCI) and relevant to ferroptosis. Ubiquitin-specific-processing protease 7 (USP7) has unveiled its role in regulating HMOX-1 stabilization while its function in SCI remains unknown. This study is to explore the potential molecular mechanism of the USP7-HMOX-1 axis in ferroptosis in a SCI rat model. SCI was assessed with Basso, Beattie, Bresnahan locomotion evaluation, hematoxylin-eosin histological staining, and immunofluorescence detection of NeuN. Ferroptosis was assessed by detections of the iron content, malondialdehyde and glutathione levels, mitochondrial damage, and glutathione peroxidase 4, 4-hydroxynonenal, USP7, and HMOX-1 expression in spinal cord. Co-immunoprecipitation was used to detect the binding of USP7 to HMOX-1. The ubiquitination level of HMOX-1 was measured after USP7 overexpression. USP7 expression was downregulated and HMOX-1 expression was upregulated in SCI rat models. HMOX-1 or USP7 overexpression promoted motor function recovery, ameliorated spinal cord damage, increased NeuN expression, and blocked the occurrence of ferroptosis in SCI rat models. In SCI rats, USP7 directly bound to HMOX-1 and its overexpression promoted HMOX-1 expression via deubiquitination. To sum up, USP7 overexpression facilitated the expression of HMOX-1 through deubiquitination, thereby reducing ferroptosis and alleviating SCI.

Multimodal data fusion for supervised learning-based identification of USP7 inhibitors: a systematic comparison

Ubiquitin-specific-processing protease 7 (USP7) is a promising target protein for cancer therapy, and great attention has been given to the identification of USP7 inhibitors. Traditional virtual screening methods have now been successfully applied to discover USP7 inhibitors aiming at reducing costs and speeding up time in several studies. However, due to their unsatisfactory accuracy, it is still a difficult task to develop USP7 inhibitors. In this study, multiple supervised learning classifiers were built to distinguish active USP7 inhibitors from inactive ligands. Physicochemical descriptors, MACCS keys, ECFP4 fingerprints and SMILES were first calculated to represent the compounds in our in-house dataset. Two deep learning (DL) models and nine classical machine learning (ML) models were then constructed based on different combinations of the above molecular representations under three activity cutoff values, and a total of 15 groups of experiments (75 experiments) were implemented. The performance of the models in these experiments was evaluated, compared and discussed using a variety of metrics. The optimal models are ensemble learning models when the dataset is balanced or severely imbalanced, and SMILES-based DL performs the best when the dataset is slightly imbalanced. Meanwhile, multimodal data fusion in some cases can improve the performance of ML and DL models. In addition, SMOTE, unbiased decoy selection and SMILES enumeration can improve the performance of ML and DL models when the dataset is severely imbalanced, and SMOTE works the best. Our study established highly accurate supervised learning classification models, which would accelerate the development of USP7 inhibitors. Some guidance was also provided for drug researchers in selecting supervised models and molecular representations as well as handling imbalanced datasets.Graphical

USP7 Enables Immune Escape of Glioma Cells by Regulating PD-L1 Expression

ABSTRACT Background Glioma is the most common primary intracranial tumor. This study investigated the mechanism of ubiquitin-specific processing protease 7 (USP7) on the immune escape of glioma cells via the regulation of programmed cell death ligand-1 (PD-L1). Methods USP7 and PD-L1 expressions in glioma and normal brain tissues were detected using quantitative reverse transcriptase polymerase chain reaction and Western blot. The glioma cells U-251 MG were transfected with si-USP7 and pcDNA3.1-PD-L1, or treated with anti-PD-L1, after which the cell viability, colony-forming ability, and apoptosis rate were evaluated using cell counting kit-8, colony formation, and TdT-mediated dUTP nick-end labeling assays. Then, CD8+ T cells were purified, extracted, then co-cultured with U-251 MG cells. CD8+ T cell proliferation and the concentrations of interferon (IFN-γ), tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, interleukin (IL)-1β, and IL-10 were verified using carboxyfluorescein succinimidyl amino ester proliferation assay and enzyme-linked immunosorbent assay. Afterwards, the interaction between USP7 and PD-L1 and the ubiquitination level of PD-L1 were also assessed. Results USP7 was highly-expressed and PD-L1 mRNA levels did not change, while PD-L1 protein levels were up-regulated in the glioma cells. Silencing USP7 in U-251 MG cells limited the growth of the glioma cells, promoted glioma cell apoptosis, and facilitated the proliferation of CD8+ T cells, thus inhibiting immune escape. USP7 stabilized PD-L1 expression through deubiquitination. PD-L1 overexpression reversed the inhibitory effect of silencing USP7 on the immune escape of glioma cells. Conclusion USP7 stabilized PD-L1 through deubiquitination and accelerated the immune escape of glioma cells.

