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Myasthenia gravis (MG) presents with symptoms that significantly affect patients' daily lives. Long‐term MG therapies may lead to substantial side effects, predominantly due to prolonged immune suppression. Sirt6, which plays a vital role in maintaining cellular homeostasis and is recognised for its involvement in cytokine production in immune cells, has not yet been explored in relation to MG. PBMCs and CD4+ T cells were isolated from blood samples. RT‐qPCR, western blot and ELISA were used to assess the expression of target genes and proteins. Flow cytometry was used to identify the subsets of T helper cells. Co‐IP was conducted to investigate the interaction between USP9X and Sirt6. Finally, the experimental autoimmune myasthenia gravis (EAMG) model was established. In MG patients, Sirt6 levels were downregulated compared to healthy controls. Sirt6 overexpression led to a reduction in Th1 and Th17 cell populations while augmenting Treg cells in PBMCs. USP9X interacted with Sirt6, leading to its deubiquitination and stabilisation. Elevated Sirt6 levels subsequently mitigated symptoms in the EAMG model. The stabilisation of Sirt6, mediated by USP9X, has been found to relieve symptoms of EAMG by influencing the subtypes of T helper cells. This highlights the promising potential of Sirt6 as a viable therapeutic target in the treatment of MG. [ABSTRACT FROM AUTHOR]

As a pattern recognition receptor, Toll-like receptor 4 (TLR4) is crucial for the development and progression of acute kidney injury (AKI). This study aims to explore whether the deubiquitinase Usp9x influences the TLR4/NF-B pathway to cause sepsis-induced acute kidney injury (S-AKI). The model of AKI was established in Sprague-Dawley rats using the cecal ligation and puncture (CLP) method, while renal tubular epithelial cell NRK-52E was stimulated with lipopolysaccharide (LPS) in vitro. All plasmids were transfected into NRK-52E cells according to the indicated group. The deubiquitinase of TLR4 was predicted by the online prediction software Ubibrowser. Subsequently, Western blot and Pearson correlation analysis identified Usp9x protein as a potential candidate. Co-IP analysis verified the interaction between TLR4 and Usp9x. Further research revealed that overexpression of Usp9x inhibited degradation of TLR4 protein by downregulating its ubiquitination modification levels. Both in vivo and in vitro experiments observed that interference with Usp9x effectively alleviated the inflammatory response and apoptosis of renal tubular epithelial cells (RTECs) induced by CLP or LPS, whereas overexpression of TLR4 reversed this situation. Transfection with sh-Usp9x in NRK-52E cells suppressed the expression of proteins associated with the TLR4/NF-κB pathway induced by LPS. Moreover, the overexpression of TLR4 reversed the effect of sh-Usp9x transfection. Therefore, the deubiquitinase Usp9x interacts with TLR4, leading to the upregulation of its expression through deubiquitination modification, and the activation of the TLR4/NF-κB signaling pathway, thereby promoting inflammation and apoptosis in renal tubular epithelial cells and contributing to sepsis-induced acute kidney injury. [ABSTRACT FROM AUTHOR]

Introduction: USP9X has been associated with neurodevelopmental disorders due to its role in synaptic development and neural function. This study aimed to compare USP9X and TGF-β levels in children with autism and healthy controls, and explore their relationship with autism severity. Methods: Serum USP9X and TGF-β levels were measured in 41 healthy control children (aged 3–12 years) and 41 children with autism. Results: Our study revealed a significant increase in USP9X levels (p=0.001) among children with autism compared to controls. However, TGF-β levels showed no significant difference between the two groups. Furthermore, we observed a positive correlation between difficulty in making eye contact subscale and blood levels of both USP9X and TGF-β. Conclusions: This study is the first to compare serum USP9X levels in children with autism to healthy controls. Our findings suggest USP9X's potential role in autism development, emphasizing the need for further research on its involvement in neurodevelopmental processes. [ABSTRACT FROM AUTHOR]

Background: Tauopathies, including Alzheimer's disease, are characterized by the pathological aggregation of tau protein, which is strongly linked to dysregulation of the autophagy-lysosomal degradation pathway. However, therapeutic strategies targeting this pathway remain limited. Methods: We used both in vitro and in vivo models to investigate the role of Raptor in tau pathology. Knockdown of Raptor was performed to assess its impact on mTORC1 activation, autophagy, and tau accumulation. The relationship between USP9X and Raptor was also examined. Pharmacological inhibition of USP9X with WP1130 was employed to further confirm the involvement of the USP9X-Raptor-mTORC1 axis in tau degradation. Results: Elevated Raptor levels in the hippocampus of P301S mice led to hyperactivation of mTORC1, impairing autophagy flux. Knockdown of Raptor effectively suppressed mTORC1 activation, promoted autophagy, and mitigated the accumulation of tau and its phosphorylated isoforms. This reduction in tau pathology was accompanied by decreased neuronal loss in the hippocampus, amelioration of synaptic damage, and improvement in cognitive function. The increased Raptor protein observed in the hippocampus of P301S mice was likely attributable to elevated USP9X content, which enhanced Raptor deubiquitination and protected it from proteasomal degradation. Pharmacological inhibition of USP9X with WP1130 in vitro effectively suppressed Raptor, promoted autophagy, and accelerated the degradation of tau and phosphorylated tau. Conclusions: Our findings highlight Raptor and USP9X as promising molecular targets for therapeutic intervention in tauopathies. Targeting the USP9X-Raptor-mTORC1 axis may provide a novel strategy for promoting autophagy and mitigating tau pathology in Alzheimer's disease and other tauopathies. [ABSTRACT FROM AUTHOR]

Background: MutT homolog 1 (MTH1) sanitizes oxidized dNTP pools to promote the survival of cancer cells and its expression is frequently upregulated in cancers. Polyubiquitination stabilizes MTH1 to facilitate the proliferation of melanoma cells, suggesting the ubiquitin system controls the stability and function of MTH1. However, whether ubiquitination regulates MTH1 in gastric cancers has not been well defined. This study aims to investigate the interaction between MTH1 and a deubiquitinase, USP9X, in regulating the proliferation, survival, migration, and invasion of gastric cancer cells. Methods: The interaction between USP9X and MTH1 was evaluated by co-immunoprecipitation (co-IP) in HGC-27 gastric cancer cells. siRNAs were used to interfere with USP9X expression in gastric cancer cell lines HGC-27 and MKN-45. MTT assays were carried out to examine the proliferation, propidium iodide (PI) and 7-AAD staining assays were performed to assess the cell cycle, Annexin V/PI staining assays were conducted to examine the apoptosis, and transwell assays were used to determine the migration and invasion of control, USP9X-deficient, and USP9X-deficient plus MTH1-overexpressing HGC-27 and MKN-45 gastric cancer cells. Results: Co-IP data show that USP9X interacts with and deubiquitinates MTH1. Overexpression of USP9X elevates MTH1 protein level by downregulating its ubiquitination, while knockdown of USP9X has the opposite effect on MTH1. USP9X deficiency in HGC-27 and MKN-45 cells causes decreased proliferation, cell cycle arrest, extra apoptosis, and defective migration and invasion, which could be rescued by excessive MTH1. Conclusion: USP9X interacts with and stabilizes MTH1 to promote the proliferation, survival, migration and invasion of gastric cancer cells. [ABSTRACT FROM AUTHOR]

