Drug resistance mechanisms and treatment strategies mediated by Ubiquitin-Specific Proteases (USPs) in cancers: new directions and therapeutic options.

Drug resistance represents a significant obstacle in cancer treatment, underscoring the need for the discovery of novel therapeutic targets. Ubiquitin-specific proteases (USPs), a subclass of deubiquitinating enzymes, play a pivotal role in protein deubiquitination. As scientific research advances, USPs have been recognized as key regulators of drug resistance across a spectrum of treatment modalities, including chemotherapy, targeted therapy, immunotherapy, and radiotherapy. This comprehensive review examines the complex relationship between USPs and drug resistance mechanisms, focusing on specific treatment strategies and highlighting the influence of USPs on DNA damage repair, apoptosis, characteristics of cancer stem cells, immune evasion, and other crucial biological functions. Additionally, the review highlights the potential clinical significance of USP inhibitors as a means to counter drug resistance in cancer treatment. By inhibiting particular USP, cancer cells can become more susceptible to a variety of anti-cancer drugs. The integration of USP inhibitors with current anti-cancer therapies offers a promising strategy to circumvent drug resistance. Therefore, this review emphasizes the importance of USPs as viable therapeutic targets and offers insight into fruitful directions for future research and drug development. Targeting USPs presents an effective method to combat drug resistance across various cancer types, leading to enhanced treatment strategies and better patient outcomes.

Role of Ubiquitin-specific Proteases in Hepatocellular Carcinoma Pathogenesis.

Signaling pathways in hepatocellular carcinoma are primarily mediated by the phosphorylation and ubiquitination of post-translational proteins. In mammalian cells, ubiquitin-specific proteases (USPs) account for the majority of protein deubiquitination activities. In addition to transcriptional and post-translational regulation, ubiquitination plays an important role in the regulation of key proteins. There is a possibility that altered biological processes may lead to serious human diseases, including cancer. Recent studies have revealed the role of USPs in hepatocellular carcinoma tumorigenesis. The purpose of this review is to summarize the involvement of this class of enzymes in the regulation of cell signaling in hepatocellular carcinoma and the therapeutic development of inhibitors that target USPs, which may lead to novel therapies to treat hepatocellular carcinoma.

Development of an OTUD1 ubiquitin variant inhibitor.

OTUD1 (Ovarian tumor domain-containing deubiquitinase 1) is a member of the OTU domain-containing deubiquitinase family of enzymes involved in immunoregulation and tumorigenesis pathways. OTUD1 consists of three distinct regions: an unstructured N-terminal region, an OTU-fold catalytic domain, and a ubiquitin-interacting motif (UIM) containing region. Enhanced enzymatic activity and a strong preference for K63-linked substrates are imparted by the UIM containing region. We used phage display with a ubiquitin variant (UbV) library to identify binders for OTUD1 lacking the unstructured N-terminal region (OTUD1OTU + UIM) in an attempt to identify inhibitors bridging the catalytic domain and the UIM containing region. Two UbVs were identified (UbVOD.1 and UbVOD.2) with high affinity and specificity for OTUD1. Of the UbVs identified, UbVOD.1 inhibited OTUD1 activity towards mono-Ub and K63-linked di-Ub substrates in vitro with single-digit nanomolar IC50 and potently inhibited deubiquitinase activity with poly-Ub chains of other linkages. In vivo expression of UbVOD.1 alone was unstable, however as a di-UbV, global deubiquitination and deubiquitinase activity with the OTUD1 substrate RIPK1 were inhibited. Herein we describe the development of molecular tools for exploring the activity of OTUD1 in a cellular context, towards protein-based therapeutics.

Inhibition of USP1 enhances anticancer drugs-induced cancer cell death through downregulation of survivin and miR-216a-5p-mediated upregulation of DR5.