Proteasomal turnover of the RhoGAP tumor suppressor DLC1 is regulated by HECTD1 and USP7

The Rho GTPase activating protein Deleted in Liver Cancer 1 (DLC1) is frequently downregulated through genetic and epigenetic mechanisms in various malignancies, leading to aberrant Rho GTPase signaling and thus facilitating cancer progression. Here we show that in breast cancer cells, dysregulation of DLC1 expression occurs at the protein level through rapid degradation via the ubiquitin–proteasome system. Using mass spectrometry, we identify two novel DLC1 interaction partners, the ubiquitin-ligase HECTD1 and the deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7). While DLC1 protein expression was rapidly downregulated upon pharmacological inhibition of USP7, siRNA-mediated knockdown of HECTD1 increased DLC1 protein levels and impaired its degradation. Immunofluorescence microscopy analyses revealed that the modulation of HECTD1 levels and USP7 activity altered DLC1 abundance at focal adhesions, its primary site of action. Thus, we propose opposing regulatory mechanisms of DLC1 protein homeostasis by USP7 and HECTD1, which could open up strategies to counteract downregulation and restore DLC1 expression in cancer.

Targeting USP7 Suppresses Tumor Development By Promoting Ubiquitination of PHF8 and Suppressing SNAI1/Wnt Signaling in T-Cell Acute Lymphoblastic Leukemia

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy with a high unfavorable prognosis. Ubiquitin-specific-processing protease 7 (USP7) is one of the deubiquitinating enzymes attracting concentrated attention in current studies for cancers; it is also involved in regulation of oncogenic transcriptional program in T-ALL, and serves as a potential target to treat T-ALL. USP7 is interacted with PHD Finger protein 8 (PHF8), a histone lysine demethylase in T-ALL. However, the impact of the interaction in T-ALL development is undetermined. In this study, we explored the anti-tumor effect of targeting USP7 and its downstream substrate in T-ALL and underlying mechanism.MethodsIn vitro GST pull-down assay, co-immunoprecipitation assay, protein deubiquitination and stability assays were used to detect the interaction of USP7 with its direct substrate and the ubiquitination level. Gain-of-function(overexpression) and loss-of-function (lentiviral shRNA knockdown) approaches for USP7 were conducted for gene expression, CCK-8 cell proliferation assay, and apoptosis assay with annexin V + PI staining. ChIP-qPCR assay was used to explore the enrichment of histone markers in promoter region of target gene. In vivo human T-ALL xenograft mouse model was developed in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse by tail vein injection of 2.5 × 10 6 /mouse and the leukemia engraftment was evaluated with human CD45+ cells by flow cytometry. The qPCR and western blot were carried out for detection mRNA level and protein level of related genes. The mRNA level of USP7 and its substrate PHF8 was examined by qPCR in 43 newly-diagnosed T-ALL patients with an approval of the Ethics Committee. Microarray datasets of T-ALL patients from GEO database were used to determine the USP7 and its substrate -related genes with web-based platform MEM and Limma.ResultsResults showed that USP7 directly interacted with PHF8; and overexpressed USP7 deubiquitinated and stabilized PHF8, and enhanced cell proliferation of JURKAT T-ALL cells. These data indicates targeting USP7 promotes cell proliferation of T-ALL cells through its substrate PHF8. Moreover, SNAI1 was identified as one of key co-expressed genes with PHF8 in microarray datasets from T-ALL patients, and knock-down of PHF8 suppressed the SNAL1 expression and increased the recruitment of repressive histone marker, H3K9me2 but decreased the enrichment of H3K4me3 in SNAI1 promoter. SNAI1 knockdown significantly induced the cell proliferation arrest and apoptosis of T-ALL cells (Fig.1A & 1B). With in vivo human leukemia xenograft mouse model, we further demonstrated that SNAI1 knockdown significantly reduce the spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity and also the inflammatory cell infiltration compared to the scramble shRNA control (Fig.1C-1F). Moreover, Wnt signaling was identified as SNAI1 interaction partners by the pathway analysis in the SNAI1 interaction genes in the microarray data from T-ALL patients. SNAI1 knockdown significantly suppressed the expression of the Axin2 and Survivn, the key components of Wnt signaling. These data suggested oncogenic role of USP7/PHF8/SNAI1/Wnt signaling in T-ALL. Next, we explored the effect of SNAI1 on USP7 knockdown-induced anti-tumor effect. We found that USP7 knockdown induced cell proliferation arrest and apoptosis, and overexpression of SNAI1 could block the effect in vitro. Furthermore, the in vivo data showed that USP7 knockdown significantly suppressed spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity, inflammatory cell infiltration, and the protein expression of PHF8, SNAI1, Axin2 and Survivin; and SNAI1 overexpression completely rescue the USP7 knockdown-mediated antitumor effect and restored the expression of Axin2 and Survivin in vivo (Fig.2).Conclusion:Our results demonstrated that targeting USP7 by shRNA induces the cell proliferation arrest and apoptosis by promoting ubiquitination of PHF8 and suppressing SNAI1/Wnt signaling in T-ALL. Our data also revealed the oncogenic roles of USP7/PHF8/SNAI1/Wnt signaling in T-ALL and suggested targeting the signaling pathway as potential therapy in T-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Deubiquitylation and stabilization of ARMC5 by ubiquitin-specific processing protease 7 (USP7) are critical for RCC proliferation.