Background: Radioresistance is a key clinical constraint on the efficacy of radiotherapy in lung cancer patients. REV1 DNA directed polymerase (REV1) plays an important role in repairing DNA damage and maintaining genomic stability. However, its role in the resistance to radiotherapy in lung cancer is not clear. This study aims to clarify the role of REV1 in lung cancer radioresistance, identify the intrinsic mechanisms involved, and provide a theoretical basis for the clinical translation of this new target for lung cancer treatment. Methods: The effect of targeting REV1 on the radiosensitivity was verified by in vivo and in vitro experiments. RNA sequencing (RNA-seq) combined with nontargeted metabolomics analysis was used to explore the downstream targets of REV1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify the content of specific amino acids. The coimmunoprecipitation (co-IP) and GST pull-down assays were used to validate the interaction between proteins. A ubiquitination library screening system was constructed to investigate the regulatory proteins upstream of REV1. Results: Targeting REV1 could enhance the radiosensitivity in vivo, while this effect was not obvious in vitro. RNA sequencing combined with nontargeted metabolomics revealed that the difference result was related to metabolism, and that the expression of glycine, serine, and threonine (Gly/Ser/Thr) metabolism signaling pathways was downregulated following REV1 knockdown. LC-MS/MS demonstrated that REV1 knockdown results in reduced levels of these three amino acids and that cystathionine γ-lyase (CTH) was the key to its function. REV1 enhances the interaction of CTH with the E3 ubiquitin ligase Rad18 and promotes ubiquitination degradation of CTH by Rad18. Screening of the ubiquitination compound library revealed that the ubiquitin-specific peptidase 9 X-linked (USP9X) is the upstream regulatory protein of REV1 by the ubiquitin-proteasome system, which remodels the intracellular Gly/Ser/Thr metabolism. Conclusion: USP9X mediates the deubiquitination of REV1, and aberrantly expressed REV1 acts as a scaffolding protein to assist Rad18 in interacting with CTH, promoting the ubiquitination and degradation of CTH and inducing remodeling of the Gly/Ser/Thr metabolism, which leads to radioresistance. A novel inhibitor of REV1, JH-RE-06, was shown to enhance lung cancer cell radiosensitivity, with good prospects for clinical translation. [ABSTRACT FROM AUTHOR]

Axenfeld–Rieger anomaly (ARA) is a specific ocular disorder that is frequently associated with other systemic abnormalities. PITX2 and FOXC1 variants explain the majority of individuals with Axenfeld–Rieger syndrome (ARS) but leave ~30% unsolved. Here, we present pathogenic/likely pathogenic variants in nine families with ARA/ARS or similar phenotypes affecting five different genes/regions. USP9X and JAG1 explained three families each. USP9X was recently linked with syndromic cognitive impairment that includes hearing loss, dental defects, ventriculomegaly, Dandy–Walker malformation, skeletal anomalies (hip dysplasia), and other features showing a significant overlap with FOXC1-ARS. Anterior segment anomalies are not currently associated with USP9X, yet our cases demonstrate ARA, congenital glaucoma, corneal neovascularization, and cataracts. The identification of JAG1 variants, linked with Alagille syndrome, in three separate families with a clinical diagnosis of ARA/ARS highlights the overlapping features and high variability of these two phenotypes. Finally, intragenic variants in CDK13, BCOR, and an X chromosome deletion encompassing HCCS and AMELX (linked with ocular and dental anomalies, correspondingly) were identified in three additional cases with ARS. Accurate diagnosis has important implications for clinical management. We suggest that broad testing such as exome sequencing be applied as a second-tier test for individuals with ARS with normal results for PITX2/FOXC1 sequencing and copy number analysis, with attention to the described genes/regions. [ABSTRACT FROM AUTHOR]

Simple Summary: Radiotherapy constitutes an important therapeutic option for prostate cancer. However, prostate cancer cells often acquire resistance during cancer progression, limiting the cytotoxic effects of radiotherapy. Among factors regulating sensitivity to radiotherapy are members of the Bcl-2 protein family, known to regulate apoptosis at the mitochondrial level. We demonstrate that protein levels of deubiquitinase USP9x and anti-apoptotic Mcl-1 increased during prostate cancer progression. Downregulation of Mcl-1 or USP9x levels improved the response of prostate cancer cells to radiotherapy. Moreover, radiotherapy itself was able to regulate Mcl-1 protein stability in prostate cancer cells. Background: Radiotherapy constitutes an important therapeutic option for prostate cancer. However, prostate cancer cells often acquire resistance during cancer progression, limiting the cytotoxic effects of radiotherapy. Among factors regulating sensitivity to radiotherapy are members of the Bcl-2 protein family, known to regulate apoptosis at the mitochondrial level. Here, we analyzed the role of anti-apoptotic Mcl-1 and USP9x, a deubiquitinase stabilizing Mcl-1 protein levels, in prostate cancer progression and response to radiotherapy. Methods: Changes in Mcl-1 and USP9x levels during prostate cancer progression were determined by immunohistochemistry. Neutralization of Mcl-1 and USP9x was achieved by siRNA-mediated knockdown. We analyzed Mcl-1 stability after translational inhibition by cycloheximide. Cell death was determined by flow cytometry using an exclusion assay of mitochondrial membrane potential-sensitive dye. Changes in the clonogenic potential were examined by colony formation assay. Results: Protein levels of Mcl-1 and USP9x increased during prostate cancer progression, and high protein levels correlated with advanced prostate cancer stages. The stability of Mcl-1 reflected Mcl-1 protein levels in LNCaP and PC3 prostate cancer cells. Moreover, radiotherapy itself affected Mcl-1 protein turnover in prostate cancer cells. Particularly in LNCaP cells, the knockdown of USP9x expression reduced Mcl-1 protein levels and increased sensitivity to radiotherapy. Conclusion: Posttranslational regulation of protein stability was often responsible for high protein levels of Mcl-1. Moreover, we demonstrated that deubiquitinase USP9x as a factor regulating Mcl-1 levels in prostate cancer cells, thus limiting cytotoxic response to radiotherapy. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIVrelated malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIVrelated malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X. [ABSTRACT FROM AUTHOR]

Alveolar epithelial cells (AECs) function as a vital defense barrier avoiding the invasion of exogenous agents and preserving the functional and structural integrity of lung tissues, while damage/breakdown of this airway epithelial barrier is frequently associated with the pathogenesis of acute lung injury (ALI). NOD‐like receptor family, pyrindomain‐containing 3 (NLRP3) inflammasome activation‐associated pyroptosis is involved in the development of ALI. Yet, how the activity of NLRP3 inflammasome is regulated in the context of ALI remains unknown. Herein we hypothesized that USP9X, an important deubiquitinase, participates in modulating the activation of NLRP3 inflammasome, thereby affecting the phenotypes in a lipopolysaccharide (LPS)‐stimulated AEC model. Human pulmonary AECs were subjected to LPS/adenosine triphosphate (ATP) treatment to induce NLRP3 inflammasome activation and cell pyroptosis. Knockdown and overexpression of USP9X were applied to validate the function of USP9X. Inhibitors of proteinase and protein synthesis, as well as approach of co‐immunoprecipitation coupled with Western blot, were utilized to explore the molecular mechanism. LPS/ATP challenge resulted in pronouncedly increased pyroptosis of AECs, activation of NLRP3 inflammasome and release of interleukin (IL)‐1β and IL‐18 cytokines, while downregulation of USP9X could reverse these alterations. USP9X was found to have marked impact on NLRP3 protein instead of mRNA level. Furthermore, increased ubiquitination of NLRP3 was observed upon downregulating USP9X. Additionally, the inhibitory effect of USP9X downregulation was reversed by NLRP3 overexpression, while the promoting impact of USP9X overexpression was dampened by NLRP3 inhibitor in terms of cell pyroptosis and cytokine secretion. USP9X modulated the activity of NLRP3 inflammasome and pyroptosis of AECs via its deubiquitination function. [ABSTRACT FROM AUTHOR]