Ubiquitin-specific protease 1 (USP1) is a deubiquitinase involved in DNA damage repair by modulating the ubiquitination of major regulators, such as PCNA and FANCD2. Because USP1 is highly expressed in many cancers, dysregulation of USP1 contributes to cancer therapy. However, the role of USP1 and the mechanisms underlying chemotherapy remain unclear. In this study, we found high USP1 expression in tumor tissues and that it correlated with poor prognosis in RCC. Mechanistically, USP1 enhanced survivin stabilization by removing ubiquitin. Pharmacological inhibitors (ML23 and pimozide) and siRNA targeting USP1 induced downregulation of survivin expression. In addition, ML323 upregulated DR5 expression by decreasing miR-216a-5p expression at the post-transcriptional level, and miR-216a-5p mimics suppressed the upregulation of DR5 by ML323. Inhibition of USP1 sensitized cancer cells. Overexpression of survivin or knockdown of DR5 markedly prevented the co-treatment with ML323 and TRAIL-induced apoptosis. These results of in vitro were proved in a mouse xenograft model, in which combined treatment significantly reduced tumor size and induced survivin downregulation and DR5 upregulation. Furthermore, USP1 and survivin protein expression showed a positive correlation, whereas miR-216a-5p and DR5 were inversely correlated in RCC tumor tissues. Taken together, our results suggest two target substrates of USP1 and demonstrate the involvement of survivin and DR5 in USP1-targeted chemotherapy.

A Synthetically Accessible Small-Molecule Inhibitor of USP5-Cav3.2 Calcium Channel Interactions with Analgesic Properties.

Cav3.2 calcium channels are important mediators of nociceptive signaling in the primary afferent pain pathway, and their expression is increased in various rodent models of chronic pain. Previous work from our laboratory has shown that this is in part mediated by an aberrant expression of deubiquitinase USP5, which associates with these channels and increases their stability. Here, we report on a novel bioactive rhodanine compound (II-1), which was identified in compound library screens. II-1 inhibits biochemical interactions between USP5 and the Cav3.2 domain III-IV linker in a dose-dependent manner, without affecting the enzymatic activity of USP5. Molecular docking analysis reveals two potential binding pockets at the USP5-Cav3.2 interface that are distinct from the binding site of the deubiquitinase inhibitor WP1130 (a.k.a. degrasyn). With an understanding of the ability of some rhodanines to produce false positives in high-throughput screening, we have conducted several orthogonal assays to confirm the validity of this hit, including in vivo experiments. Intrathecal delivery of II-1 inhibited both phases of formalin-induced nocifensive behaviors in mice, as well as abolished thermal hyperalgesia induced by the delivery of complete Freund's adjuvant (CFA) to the hind paw. The latter effects were abolished in Cav3.2 null mice, thus confirming that Cav3.2 is required for the action of II-1. II-1 also mediated a robust inhibition of mechanical allodynia induced by injury to the sciatic nerve. Altogether, our data uncover a novel class of analgesics─well suited to rapid structure-activity relationship studies─that target the Cav3.2/USP5 interface.

Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFRß and affect PDGF-BB-induced STAT3 signalling.

Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor ß (PDGFRß) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRß did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRß lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.

Ubiquitin-specific proteases as therapeutic targets in paediatric primary bone tumours?

In children and young adults, primary malignant bone tumours are mainly composed of osteosarcoma and Ewing's sarcoma. Despite advances in treatments, nearly 40% of patients succumb to these diseases. In particular, the clinical outcome of metastatic osteosarcoma or Ewing's sarcoma remains poor, with less than 30% of patients who develop metastases surviving five years after initial diagnosis. Over the last decade, the cancer research community has shown considerable interest in the processes of protein ubiquitination and deubiquitination. In particular, a growing number of studies show the relevance to target the ubiquitin-specific protease (USP) family in various cancers. This review provides an update on the current knowledge regarding the implication of these USPs in the progression of bone sarcoma: osteosarcoma and Ewing's sarcoma.

Overexpression of the Ubiquitin Specific Proteases USP43, USP41, USP27x and USP6 in Osteosarcoma Cell Lines: Inhibition of Osteosarcoma Tumor Growth and Lung Metastasis Development by the USP Antagonist PR619.