The ubiquitin‐proteasome system is an essential regulator of ARMC5, which serves as a new tumour suppressor protein for inhibiting meningiomas and hereditary adrenocortical tumorigenesis. However, the precise mechanism for the deubiquitination of ARMC5 is still not fully understood. A Western blot analysis of ARMC5 was performed and showed that the expression of ARMC5 was decreased in the renal cancer cell tissues and lines. By screening a deubiquitinase library, we identified USP7 as a potential ARMC5 associated deubiquitinase. In this paper, we demonstrated that there was an interaction between USP7 and ARMC5 in vivo and in vitro. Employing the overexpression and knockdown assay indicated that USP7 could greatly increase the steady state of ARMC5 through the ubiquitin‐proteasome pathway and regulate ARMC5 ubiquitination. Moreover, USP7 altered cell cycle G1/S phases and regulated renal cancer cell proliferation by targeting ARMC5. Together, these results suggest that USP7 plays an important role in the RCC proliferation through modulating ARMC5 stability.

Upregulation of deubiquitinase USP7 by transcription factor FOXO6 promotes EC progression via targeting the JMJD3/CLU axis

Esophageal carcinoma (EC) is recognized as one of the most frequently occurring malignancies worldwide, and its high morbidity rate motivates efforts to identify potential therapeutic targets. Notably, forkhead box (FOX) family genes are highlighted as possible biomarkers for diagnostics, prognostics, and therapeutics of various malignancies, including EC. Our present study was performed to investigate the underlying mechanism of FOXO6 on the development of EC. We observed a significant upregulation of FOXO6 in EC tissues, contributing to the migration and proliferation in EC cells through gain- and loss-of-function assays. FOXO6 directly interacted with the ubiquitin-specific processing protease 7 (USP7) gene promoter and enhanced its transcriptional activity, which resulted in suppressed cancer cell apoptosis as revealed by chromatin immunoprecipitation (ChIP)-qPCR. USP7 enhanced the ubiquitination of Jumonji domain-containing protein D3 (JMJD3), elevated JMJD3-promoted growth of EC cells, and transcriptionally activated clusterin (CLU) expression at the promoter region via histone H3 lysine 27 tri-methyl (H3K27me3) demethylation, according to immunoprecipitation and ubiquitination assays. Finally, we verified that FOXO6 mediated effects on the USP7/JMJD3/CLU axis to exert an oncogenic role in vivo, which was blocked by USP7 and JMJD3 inhibitor. Our findings demonstrate an important role of the FOXO6/USP7/JMJD3/CLU pathway in EC progression and thus provide attractive potential therapeutic targets for EC patients.