Pathological angiogenesis of liver sinusoidal endothelial cells (LSEC) plays a crucial role in the progression of metabolic dysfunction-associated steatohepatitis (MASH)-induced liver fibrosis. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) have shown promising therapeutic potential against MASH. This study aimed to investigate the impact of MSC-sEV on LSEC angiogenesis and elucidate the underlying molecular mechanisms. The effects of MSC-sEV on LSEC angiogenesis were evaluated in Tumor Necrosis Factor- alpha (TNF-α)-treated LSECs in vitro and in Methionine and Choline Deficient Diet (MCD)-induced MASH mice in vivo. Herein, we found that MSC-sEV effectively suppressed LSEC angiogenesis by targeting the angiogenesis marker Angiogenin 2 (Ang-2) in both TNF-α-treated LSECs and MASH mice. Gene manipulation experiments revealed that the primary mechanism by which MSC-sEV inhibited LSEC angiogenesis was through the modulation of nuclear factor kappa B inhibitor alpha (IκBα) / nuclear factor kappa B (NF-κB) / Ang-2 pathway. Additionally, mass spectrometry and co-immunoprecipitation (Co-IP) data suggested that MSC-sEV delivered the ubiquitin specific peptidase 9 X-linked (USP9X) protein to LSECs, leading to enhanced IκBα deubiquitination and NF-κB in activation, ultimately resulting in the inhibition of Ang-2-mediated LSEC angiogenesis. Knockdown of USP9X attenuated the regulatory effects of MSC-sEV on Ang-2 expression, LSEC angiogenesis, and the progression of MASH. In conclusion, our findings indicate that USP9X delivered via MSC-sEV can suppress LSEC angiogenesis and alleviate MASH-induced liver fibrosis through the IκBα/NF-κB/Ang-2 signaling pathway. [Display omitted] • MSC-sEV suppresses LSEC angiogenesis and alleviates MASH in mice. • MSC-sEV-transferred USP9X is responsible for reducing LSEC angiogenesis. • USP9X inhibits LSEC angiogenesis via IκBα/NF-κB/Ang-2 pathway. [ABSTRACT FROM AUTHOR]

The ubiquitin‐specific peptidase 9 X‐linked (USP9X) is one of the highly conserved members belonging to the ubiquitin‐specific proteases (USPs) family, which has been reported to control substrates‐mediated biological functions through deubiquitinating and stabilizing substrates. Here, we have found that TGFBR2, the type II receptor of the transforming growth factor beta (TGF‐β) signaling pathway, is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells (GCs). Mechanically, USP9X positively influences the expression of TGFBR2 at different levels through two independent ways: (i) directly targets and deubiquitinates TGFBR2, which maintains the protein stability of TGFBR2 through avoiding degradation mediated by ubiquitin‐proteasome system; (ii) indirectly maintains TGFBR2 messenger RNA (mRNA) expression via SMAD4/miR‐143 axis. Specifically, SMAD4, another substrate of USP9X, acts as a transcription factor and suppresses miR‐143 which inhibits the mRNA level of TGFBR2 by directly binding to its 3′‐untranslated region. Functionally, the maintenance of TGFBR2 by USP9X activates the TGF‐β signaling pathway, which further represses GC apoptosis. Our study highlights a functional micro‐regulatory network composed of deubiquitinase (USP9X), small noncoding RNA (miR‐143) and the TGF‐β signaling pathway, which plays a crucial role in the regulation of GC apoptosis and female fertility. [ABSTRACT FROM AUTHOR]

Breast cancer is one of the most common malignancies in women worldwide. Triple‐negative breast cancer (TNBC) is a highly aggressive and metastatic subtype that has the characteristics of easy recurrence, poor prognosis as well as lack of targeted therapeutics. Snail1, a key factor regulating epithelial–mesenchymal transition (EMT) process, contributing to metastasis and chemoresistance in human cancers. However, the molecular mechanism of Snail1 stabilization in cancers is not fully understood. Here, we demonstrate that the deubiquitinating enzyme USP9X deubiquitinates and stabilizes Snail1, thereby promoting metastasis and chemoresistance. The depletion and pharmacological inhibition of USP9X by WP1130, an inhibitor of USP9X, downregulate endogenous Snail1 protein, inhibit cell migration, invasion, metastasis, and increase cellular sensitivity to cisplatin and paclitaxel both in vitro and in vivo, whereas the reconstitution of Snail1 in cells with USP9X depletion at least partially reverses these phenotypes. Overall, our study establishes the USP9X‐Snail1 axis as an important regulatory mechanism of breast cancer metastasis and chemoresistance and provides a rationale for potential therapeutic interventions in the treatment of TNBC. [ABSTRACT FROM AUTHOR]

Here, we describe a novel interaction between the RNA helicase DDX3 and the deubiquitinase ubiquitin‐specific peptidase 9 X‐linked (USP9X) in human cells. Domain mapping studies reveal that the C‐terminal region of DDX3 interacted with the N terminus of USP9X. USP9X was predominantly localized in the cytoplasm where the interaction between DDX3 and USP9X occurred. USP9X was not visibly enriched in cytoplasmic stress granules (SGs) under oxidative stress conditions, whereas overexpression of GFP‐DDX3 induced SG formation and recruited USP9X to SGs in HeLa cells. Luciferase reporter assays showed that depletion of USP9X had no significant effect on DDX3‐mediated translation. Given that DDX3 is not ubiquitinated upon ubiquitin overexpression, it is unlikely that DDX3 serves as a substrate of USP9X. Importantly, we found that ubiquitinated MCL1 was accumulated upon depletion of USP9X and/or DDX3 in MG132‐treated cells, suggesting that USP9X and DDX3 play a role in regulating MCL1 protein stability and anti‐apoptotic function. This study indicates that DDX3 exerts anti‐apoptotic effects probably by coordinating with USP9X in promoting MCL1 deubiquitination. [ABSTRACT FROM AUTHOR]

Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy with a poor prognosis. Ubiquitin-specific peptidase 9, X-linked (USP9X), has been associated with multiple physiological pathways and regulates various cellular activities. In this study, we explored the role of USP9X in MCL in vitro and in vivo. USP9X was verified to be increased in peripheral blood mononuclear cells (PBMCs) of MCL patients and MCL cells. Moreover, CCND1 and SOX11 were also upregulated in PBMCs of MCL patients. The positive correlation between USP9X and CCND1, USP9X and SOX11, and CCND1 and SOX11 were identified. Further, USP9X overexpression and knockdown were performed in MCL cells. We proved that USP9X overexpression promoted proliferation and cell cycle and suppressed cell apoptosis in MCL cells. Upregulation of angiogenesis and cell migration were induced by USP9X overexpression in MCL cells. However, the USP9X knockdown showed opposite effects. In addition, USP9X was discovered to decrease Cyclin D1 (CCND1)-mediated SOX11 expression in MCL cells. We demonstrated that SOX11 overexpression reversed USP9X knockdown-mediated angiogenesis in MCL cells. Besides, tumor formation was inhibited by USP9X knockdown in mice in vivo. In conclusion, these results revealed that USP9X promoted tumor angiogenesis in MCL via increasing CCND1-mediated SOX11. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is a breast cancer subtype that lacks targeted treatment options. The activation of the Notch developmental signaling pathway, which is a feature of TNBC, results in the secretion of proinflammatory cytokines and the recruitment of protumoral macrophages to the tumor microenvironment. While the Notch pathway is an obvious therapeutic target, its activity is ubiquitous, and predictably, anti-Notch therapies are burdened with significant on-target side effects. Previously, we discovered that, under conditions of cellular stress commonly found in the tumor microenvironment, the deubiquitinase USP9x forms a multiprotein complex with the pseudokinase tribbles homolog 3 (TRB3) that together activate the Notch pathway. Herein, we provide preclinical studies that support the potential of therapeutic USP9x inhibition to deactivate Notch. Using a murine TNBC model, we show that USP9x knockdown abrogates Notch activation, reducing the production of the proinflammatory cytokines, C-C motif chemokine ligand 2 (CCL2) and interleukin-1 beta (IL-1β). Concomitant with these molecular changes, a reduction in tumor inflammation, the augmentation of antitumor immune response, and the suppression of tumor growth were observed. The pharmacological inhibition of USP9x using G9, a partially selective, small-molecule USP9x inhibitor, reduced Notch activity, remodeled the tumor immune landscape, and reduced tumor growth without associated toxicity. Proving the role of Notch, the ectopic expression of the activated Notch1 intracellular domain rescued G9-induced effects. This work supports the potential of USP9x inhibition to target Notch in metabolically vulnerable tissues like TNBC, while sparing normal Notch-dependent tissues. [ABSTRACT FROM AUTHOR]

Background: Cholangiocarcinoma represents the second most common primary liver malignancy. The incidence rate has constantly increased over the last decades. Cholangiocarcinoma silent nature limits early diagnosis and prevents efficient treatment. Methods: Immunoblotting and immunohistochemistry were used to assess the expression profiling of USP9X and EGLN3 in cholangiocarcinoma patients. ShRNA was used to silence gene expression. Cell apoptosis, cell cycle, CCK8, clone formation, shRNA interference and xenograft mouse model were used to explore biological function of USP9X and EGLN3. The underlying molecular mechanism of USP9X in cholangiocarcinoma was determined by immunoblotting, co-immunoprecipitation and quantitative real time PCR (qPCR). Results: Here we demonstrated that USP9X is downregulated in cholangiocarcinoma which contributes to tumorigenesis. The expression of USP9X in cholangiocarcinoma inhibited cell proliferation and colony formation in vitro as well as xenograft tumorigenicity in vivo. Clinical data demonstrated that expression levels of USP9X were positively correlated with favorable clinical outcomes. Mechanistic investigations further indicated that USP9X was involved in the deubiquitination of EGLN3, a member of 2-oxoglutarate and iron-dependent dioxygenases. USP9X elicited tumor suppressor role by preventing degradation of EGLN3. Importantly, knockdown of EGLN3 impaired USP9X-mediated suppression of proliferation. USP9X positively regulated the expression level of apoptosis pathway genes de through EGLN3 thus involved in apoptosis of cholangiocarcinoma. Conclusion: These findings help to understand that USP9X alleviates the malignant potential of cholangiocarcinoma through upregulation of EGLN3. Consequently, we provide novel insight into that USP9X is a potential biomarker or serves as a therapeutic or diagnostic target for cholangiocarcinoma. [ABSTRACT FROM AUTHOR]

Since its discovery forty years ago, protein ubiquitination has been an ever-expanding field. Virtually all biological processes are controlled by the post-translational conjugation of ubiquitin onto target proteins. In addition, since ubiquitin controls substrate degradation through the action of hundreds of enzymes, many of which represent attractive therapeutic candidates, harnessing the ubiquitin system to reshape proteomes holds great promise for improving disease outcomes. Among the numerous physiological functions controlled by ubiquitin, the cell cycle is among the most critical. Indeed, the discovery that the key drivers of cell cycle progression are regulated by the ubiquitin-proteasome system (UPS) epitomizes the connection between ubiquitin signaling and proliferation. Since cancer is a disease of uncontrolled cell cycle progression and proliferation, targeting the UPS to stop cancer cells from cycling and proliferating holds enormous therapeutic potential. Ubiquitination is reversible, and ubiquitin is removed from substrates by catalytic proteases termed deubiquitinases or DUBs. While ubiquitination is tightly linked to proliferation and cancer, the role of DUBs represents a layer of complexity in this landscape that remains poorly captured. Due to their ability to remodel the proteome by altering protein degradation dynamics, DUBs play an important and underappreciated role in the cell cycle and proliferation of both normal and cancer cells. Moreover, due to their enzymatic protease activity and an open ubiquitin binding pocket, DUBs are likely to be important in the future of cancer treatment, since they are among the most druggable enzymes in the UPS. In this review we summarize new and important findings linking DUBs to cell cycle and proliferation, as well as to the etiology and treatment of cancer. We also highlight new advances in developing pharmacological approaches to attack DUBs for therapeutic benefit. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific proteases (USPs) play important roles in the regulation of many cancer-related biological processes. USPs copy number variation (CNVs) may affect the risk and prognosis of colorectal cancer (CRC). We detected CNVs of USPs genes in 468 matched CRC patients and controls, estimated the associations between the USPs genes CNVs and CRC risk and prognosis and their interactions with environmental factors on CRC risk. Finally, we generated five CRC risk predictive models with different CNVs patterns combining with environmental factors (EF). We identified significant association between CYLD deletion and CRC risk (ORadj = 4.18, 95% CI: 2.03–8.62), significant association between USP9X amplification and CRC risk (ORadj = 2.30, 95% CI: 1.48–3.57), and significant association between USP11 deletion and CRC risk (ORadj = 3.49, 95% CI: 1.49–8.64). There were significant gene-environment and gene–gene interactions on CRC risk. The area under the receiver operating characteristic curve (AUC) of EF + SIG (deletion of CYLD and USP11, amplification of USP9X) model was significantly larger than any other models (AUC = 0.75, 95% CI: 0.74–0.77). We did not identify significant associations between CNVs of the three genes and CRC prognosis. CNVs of CYLD, USP9X, and USP11 are significantly associated with the risk of CRC. Gene-gene and gene–environment interactions might also play an important role in the development of CRC. [ABSTRACT FROM AUTHOR]

Protein ubiquitination, one of the most significant post-translational modifications, plays an important role in controlling the proteins activity in diverse cellular processes. The reversible process of protein ubiquitination, known as deubiquitination, has emerged as a critical mechanism for maintaining cellular homeostasis. The deubiquitinases (DUBs), which participate in deubiquitination process are increasingly recognized as potential candidates for drug discovery. Among these DUBs, ubiquitin-specific protease 9× (USP9X), a highly conserved member of the USP family, exhibits versatile functions in various cellular processes, including the regulation of cell cycle, protein endocytosis, apoptosis, cell polarity, immunological microenvironment, and stem cell characteristics. The dysregulation and abnormal activities of USP9X are influenced by intricate cellular signaling pathway crosstalk and upstream non-coding RNAs. The complex expression patterns and controversial clinical significance of USP9X in cancers suggest its potential as a prognostic biomarker. Furthermore, USP9X inhibitors has shown promising antitumor activity and holds the potential to overcome therapeutic resistance in preclinical models. However, a comprehensive summary of the role and molecular functions of USP9X in cancer progression is currently lacking. In this review, we provide a comprehensive delineation of USP9X participation in numerous critical cellular processes, complicated signaling pathways within the tumor microenvironment, and its potential translational applications to combat therapeutic resistance. By systematically summarizing the updated molecular mechanisms of USP9X in cancer biology, this review aims to contribute to the advancement of cancer therapeutics and provide essential insights for specialists and clinicians in the development of improved cancer treatment strategies. [ABSTRACT FROM AUTHOR]

In order to prevent the deleterious effects of genotoxic agents, cells have developed complex surveillance mechanisms and DNA repair pathways that allow them to maintain genome integrity. The ubiquitinspecific protease 9X (USP9X) contributes to genome stability during DNA replication and chromosome segregation. Depletion of USP9X leads to DNA double-strand breaks, some of which are triggered by replication fork collapse. Here, we identify USP9X as a novel regulator of homologous recombination (HR) DNA repair in human cells. By performing cellular HR reporter, irradiation-induced focus formation and colony formation assays, we show that USP9X is required for efficient HR. Mechanistically, we show USP9X is important to sustain the expression levels of key HR factors, namely BRCA1 and RAD51 through a non-canonical regulation of their mRNA abundance. Intriguingly, we find that the contribution of USP9X to BRCA1 and RAD51 expression is independent of its known catalytic activity. Thus, this work identifies USP9X as a regulator of HR, demonstrates a novel mechanism by which USP9X can regulate protein levels, and provides insights in to the regulation of BRCA1 and RAD51 mRNA. [ABSTRACT FROM AUTHOR]