Abrogation of USP7 is an alternative strategy to downregulate PD-L1 and sensitize gastric cancer cells to T cells killing

Targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for treating melanoma, gastric cancer (GC) and bladder cancer with clinical benefit. Nevertheless, many patients failed to respond to anti-PD-1/PD-L1 treatment, so it is necessary to seek an alternative strategy for traditional PD-1/PD-L1 targeting immunotherapy. Here with the data from The Cancer Genome Atlas (TCGA) and our in-house tissue library, PD-L1 expression was found to be positively correlated with the expression of ubiquitin-specific processing protease 7 (USP7) in GC. Furthermore, USP7 directly interacted with PD-L1 in order to stabilize it, while abrogation of USP7 attenuated PD-L1/PD-1 interaction and sensitized cancer cells to T cell killing in vitro and in vivo. Besides, USP7 inhibitor suppressed GC cells proliferation by stabilizing P53 in vitro and in vivo. Collectively, our findings indicate that in addition to inhibiting cancer cells proliferation, USP7 inhibitor can also downregulate PD-L1 expression to enhance anti-tumor immune response simultaneously. Hence, these data posit USP7 inhibitor as an anti-proliferation agent as well as a novel therapeutic agent in PD-L1/PD-1 blockade strategy that can promote the immune response of the tumor.

Nuclear expression of MDM2 in hibernoma: a potential diagnostic pitfall.

Hibernoma is a rare benign adipocytic tumor composed of a proliferation of brown and white fat cells varying in their proportions. The tumor may also contain fat cells resembling lipoblasts, which makes it difficult to distinguish it from atypical lipomatous tumor/well differentiated liposarcoma (ALT/WDLS). Although nuclear expressions of murine double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) are widely used as immunohistochemical surrogate markers for ALT/WDLS, the utility of these proteins in distinguishing between hibernoma and ALT/WDLS still remains to be elucidated. We evaluated immunohistochemical expressions of MDM2 and CDK4 in 10 hibernomas expressing uncoupling protein-1 (UCP-1), a mitochondrial protein transporter consistently expressed in brown fat cells, and lacking MDM2 gene amplification, which was analyzed by fluorescence in situ hybridization (FISH). In contrast to the data previously obtained, nuclear expression of MDM2 was observed in 100% (10/10 cases) of the hibernomas irrespective of the proportion of brown fat cells, whereas no cases were positive for CDK4. The tumors also showed almost concurrent expression of p53 (in 9/10 cases) and ubiquitin-specific-processing protease 7 (USP7) (in 10/10 cases), which deubiquitinates and stabilizes MDM2, potentially resulting in its nuclear expression without MDM2 gene amplification. MDM2 expression may thus be a diagnostic pitfall for hibernoma particularly in differentiating it from ALT/WDLS.

Abstract 2919: p16 modulates a novel ubiquitin signaling cascade which regulates radioresponse and offers clinically relevant therapeutic targets in squamous cell carcinoma

Abstract Human papilloma virus (HPV) is associated with multiple types of squamous cell carcinoma, including that of the head and neck (HNSCC). The presence of HPV leads to increased expression of p16, to the extent that p16 is used as a surrogate marker of HPV positivity. HPV/p16 expression is associated with improved response to genotoxic therapy and improved outcome; however, the mechanism of this phenomenon is unclear. Previously, we showed that HPV/p16 expression repressed the DNA damage repair protein TRIP12 and led to radiosensitivity. In the current project we identify USP7 and HUWE1 as key links in this signaling pathway. Ubiquitin-specific-processing protease 7 (USP7) is a deubiquitinating enzyme that has previously been shown to bind to TRIP12, which we confirmed in HPV (-) HNSCC cell lines. We found that forced p16 overexpression in a panel of SCC cell lines led to decreased levels of USP7 and TRIP12 protein, while inhibition of p16 in HPV (+) HNSCC cell lines led to their increase. Forced p16 expression did not affect USP7 mRNA expression, however did lead to increased K48-linked ubiquitinylation and degradation of USP7. Chemical or shRNA mediated inhibition of USP7 in p16/HPV (-) cells led to decreased 53BP1 and BRCA1 foci and increased cell death following radiation. Expression of USP7 shRNA in a HPV (-), radioresistant cell derived xenograft (HN5) lead to a tumor growth delay (TGD) of 10 days following radiation (4 Gy x 5d), compared to a TGD of 1.4 days in control tumors (p<1x10-15). To determine how p16 regulates USP7 ubquitinylation and degradation, we performed immunoprecipitation/mass spectrography (IP/MS) analysis. In 3 HPV (-) and 3 HPV (+) cell lines, pulldown of USP7 identified several E3 ubquitin ligases, however only HUWE1 and TRIM21 were identified in all cell lines tested. We then examined HUWE1 expression following forced expression of p16 and found that both HUWE1 mRNA and protein were increased. Moreover, inhibition of p16 expression in HPV (+) HNSCC cell lines led to decreased HUWE1 expression. Moreover, inhibition of HUWE1 via shRNA could partially rescue the effects of p16 overexpression on expression of DNA damage repair proteins. As HUWE1 is known to be mutated in ~10% of HNSCC, we then examined the effects of HUWE1 on outcome in the TCGA HNSCC patient cohort. In HPV (+) patients, neither HUWE1 mutation nor its gene expression were associated with survival. However, in HPV (-) patients, low HUWE1 expression was associated with worse disease-free survival (DFS)(p=0.048). Additionally, truncating mutations in HUWE1 led to a median survival of 9.4 mos, compared to 67.7 mos in the remaining patients (p=0.008). In conclusion, p16-HUWE1-USP7 signaling can modulate response to radiation. These data suggest that agents under investigation to target USP7, or other members of this signaling cascade, have the potential to improve outcomes in HNSCC. Citation Format: David Molkentine, Kathleen Bridges, Aakash Sheth, David Valdecanas, Curtis Pickering, Heath D. Skinner. p16 modulates a novel ubiquitin signaling cascade which regulates radioresponse and offers clinically relevant therapeutic targets in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2919.

Overexpression of UHRF1 promotes silencing of tumor suppressor genes and predicts outcome in hepatoblastoma

BackgroundHepatoblastoma (HB) is the most common liver tumor of childhood and occurs predominantly within the first 3 years of life. In accordance to its early manifestation, HB has been described to display an extremely low mutation rate. As substitute, epigenetic modifiers seem to play an exceptional role in its tumorigenesis, which holds promise to develop targeted therapies and establish biomarkers for patient risk stratification.ResultsWe examined the role of a newly described protein complex consisting of three epigenetic regulators, namely E3 ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), ubiquitin-specific-processing protease 7 (USP7), and DNA methyltransferase 1 (DNMT1), in HB. We found the complex to be located on the promoter regions of the pivotal HB-associated tumor suppressor genes (TSGs) HHIP, IGFBP3, and SFRP1 in HB cells, thereby leading to strong repression through DNA methylation and histone modifications. Consequently, knockdown of UHRF1 led to DNA demethylation and loss of the repressive H3K9me2 histone mark at the TSG loci with their subsequent transcriptional reactivation. The observed growth impairment of HB cells upon UHRF1 knockdown could be attributed to reduced expression of genes involved in cell cycle progression, negative regulation of cell death, LIN28B signaling, and the adverse 16-gene signature, as revealed by global RNA sequencing. Clinically, overexpression of UHRF1 in primary tumor tissues was significantly associated with poor survival and the prognostic high-risk 16-gene signature.ConclusionThese findings suggest that UHRF1 is critical for aberrant TSG silencing and sustained growth signaling in HB and that UHRF1 overexpression levels might serve as a prognostic biomarker and potential molecular target for HB patients.

BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage

BRCA2 is essential for maintaining genomic integrity. BRCA2-deficient primary cells are either not viable or exhibit severe proliferation defects. Yet, BRCA2 deficiency contributes to tumorigenesis. It is believed that mutations in genes such as TRP53 allow BRCA2 heterozygous cells to overcome growth arrest when they undergo loss of heterozygosity. Here, we report the use of an insertional mutagenesis screen to identify a role for BRE (Brain and Reproductive organ Expressed, also known as BRCC45), known to be a part of the BRCA1-DNA damage sensing complex, in the survival of BRCA2-deficient mouse ES cells. Cell viability by BRE overexpression is mediated by deregulation of CDC25A phosphatase, a key cell cycle regulator and an oncogene. We show that BRE facilitates deubiquitylation of CDC25A by recruiting ubiquitin-specific-processing protease 7 (USP7) in the presence of DNA damage. Additionally, we uncovered the role of CDC25A in BRCA-mediated tumorigenesis, which can have implications in cancer treatment.