The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function. [ABSTRACT FROM AUTHOR]

BRCA1, a multifunctional protein with an important role in DNA double‐strand break repair by homologous recombination (HR), is subjected to ubiquitin‐dependent degradation. To date, several E3 ubiquitin ligases have been identified to govern BRCA1 stability, but the deubiquitinase that counteracts its turnover remains undefined. In this study, we report that the ubiquitin‐specific protease 9X (USP9X) is a bona fide deubiquitinase for BRCA1 in human cancer cells. Reciprocal immunoprecipitation assays demonstrated that USP9X interacted with BRCA1. Depletion of USP9X by short interfering RNAs or inhibition of USP9X by the small‐molecular inhibitor WP1130 significantly reduced BRCA1 protein abundance, without affecting its mRNA levels. In contrast, overexpression of wild‐type USP9X, but not its deubiquitinase activity‐defective mutant (C1566S), resulted in an upregulation of BRCA1 protein levels. Moreover, USP9X depletion reduced the half‐life of BRCA1, accompanied by an increase in its ubiquitination. HR assays showed that knockdown of USP9X significantly reduced HR efficiency, which was partially rescued by reintroduction of BRCA1 into USP9X‐depleted cells. In support of these findings, USP9X knockdown significantly enhanced sensitivity to PARP inhibitor Olaparib and methyl methanesulfonate (MMS). Collectively, these results establish USP9X as a deubiquitinase for BRCA1 and reveal a previously unrecognized role of USP9X in the regulation of HR repair and the sensitivity of cancer cells to DNA‐damaging agents. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC) is one of the most common cancers and the second leading cause of cancer-related death worldwide. Long noncoding RNAs (lncRNAs) are associated with GC development and progression. However, the functional roles and underlying mechanism of LINC01433 on GC progression remain elusive. Methods: Firstly, the expression of LINC01433 was examined in 76 pairs of primary GC and corresponding adjacent non-tumorous tissues. Next, overexpression and knockdown experiments were conducted in GC cells to explore the effect of LINC01433 on the malignant behaviors of GC cells. Then, the interaction between LINC01433 and YAP was detected by RNA immunoprecipitation (RIP) and RNA pull-down assays. Results: We found that LINC01433 was significantly upregulated in GC tissues and cell lines and correlated with poor prognosis. Through gain- and loss-of-function experiments, we demonstrated that LINC01433 promoted proliferation, migration, invasion and chemotherapy resistance in GC cells. Further mechanistic investigation revealed that LINC01433 could stabilize oncoprotein YAP through enhancing the interaction between deubiquitinase USP9X and YAP. LINC01433 decreased the phosphorylation of YAP via suppressing YAP-LATS1 association. Intriguingly, YAP directly bound to LINC01433 promoter region and activated its transcription. Thus, LINC01433 and YAP formed a positive feedback loop. Conclusion: Collectively, our study demonstrates that the positive feedback loop between LINC01433 and YAP promotes GC progression, and implies that the LINC01433-YAP feedback loop may be a promising therapeutic target for GC treatment. [ABSTRACT FROM AUTHOR]

NF-κB is a central regulator of inflammatory and immune responses and has been shown to regulate transcription of several inflammatory factors as well as promote acute lung injury. However, the regulation of NF-κB signaling in acute lung injury has yet to be investigated. Human pulmonary alveolar epithelial cells (HPAEpiC) were treated with LPS to establish an acute lung injury model in vitro in which LPS stimulation resulted in pulmonary epithelial barrier breakdown and hyperpermeability. Cell viability was measured by CCK-8, and the transepithelial permeability was examined by measurement of transepithelial electrical resistance (TEER) and the transepithelial flux. Expression of ubiquitin-specific peptidase 9 X-linked (USP9X), zonula occludens (ZO-1), occludin and NF-κBp65, and the secretion of TNF-α and IL-1β were measured by Western blotting and ELISA, respectively. For in vivo studies, mice were intraperitoneally injected with LPS and/or NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Lung tissues were harvested for hematoxylin-eosin staining and Western blotting, and bronchoalveolar lavage fluid (BALF) was harvested for ELISA. We found that treatment with LPS in HPAEpiC inhibited cell viability and induced the expression of USP9X. Interestingly, knockdown of USP9X and treatment with PDTC suppressed LPS-induced HPAEpiC injury. USP9X overexpression promoted NF-κB activation, while NF-κB inactivation inhibited USP9X transcription and HPAEpiC injury induced by USP9X overexpression. Furthermore, LPS also induced the expression of USP9X in lungs, which was inhibited by PDTC. Taken together, these results demonstrate a critical role of USP9X-NF-κBp65 loop in mediating LPS-induced acute lung injury and may serve as a potential therapeutic target in acute lung injury. [ABSTRACT FROM AUTHOR]

Purpose: Anti-apoptotic and pro-migratory phenotypes are hallmarks of neoplastic diseases, including primary brain malignancies. In this work, we examined whether reprogramming of the apoptotic and migratory machineries through a multi-targeting approach would induce enhanced cell death and enhanced inhibition of the migratory capacity of glioblastoma cells. Methods: Preclinical testing and molecular analyses of combined inhibition of Bcl-2/Bcl-xL and RAC1 were performed in established, primary cultured and stem-like glioblastoma cell systems. Results: We found that the combined inhibition of Bcl-2/Bcl-xL and RAC1 resulted in synergistic pro-apoptotic and anti-migratory effects in a broad range of different glioblastoma cells. At the molecular level, we found that RAC1 inhibition led to a decreased expression of the deubiquitinase Usp9X, followed by a decreased stability of Mcl-1. We also found that the combined inhibition led to a significantly decreased migratory activity and that tumor formation of glioblastoma cells on chorion allantoic membranes of chicken embryos was markedly impaired following the combined inhibition. Conclusions: Our data indicate that concomitant inhibition of RAC1 and Bcl-2/Bcl-xL induces pro-apoptotic and anti-migratory glioblastoma phenotypes as well as synergistic anti-neoplastic activities. The clinical efficacy of this inhibitory therapeutic strategy warrants further evaluation. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 9X (USP9X) is one of the ubiquitin-specific protease(USPs) families of deubiquitinating enzymes (DUBs). USP9X is DUBs of X chromosome chain, which can play corresponding biological effects by stabilizing different substrates. USP9X plays an important role in cell signaling transduction, regulation of cell growth and migration, immune response, self-renewal and differentiation of stem cells, neurological function and carcinogenesis. Because of its important role in the development of digestive tract tumors and its feasibility as a drug target, this paper reviews the research progress of USP9X in the occurrence and development of common malignant tumors of digestive system. [ABSTRACT FROM AUTHOR]

USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability. Here, we report the crystal structure of the USP9X catalytic domain at 2.5-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual β-hairpin insertion. The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assayswith di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains. Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains. The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the β-hairpin insertion both play important roles in polyUb chain processing and linkage specificity. Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition. The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs. [ABSTRACT FROM AUTHOR]

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a destructive cancer with poor prognosis. Both novel therapeutic targets and approaches are needed to improve the overall survival of PDAC patients. MicroRNA-212 (miR-212) has been reported as a tumor suppressor in multiple cancers, but its definitive role and exact mechanism in the progression of pancreatic cancer is unclear. In this study, we developed a new chimeric peptide (PL-1) composed of plectin-1-targeted PDAC-specific and arginine-rich RNA-binding motifs which could condense miRNA to self-assemble supramolecular nanoparticles. These nanoparticles could deliver miR-212 into PDAC cells specifically and efficiently which also showed good stability in RNase and serum. Moreover, we demonstrated that PL-1/miR-212 nanoparticles could dramatically enhance the chemotherapeutic effect of doxorubicin for PDAC both in vitro and in vivo. In terms of mechanism, combined miR-212 intervention by PL-1/miR-212 nanoparticles resulted in obvious decrease of USP9X expression (ubiquitin specific peptidase 9, X-linked, USP9X) and eventually enhanced the doxorubicin induced apoptosis and autophagy of PDAC cells. These findings provide a new promising anti-cancer strategy via PL-1/miR-212 nanoparticles and identify miR-212/USP9X as a new potential target for future systemic therapy against human PDAC. [ABSTRACT FROM AUTHOR]

Centriolar satellites are small cytoplasmic granules that play important roles in regulating the formation of centrosomes and primary cilia. Ubiquitylation of satellite proteins, including the core satellite scaffold protein pericentriolar material 1 (PCM1), regulates centriolar satellite integrity. Currently, deubiquitylases that control centriolar satellite integrity have not been identified. In this study, we find that the deubiquitylase USP9X binds PCM1, and antagonizes PCM1 ubiquitylation to protect it from proteasomal degradation. Knockdown of USP9X in human cell lines reduces PCM1 protein levels, disrupts centriolar satellite particles and causes localization of satellite proteins, such as CEP290, to centrosomes. Interestingly, knockdown of mindbomb 1 (MIB1), a ubiquitin ligase that promotes PCM1 ubiquitylation and degradation, in USP9X-depleted cells largely restores PCM1 protein levels and corrects defects caused by the loss of USP9X. Overall, our study reveals that USP9X is a constituent of centriolar satellites and functions to maintain centriolar satellite integrity by stabilizing PCM1. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the expression level of ubiquitin specific peptidase 9X (USP9X) and its clinical significance in Chinese patients with pancreatic ductal adenocarcinoma (PDAC). The mRNA expression levels of USP9X in 30 paired PDAC tissue samples were examined by reverse transcription‑quantitative polymerase chain reaction. The protein expression of USP9X was analyzed by immunohistochemistry (IHC) in a tissue microarray containing 205 PDAC specimens. All analyses were performed by SPSS 20.0 and GraphPad Prism 5.0 software. The USP9X mRNA level was significantly decreased in 18/30 (60.0%) PDAC tissue samples compared with matched surrounding non‑tumor tissue samples. The results of IHC revealed that decreased expression of USP9X was inversely associated with liver metastasis (P=0.032). Kaplan‑Meier survival curves indicated that patients with high expression of USP9X presented a longer clinical overall survival time (P<0.001). Univariate and multivariate COX regression analysis revealed that USP9X protein expression level was a significant, and independent prognostic factor for the overall survival rate of patients with PDAC. The results of the present study indicate that USP9X may serve as a candidate tumor suppressor and prognostic biomarker in PDAC. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Recent studies reported that lncRNA LINC00473 (LNC473) was involved in cancer progression. However, the clinical significance and functional role of LNC473 in HCC progression is still unknown. In the present study, we found that the LNC473 expression was markedly elevated in HCC tissues and correlated with bigger tumor size, higher BCLC stage, vascular invasion and poor prognosis. Gain- and loss-of-function assay showed that LNC473 enhanced HCC cell proliferation and invasion and induced epithelial–mesenchymal transition (EMT) process. Mechanistically, LNC473 associated with oncoprotein survivin and regulates its stability. Moreover, LNC473 could recruit deubiquitinase USP9X to inhibit the ubiquitination level of survivin and then increase survivin expression. Therefore, our results suggest that LNC473 exerts its functions as an oncogene in HCC progression and may be a therapeutic target for HCC treatment. [ABSTRACT FROM AUTHOR]

publisher‐imprint‐name Springer volume‐issue‐count 1 issue‐article‐count 0 issue‐toc‐levels 0 issue‐pricelist‐year 2017 issue‐copyright‐holder The Author(s) issue‐copyright‐year 2017 article‐contains‐esm No article‐numbering‐style Unnumbered article‐registration‐date‐year 2017 article‐registration‐date‐month 4 article‐registration‐date‐day 20 article‐toc‐levels 0 toc‐levels 0 volume‐type Regular journal‐product ArchiveJournal numbering‐style Unnumbered article‐grants‐type OpenChoice metadata‐grant OpenAccess abstract‐grant OpenAccess bodypdf‐grant OpenAccess bodyhtml‐grant OpenAccess bibliography‐grant OpenAccess esm‐grant OpenAccess online‐first false pdf‐file‐reference BodyRef/PDF/40169_2017_Article_147.pdf target‐type OnlinePDF issue‐type Regular article‐type ReviewPaper journal‐subject‐primary Medicine & Public Health journal‐subject‐secondary Medicine/Public Health, general journal‐subject‐collection SC11 open‐access true --> Background: The RAS/MAPK pathway has been intensively studied in cancer. Constitutive activation of ERK1 and ERK2 is frequently found in cancer cells from a variety of tissues. In clinical practice and clinical trials, small molecules targeting receptor tyrosine kinases or components in the MAPK cascade are used for treatment. MEK1 and MEK2 are ideal targets because these enzymes are physiologically important and have narrow substrate specificities and distinctive structural characteristics. Despite success in pre‐clinical testing, only two MEK inhibitors, trametinib and cobimetinib, have been approved, both for treatment of BRAF‐mutant melanoma. Surprisingly, the efficacy of MEK inhibitors in other tumors has been disappointing. These facts suggest the need for a different approach. We here consider transcription factor ETS1 and ETS2 as alternate therapeutic targets because they are major MAPK downstream effectors. Main text: The lack of clinical efficacy of MEK inhibitors is attributed mostly to a subsequent loss of negative feedback regulation in the MAPK pathway. To overcome this obstacle, second‐generation MEK inhibitors, so‐called "feedback busters," have been developed. However, their efficacy is still unsatisfactory in the majority of cancers. To substitute ETS‐targeted therapy, therapeutic strategies to modulate the transcription factor in cancer must be considered. Chemical targeting of ETS1 for proteolysis is a promising strategy; Src and USP9X inhibitors might achieve this by accelerating ETS1 protein turnover. Targeting the ETS1 interface might have great therapeutic value because ETS1 dimerizes itself or with other transcription factors to regulate target genes. In addition, transcriptional cofactors, including CBP/p300 and BRD4, represent intriguing targets for both ETS1 and ETS2. Conclusions: ETS‐targeted therapy appears to be promising. However, it may have a potential problem. It might inhibit autoregulatory negative feedback loops in the MAPK pathway, with consequent resistance to cell death by ERK1 and ERK2 activation. Further research is warranted to explore clinically applicable ways to inhibit ETS1 and ETS2. [ABSTRACT FROM AUTHOR]

The Bcl-2 family protein, Mcl-1 is known to have anti-apoptotic functions, and depletion of Mcl-1 by cellular stresses favors the apoptotic process. Moreover, Mcl-1 levels are frequently increased in various cancer cells, including non-small cell lung cancer (NSCLC), and is implicated in resistance to conventional chemotherapy and in cancer metastasis. In this study, we demonstrated that KRIBB11 accelerates the proteasomal degradation of Mcl-1 in the NSCLC cell line, A549. While KRIBB11 is an inhibitor of HSF1, we found that KRIBB11 induced Mcl-1 degradation in an HSF1-independent manner. Furthermore, this process was triggered via increase ubiquitination by the E3 ligase, Mule, rather than via de-ubiquitination by USP9X. Additionally, we found that Mcl-1 levels were only transiently reduced by KRIBB11: Mcl-1 levels were gradually restored as KRIBB11 activity diminished. However, we found that this effect was blocked in BIS (Bcl-2 interacting cell death suppressor, also called BAG3)-depleted cells, and that BIS prevents Mcl-1 from undergoing HSP70-driven proteasomal degradation, through an interaction with HSP70. Taken together, our results suggest that targeting Mcl-1 with KRIBB11 treatment, while simultaneously downregulating BIS, could be a therapeutic strategy in NSCLC. [ABSTRACT FROM AUTHOR]