USP7 is associated with greater disease activity in systemic lupus erythematosus via stabilization of the IFNα receptor

An improved understanding of the mechanism of interferon (IFN)α activation in systemic lupus erythematosus (SLE) is likely to aid the identification of effective therapeutic targets. Increasing evidence has indicated that the activity of IFNα is mediated by the interplay of ubiquitylation/deubiquitylation enzyme regulators. The present study identified the deubiquitylation enzyme ubiquitin‑specific‑processing protease7 (USP7) as a critical regulator of the human IFNα‑2 receptor (IFNAR1) protein levels. A co‑immunoprecipitation assay was used to demonstrate that USP7 was physically associated with IFNAR1 invivo. A glutathione S‑transferase pull down assay revealed that USP7 interacted with IFNAR1 directly invitro. Furthermore, USP7 may disassemble IFNAR1 dependent poly‑ubiquitin chains and stabilize IFNAR1 invivo. The activation effects of USP7 on the IFNα pathway were confirmed by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Knockdown of USP7 expression consistently reduced the expression levels of signal transducer and activator of transcription (STAT)‑1, STAT‑2 and selected IFN‑inducible genes, including IFN‑induced protein with tetratricopeptide repeats 3, MX dynamin like GTPase 1 and 2'‑5'‑oligoadenylate synthetase 1. The present study demonstrated that USP7 was significantly overexpressed in 210 SLE patients compared with healthy controls. Furthermore, the association between USP7 levels, IFN scores, SLE disease activity index scores and anti‑double stranded DNA were analyzed and, as expected, positive correlations were demonstrated, indicating that USP7 may be associated with SLE disease activity through the stabilization of IFNAR1.

USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells

The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer.

Editorial: Molecules Balancing Immunological Surveillance against Cancer and Autoimmune Diseases.

The Editorial on the Research Topic Molecules Balancing Immunological Surveillance against Cancer and Autoimmune Diseases The innate and the adaptive immune systems work in concert to deal with pathologies introduced by internal and external factors (e.g., viruses, bacteria, and fungi) – some of which may be associated with the development of autoimmune and cancerous cells. Mechanistically, there are internal factors that are responsible for the hypo- and hyperfunction of immune tolerance apparatus. Consequently, it could lead to the development of autoimmune diseases and malignancies, respectively. In the last decade, the study of cancer and autoimmunity suggested that these two entities might reflect two opposed extreme end-points of an immune response spectrum. Accordingly, the overexpression of certain molecules could result in malignancies, whereas deficient expression of the same molecules could lead to the development of autoimmune diseases. Two journals, Frontiers in Immunology and Frontiers in Oncology, participated in this special issue in an attempt to gain the prospective of professionals in both fields of autoimmunity and cancer, hoping to identify molecules and factors that possess dual roles with opposing effects on autoimmune diseases and cancer. E3 ligase Cbl-b, as reviewed by Lutz-Nicoladoni et al., is central in the balance of activating and inhibitory inputs to immune cells. Specifically, Cbl-b negatively regulates activation signals through antigen or pattern recognition receptors and costimulatory molecules. Although cblb-deficient immune cells display lower activation thresholds and cblb knockout mice spontaneously develop autoimmunity and are highly susceptible to experimental autoimmunity, the increased activation potential of cblb-deficient cells renders them more potent to fight against malignancies or infections. In the study of multiple sclerosis (MS), Kaushansky and Ben-Nun, report that the allele DQB1*06:02 shown previously to be a disease-modifying allele, also plays a role as a disease-predisposing allele. They review a novel “humanized” model of MS-like disease, in which autoimmunity against myelin may be driven by a pathogenic HLA-DQ expression. This model also supports an additional target autoantigen, e.g., myelin-associated oligodendrocytic basic protein (MOBP) in the pathogenesis of MS. Haque et al. summarize the utilization of regulatory T cells (Treg cells) in treating rheumatoid arthritis (RA). Because Foxp3 can be polyubiquitinated, they show that the expression of Foxp3 in T cells can be facilitated through Foxp3 ubiquitination. Ubiquitin-specific-processing protease 7 (USP7) is an active enzyme in Treg cells, which may be associated with Foxp3, and leads to its deubiquitination. By contrast, diminished function of USP7 results in a decreased function of Treg cells. In RA, Treg cells do not possess normal immune suppressive activity, although they can be found in the joints of the patients. It is explained, in part, by the elevated levels of TNF-α within the inflamed synovium and the joint fluid, which may lead to Foxp3 phosphorylation, thereby interfering with the suppressive capacities of Treg cells. Recent studies demonstrated that induced pluripotent stem cell (iPSC) can be reprogramed to constitute a source for functional CD4+ Treg cells or CD8+ cytotoxic T lymphocytes. The adoptive transfer of the latter cells may be used for immunotherapy of autoimmune arthritis and cancers. Hence, generation of antigen-specific Foxp3+ Treg cells from stem cells, such as iPSCs, may open a new area in Treg cell-based immunotherapy in transplantation and autoimmune disorders. Manjili et al. describes the evolution of myeloid regulatory cells (Mregs). These cells suppress (through direct and indirect mechanisms) the host’s anti-tumor immune responses in favor of the tumor. By contrast, the ability of Mregs to limit and modulate Th1 responses or support a skewed Th2 immunity could be beneficial during parasitic and helminth infections. In addition, the immunoregulatory functions of Mregs on Th17 differentiation and inflammation were demonstrated in experimental autoimmune encephalomyelitis (EAE). In this model, treatment with gemcitabine that depleted Mregs led to a reduction in Th17 cells, IL-17A, and IL-1β in the lymphoid tissues and spinal cord and to a substantial decrease in the severity of EAE. In addition, Mregs were shown to induce Th17 differentiation in tumor-bearing mice and in patients with ovarian cancer. Also, Mregs’ levels correlated the levels of Th17 cells or IL-17 production in patients with gastrointestinal cancer. Because of the association observed between inflammation, tumorigenesis, and the progression of autoimmune diseases, these proinflammatory cells may represent a pivotal pathogenic factor in the spectrum of cancer and autoimmunity.