The genetic causes of intellectual disability (ID) are heterogeneous and include both chromosomal and monogenic etiologies. The X-chromosome is known to contain many ID-related genes and males show a marked predominance for intellectual disability. Here we report two females with syndromic intellectual disability. The first individual was relatively mild in her presentation with mild-moderate intellectual disability, hydronephrosis and altered pigmentation along the lines of Blaschko without additional congenital anomalies. A second female presented shortly after birth with dysmorphic facial features, post-axial polydactyly and, on follow-up assessment, demonstrated moderate intellectual disability. Chromosomal studies for Individual 1 identified an X-chromosome deletion due to a de novo pericentric inversion; the inversion breakpoint was associated with deletion of the 5′UTR of the USP9X , a gene which has been implicated in a syndromic intellectual disability affecting females. The second individual had a de novo frameshift mutation detected by whole-exome sequencing that was predicted to be deleterious, NM_001039590.2 ( USP9X ): c.4104_4105del (p.(Arg1368Serfs*2)). Haploinsufficiency of USP9X in females has been associated with ID and congenital malformations that include heart defects, scoliosis, dental abnormalities, anal atresia, polydactyly, Dandy Walker malformation and hypoplastic corpus callosum. The extent of the congenital malformations observed in Individual 1 was less striking than Individual 2 and other individuals previously reported in the literature, and suggests that USP9X mutations in females can have a wider spectrum of presentation than previously appreciated. [ABSTRACT FROM AUTHOR]

Botulism is characterized by flaccid paralysis, which can be caused by intoxication with any of the seven known serotypes of botulinum neurotoxin (BoNT), all of which disrupt synaptic transmission by endoproteolytic cleavage of SNARE proteins. BoNT serotype A (BoNT/A) has the most prolonged or persistent effects, which can last several months, and exerts its effects by specifically cleaving and inactivating SNAP25. A major factor contributing to the persistence of intoxication is the long half-life of the catalytic light chain, which remains enzymatically active months after entry into cells. Here we report that BoNT/A catalytic light chain binds to, and is a substrate for, the ubiquitin ligase HECTD2. However, the light chain evades proteasomal degradation by the dominant effect of a deubiquitinating enzyme, VCIP135/VCPIP1. This deubiquitinating enzyme binds BoNT/A light chain directly, with the two associating in cells through the C-terminal 77 amino acids of the light chain protease. The development of specific DUB inhibitors, together with inhibitors of BoNT/A proteolytic activity, may be useful for reducing the morbidity and public health costs associated with BoNT/A intoxication and could have potential biodefense implications. [ABSTRACT FROM AUTHOR]

Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic "Philadelphia-like" ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remission and -relapse DSALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in the JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are "relapse driving." We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2pos DSALLs by loss-of-function aberrations in USP9X, a deubiquitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2pos, Philadelphia-like ALLs. Pharmacological or genetic inhibition of USP9X, as well as treatment with low-dose ruxolitinib, enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT "hypersignaling" may be beneficial to leukemic B-cell precursors. This finding and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL. [ABSTRACT FROM AUTHOR]

Background: The RAS/MAPK pathway has been intensively studied in cancer. Constitutive activation of ERK1 and ERK2 is frequently found in cancer cells from a variety of tissues. In clinical practice and clinical trials, small molecules targeting receptor tyrosine kinases or components in the MAPK cascade are used for treatment. MEK1 and MEK2 are ideal targets because these enzymes are physiologically important and have narrow substrate specificities and distinctive structural characteristics. Despite success in pre-clinical testing, only two MEK inhibitors, trametinib and cobimetinib, have been approved, both for treatment of BRAF-mutant melanoma. Surprisingly, the efficacy of MEK inhibitors in other tumors has been disappointing. These facts suggest the need for a different approach. We here consider transcription factor ETS1 and ETS2 as alternate therapeutic targets because they are major MAPK downstream effectors. Main text: The lack of clinical efficacy of MEK inhibitors is attributed mostly to a subsequent loss of negative feedback regulation in the MAPK pathway. To overcome this obstacle, second-generation MEK inhibitors, so-called 'feedback busters,' have been developed. However, their efficacy is still unsatisfactory in the majority of cancers. To substitute ETS-targeted therapy, therapeutic strategies to modulate the transcription factor in cancer must be considered. Chemical targeting of ETS1 for proteolysis is a promising strategy; Src and USP9X inhibitors might achieve this by accelerating ETS1 protein turnover. Targeting the ETS1 interface might have great therapeutic value because ETS1 dimerizes itself or with other transcription factors to regulate target genes. In addition, transcriptional cofactors, including CBP/p300 and BRD4, represent intriguing targets for both ETS1 and ETS2. Conclusions: ETS-targeted therapy appears to be promising. However, it may have a potential problem. It might inhibit autoregulatory negative feedback loops in the MAPK pathway, with consequent resistance to cell death by ERK1 and ERK2 activation. Further research is warranted to explore clinically applicable ways to inhibit ETS1 and ETS2. [ABSTRACT FROM AUTHOR]

The neomorphic transcription factor EWS-FLI1 is a key driver of Ewing sarcoma. Ablation of EWS-FLI1 may present a promising therapeutic strategy for this malignancy. Here we found that the deubiquitinase, ubiquitin specific peptidase 9 X-linked (USP9X) stabilizes EWS-FLI1 protein expression in Ewing sarcoma. We show that USP9X binds the ETS domain of EWS-FLI1 in Ewing sarcoma cells and deubiquitinates EWS-FLI1 and that USP9X and EWS-FLI1 protein expression is correlated in clinical Ewing sarcoma specimens. We found that treatment of Ewing sarcoma cells with the USP9X inhibitor WP1130 mediates rapid EWS-FLI1 degradation in vitro and in vivo which coincides with reduced growth of Ewing sarcoma cells and tumors. Our results suggest that USP9X might be a potential therapeutic target to mediate EWS-FLI1 depletion in Ewing sarcoma. [ABSTRACT FROM AUTHOR]

Resistance to chemotherapeutic drugs is a major obstacle in hepatocellular carcinoma (HCC) therapy. However, the underlying mechanisms are not well understood. Recent evidence suggests that deubiquitinases (DUB) are key regulators in the mechanisms of cell proliferation, apoptosis and chemoresistance. The present study aimed to investigate whether WP1130, which inhibits activity of deubiquitinases, exerts synergistic cytotoxicity with doxorubicin in HCC and the underlying mechanisms. In the study, we found that Huh7, HepG2, and SNU387 HCC cells with p53 expression displayed enhanced response to the combination therapy compared with p53-deficient HCC cells (Hep3B) in the manner of inhibiting cell proliferation. Downregulation of p53 abolished the synergistic cytotoxicity of doxorubicin and WP1130 on HCC cells. Mechanistically, we found that combined treatment with WP1130 suppressed doxorubicin-mediated upregulation of p53 via promoting its ubiquitin-proteasome dependent degradation, whereas knockdown of DUB usp9x abolished this effect. Taken together, these results demonstrate that combined treatment with WP1130 sensitized HCC cells to doxorubicin via usp9x-depedent p53 degradation. [ABSTRACT FROM AUTHOR]

Although previous studies attributed a pro-survival role to USP9X in human cancer, how USP9X affects B-cell acute lymphoblastic leukemia (B-ALL) remains unclear. Here, we found that USP9X is overexpressed in B-ALL cell lines and human patients. We investigated the role of USP9X in B-ALL and found that USP9X knockdown significantly reduced leukemic cell growth and increased spontaneous apoptosis, thereby improving survival in immunodeficient mice. These effects are partially mediated by the intrinsic apoptotic pathway, as we found that USP9X-knockdown leukemic cells displayed MCL1 down-regulation, with decreased BCL-2/BCL-XL levels and increased BAX levels. In addition, we demonstrated that USP9X inhibition negatively regulates mTORC1 activity toward its substrate S6K1. Clinically, USP9X inhibition sensitized glucocorticoid-resistant ALL cells to prednisolone; this observation reveals a potential avenue for improving the treatment of drug-resistant relapses. Collectively, our findings suggest that the combination of USP9X targeting and glucocorticoids treatment has attractive utility in B-ALL. This approach represents a potential strategy for promising combination therapies for lymphoid malignancies. [ABSTRACT FROM AUTHOR]