Abstract 3952: BRE: a genetic interactor of BRCA2

Abstract Mutations in breast cancer susceptibility genes BRCA1 and BRCA2 predispose individuals to breast and ovarian cancers. Though BRCA associated tumor tissues showed inactivation of both alleles, in primary cells mutations in both copies affect cell viability. This suggests that BRCA1 or 2 loss mediated tumorigenesis process needs other factors. In this study, MSCV (Murine Stem Cell Virus) retroviral mediated insertional mutagenesis approach was used in conditional Brca2 mutant mES (mouse embryonic stem) cells to identify and characterize genetic interactors of BRCA2. One of the factors identified in this screen is brain and reproductive organ expressed protein BRE. BRE is an anti-apoptotic protein and also a component of the BRCA1-A complex that specifically recognizes ‘Lys-63′-linked ubiquitinated histones H2A and H2AX at DNA lesions sites. Overexpression of BRE results into perturbation of DNA damage induced G1/S cell cycle checkpoint by preventing DNA damage induced degradation of CDC25a phosphatase. Further it was found that the stabilization of CDC25a phosphatase in BRE overexpressing cells on DNA damage is mediated via ubiquitin-specific-processing protease 7 (USP7). We are now examining the expression of BRE and CDC25a in BRCA2 deficient tumors. Citation Format: Kajal Biswas, Suhwan Chang, Subha Philip, Shyam K. Sharan. BRE: a genetic interactor of BRCA2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3952. doi:10.1158/1538-7445.AM2015-3952

Changes and significance of ubiquitination of Foxp3 protein during the acute phase of Kawasaki dis-ease

Objective To investigate the changes and significance of ubiquitination of Foxp3 protein during the acute phage of Kawasaki disease (KD). Methods Forty-eight children with KD and twenty-eight age-matched healthy children were recruited in this study. Co-immunoprecipitation and Western blot assays were performed to determine the poly-ubiquitination status of Foxp3 in CD4+ T cells. The percentages of CD4+ CD25highFoxp3+ regulatory T cells (Treg) and the levels of IL-10, transforming growth factor-β (TGF-β), cytotoxic T lymphocyte-associated protein-4 (CTLA4), STIP1 homology and U-Box containing protein 1 (STUB1), heat shock protein 70 (HSP70), ubiquitin-specific-processing protease 7 (USP7) and pSTAT3 were analyzed by flow cytometry analysis. Quantitative real-time PCR was used to evaluate the transcription levels of TLR1-10, IL-1R1, IL-1RAP, IL-6Rα, gp130, TNFR1, MyD88 and RIP1 in CD4+ T cells. Plasma concentrations of IL-1β, IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. Results (1) The percentages of Treg cells and the levels of IL-10, TGF-β, CTLA4 and forkhead box P3 (Foxp3) in patients with acute KD were lower than those of healthy subjects (P 0.05). Conclusion Hyper-ubiquitination of Foxp3 protein might be involved in the immune dysfunction during Kawasaki disease. Key words: Kawasaki disease; Foxp3; Ubiquitination; Regulatory T cells; Immune regulation

Hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells of marine medaka Oryzias melastigma

Hypoxia is a major global problem that impairs reproductive functions and reduces the quality and quantity of gametes and the fertilization success of marine fish. Nevertheless, the detailed molecular mechanism underlying hypoxia-induced female reproductive impairment remains largely unknown. There is increasing evidence that miRNA is vital in regulating ovarian functions and is closely associated with female fertility in humans. Certain miRNAs that regulate apoptotic genes can be induced by hypoxia, resulting in cell apoptosis. Using primary ovarian follicular cells of the marine medaka, Oryzias melastigma, as a model, we investigated the response of miR-210 to hypoxic stress in ovarian tissues to see if it would interrupt reproductive functions. A significant induction of miR-210 was found in primary ovarian follicular cells exposed to hypoxia, and gene ontology analysis further highlighted the potential roles of miR-210 in cell proliferation, cell differentiation, and cell apoptosis. A number of miR-210 target apoptotic genes, including Deleted in liver cancer 1 protein (DLC1), STE20-like serine/threonine-protein kinase (SLK), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), RNA binding motif protein 25 (RBM25), and Ubiquitin-specific-processing protease 7 (USP7), were identified. We further showed that ectopic expression of miR-210 would result in down-regulation of these apoptotic genes. On the other hand, the inhibition of miR-210 promoted apoptotic cell death and the expression of apoptotic marker – caspase 3 in follicular cells under hypoxic treatment, supporting the regulatory role of miR-210 in ovarian cell apoptosis. This study provides new insights on how hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells in fish, which is fundamentally important in environmental sciences and reproductive biology.

Usp7 protects genomic stability by regulating Bub3.

USP7 (Ubiquitin Specific processing Protease-7) is a deubiquitinase which, over the past decade emerged as a critical regulator of cellular processes. Deregulation of USP7 activity has been linked to cancer, making USP7 inhibition an appealing anti-cancer strategy. The identification of novel USP7 substrates and additional USP7-dependent cellular activities will broaden our knowledge towards potential clinical application of USP7 inhibitors. Results presented in this study uncover a novel and pivotal function of USP7 in the maintenance of genomic stability. Upon USP7 depletion we observed prolonged mitosis and mitotic abnormalities including micronuclei accumulation, lagging chromosomes and karyotype instability. Inhibition of USP7 with small molecule inhibitors stabilizes cyclin B and causes mitotic abnormalities. Our results suggest that these USP7-dependent effects are mediated by decreased levels of spindle assembly checkpoint (SAC) component Bub3, which we characterized as an interacting partner and substrate of USP7. In silico analysis across the NCI-60 panels of cell lines supports our results where lower levels of USP7 strongly correlate with genomic instability. In conclusion, we identified a novel role of USP7 as regulator of the SAC component Bub3 and genomic stability.

The landscape of somatic mutations in epigenetic regulators across 1,000 paediatric cancer genomes

Studies of paediatric cancers have shown a high frequency of mutation across epigenetic regulators. Here we sequence 633 genes, encoding the majority of known epigenetic regulatory proteins, in over 1,000 paediatric tumours to define the landscape of somatic mutations in epigenetic regulators in paediatric cancer. Our results demonstrate a marked variation in the frequency of gene mutations across 21 different paediatric cancer subtypes, with the highest frequency of mutations detected in high-grade gliomas, T-lineage acute lymphoblastic leukaemia and medulloblastoma, and a paucity of mutations in low-grade glioma and retinoblastoma. The most frequently mutated genes are H3F3A, PHF6, ATRX, KDM6A, SMARCA4, ASXL2, CREBBP, EZH2, MLL2, USP7, ASXL1, NSD2, SETD2, SMC1A and ZMYM3. We identify novel loss-of-function mutations in the ubiquitin-specific processing protease 7 (USP7) in paediatric leukaemia, which result in decreased deubiquitination activity. Collectively, our results help to define the landscape of mutations in epigenetic regulatory genes in paediatric cancer and yield a valuable new database for investigating the role of epigenetic dysregulations in cancer.