The finding of USP9X variants in females has been associated with female-restricted X-linked mental retardation (MRXS99F), a rare syndrome featured by developmental delay and distinct congenital anomalies. Here, we report a female fetus with MRXS99F due to a novel frameshift variant, c.6679_6685delAAATTATinsTCCTG (p.Lys2227SerfsTer2) in USP9X , which was present in a mosaic state in the amniocytes and in the peripheral blood after birth (14% and 30%, respectively). The X methylation status analysis displayed a partially skewed X-inactivation with 80% of inactive paternal X. The first signs of the MRXS99F were prenatally detected at 20 weeks, with an enlarged posterior cranial fossa, an upward rotation of the cerebellar vermis and partial corpus callosum agenesis, together with a cardiac septal defect and a single umbilical artery. After birth and during postnatal follow-up the anal anteriorization and the presence of a bilateral membranous choanal atresia were noted, whereas the MRI revealed the hypo/aplasia of the olfactory bulbs, a widening of the subarachnoid spaces and a delay in the myelinisation. During the 18-months follow-up a severe growth and global developmental delay, together with a bilateral moderate deafness with a threshold at 40 dB, dental enamel erosions and an initial scoliosis were observed. We report the prenatal and postnatal features of a classical MRXS99F phenotype and a mosaic USP9X frameshift variant with a partially skewed X inactivation and discuss genotype/phenotype correlations in view of the published studies so far. [ABSTRACT FROM AUTHOR]

Diagnosis of rare skeletal diseases is based primarily on clinical phenotype and radiographic analysis. Genetic etiology of these heterogeneous diseases remains largely unknown. Here, we report the identification of two genomic mutations using exome sequencing from patients with multiple epiphyseal dysplasia (MED) of an unusual family in autosomal dominant and X-linked inheritance. A dominant mutation (c.2224G > A; p.Gly687Ser) in the known causal COL2A1 gene was identified in three patients with MED, deformed femoral heads and vertebral dysplasia. Furthermore, a hemizygous mutation (c.2830G > A; p.Ala944Thr) in the USP9X gene was identified in the fourth patient with short stature, MED, deformed femoral head, thoracic and lumbar platyspondyly, right ankle condyle dysplasia, and subchondral sclerosis. This is the first identification of an X-linked candidate causative gene in a patient with MED, suggesting a new clinical entity. Our findings shed a new light on the role of USP9X in MED-associated disorders. [ABSTRACT FROM AUTHOR]

BIX-01294, an euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor, has been reported to induce apoptosis in human neuroblastoma cells and inhibit the proliferation of bladder cancer cells. However, the definite mechanism of the apoptosis mediated by BIX-01294 in bladder cancer cells remains unclear. In the present study, we found that BIX-01294 induced caspase-dependent apoptosis in human bladder cancer cells. Moreover, our data show BIX-01294 stimulates endoplasmic reticulum stress (ER stress) and up-regulated expression of PMAIP1 through DDIT3 up-regulation. Furthermore, down-regulation of the deubiquitinase USP9X by BIX-01294 results in downstream reduction of MCL1 expression, leading to apoptosis eventually. Thus, our findings demonstrate PMAIP1-USP9X-MCL1 axis may contribute to BIX-01294-induced apoptosis in human bladder cancer cells. [ABSTRACT FROM AUTHOR]

Background: Metastasis is responsible for the rapid recurrence and poor survival of malignancies. Epithelial-mesenchymal transition (EMT) has a critical role in metastasis. Increasing evidence indicates that EMT can be regulated by microRNAs (miRNAs). The aim of this study was to investigate the role of miR-26b in modulating epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC), as well as to identify its underlying mechanism of action. Methods: The expression level of miR-26b was assessed in multiple HCC cell lines (HepG2, MHCC97H, Hep3B, MHCC97L, HCCC9810, BEL-7402, Huh7 and QGY-7703), as well as in liver tissue from patients with HCC. Follow-up studies examined the effects of a miR-26b mimic (increased expression) and a miR-26b inhibitor (decreased expression) on markers of EMT, wound healing and cell migration. The molecular target of miR-26b was also identified using a computer algorithm and confirmed experimentally. Results: MiR-26b expression was decreased in HCC cell lines and was inversely correlated with the grade of HCC. Increased expression of miR-26b inhibited the migration and invasiveness of HCC cell lines, which was accompanied by decreased expression of the epithelial marker E-cadherin and increased expression of the mesenchymal marker vimentin, at both the mRNA and protein expression levels. A binding site for miR-26b was theoretically identified in the 3'UTR of USP9X. Further studies revealed that overexpression of miR-26b repressed the endogenous level of USP9X protein expression. Overexpression of miR-26b also repressed Smad4 expression, whereas its inhibition elevated Smad4 expression. Conclusions: Taken together, our results indicate that miR-26b were inhibited in HCC. In HCC cell lines, miR-26b targeted the 3'UTR of USP9X, which in turn affects EMT through Smad4 and the TGF-ß signaling pathway. Our analysis of clinical HCC samples verifies that miR-26b also targets USP9X expression to inhibit the EMT of hepatocytes. Thus, miR-26b may have some effects on the EMT of HCC cells. [ABSTRACT FROM AUTHOR]

Background Metastasis is responsible for the rapid recurrence and poor survival of malignancies. Epithelial-mesenchymal transition (EMT) has a critical role in metastasis. Increasing evidence indicates that EMT can be regulated by microRNAs (miRNAs). The aim of this study was to investigate the role of miR-26b in modulating epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC), as well as to identify its underlying mechanism of action. Methods The expression level of miR-26b was assessed in multiple HCC cell lines (HepG2, MHCC97H, Hep3B, MHCC97L, HCCC9810, BEL-7402, Huh7 and QGY-7703), as well as in liver tissue from patients with HCC. Follow-up studies examined the effects of a miR-26b mimic (increased expression) and a miR-26b inhibitor (decreased expression) on markers of EMT, wound healing and cell migration. The molecular target of miR-26b was also identified using a computer algorithm and confirmed experimentally. Results MiR-26b expression was decreased in HCC cell lines and was inversely correlated with the grade of HCC. Increased expression of miR-26b inhibited the migration and invasiveness of HCC cell lines, which was accompanied by decreased expression of the epithelial marker E-cadherin and increased expression of the mesenchymal marker vimentin, at both the mRNA and protein expression levels. A binding site for miR-26b was theoretically identified in the 3'UTR of USP9X. Further studies revealed that overexpression of miR-26b repressed the endogenous level of USP9X protein expression. Overexpression of miR-26b also repressed Smad4 expression, whereas its inhibition elevated Smad4 expression. Conclusions Taken together, our results indicate that miR-26b were inhibited in HCC. In HCC cell lines, miR-26b targeted the 3'UTR of USP9X, which in turn affects EMT through Smad4 and the TGF-β signaling pathway. Our analysis of clinical HCC samples verifies that miR-26b also targets USP9X expression to inhibit the EMT of hepatocytes. Thus, miR-26b may have some effects on the EMT of HCC cells. [ABSTRACT FROM AUTHOR]

Background: It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survivalprotein in a variety of cell lineages and is critically regulated via ubiquitination. Methods: The Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay. Results: In our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells.Conclusion: Mcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective againstchemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors. [ABSTRACT FROM AUTHOR]