Osteosarcoma (OS) is the most common malignant bone tumor in children and teenagers. In many cases, such as poor response to treatment or the presence of metastases at diagnosis, the survival rate of patients remains very low. Although in the literature, more and more studies are emerging on the role of Ubiquitin-Specific Proteases (USPs) in the development of many cancers, few data exist regarding OS. In this context, RNA-sequencing analysis of OS cells and mesenchymal stem cells differentiated or not differentiated into osteoblasts reveals increased expression of four USPs in OS tumor cells: USP6, USP27x, USP41 and USP43. Tissue microarray analysis of patient biopsies demonstrates the nucleic and/or cytoplasmic expression of these four USPs at the protein level. Interestingly, Kaplan-Meyer analysis shows that the expression of two USPs, USP6 and USP41, is correlated with patient survival. In vivo experiments using a preclinical OS model, finally demonstrate that PR619, a USP inhibitor able to enhance protein ubiquitination in OS cell lines, reduces primary OS tumor growth and the development of lung metastases. In this context, in vitro experiments show that PR619 decreases the viability of OS cells, mainly by inducing a caspase3/7-dependent cell apoptosis. Overall, these results demonstrate the relevance of targeting USPs in OS.

MKP-1 modulates ubiquitination/phosphorylation of TLR signaling.

Ubiquitination and phosphorylation are reversible posttranslational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK MAPKs. We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1-/- showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1, and an increased K63 polyubiquitination on TRAF6. Increased K63 polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, ubiquitin-specific protease-13 (USP13), which cleaves polyubiquitin-chains on client proteins, was substantially enhanced in murine MKP-1-deficient BMDMs. An inhibitor of USP13 decreased the K63 polyubiquitination on TRAF6, TAK1 phosphorylation, IL-1ß, and TNF-α induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

Advances in the Development Ubiquitin-Specific Peptidase (USP) Inhibitors.

Ubiquitylation and deubiquitylation are reversible protein post-translational modification (PTM) processes involving the regulation of protein degradation under physiological conditions. Loss of balance in this regulatory system can lead to a wide range of diseases, such as cancer and inflammation. As the main members of the deubiquitinases (DUBs) family, ubiquitin-specific peptidases (USPs) are closely related to biological processes through a variety of molecular signaling pathways, including DNA damage repair, p53 and transforming growth factor-ß (TGF-ß) pathways. Over the past decade, increasing attention has been drawn to USPs as potential targets for the development of therapeutics across diverse therapeutic areas. In this review, we summarize the crucial roles of USPs in different signaling pathways and focus on advances in the development of USP inhibitors, as well as the methods of screening and identifying USP inhibitors.

Inhibition of USP1 induces apoptosis via ID1/AKT pathway in B-cell acute lymphoblastic leukemia cells.

Deubiquitylating enzyme ubiquitin-specific protease 1 (USP1) has been reported to be aberrantly overexpressed in cancers, and it plays a critical role in regulating various cellular processes, such as cell proliferation, apoptosis, and cell differentiation. However, the role of USP1 in B-cell acute lymphoblastic leukemia (B-ALL) remains largely undefined. USP1 expression in 30 newly diagnosed B-ALL patients was detected by real-time PCR and western blot. We found that USP1 was generally upregulated in the bone marrow cells derived from B-ALL patients. Knockdown of USP1 by siRNA decreased B-ALL cell growth and induced apoptosis. Similarly, pharmacological inhibition of USP1 by SJB3-019A significantly repressed cell proliferation and triggered B-ALL cell apoptosis. Finally, we found that inhibition of USP1 downregulated the expression of ID1 and p-AKT, and upregulated ID1 expression could reverse the suppressive effects of USP1 inhibitor in B-ALL cells. Taken together, these results demonstrate that USP1 promote B-ALL progression at least partially via the ID1/AKT signaling pathway, and USP1 inhibitors might be promising therapeutic application for B-ALL.

Ubiquitously specific protease 4 inhibitor-Vialinin A attenuates inflammation and fibrosis in S100-induced hepatitis mice through Rheb/mTOR signalling.

Inflammation and fibrosis are major consequences of autoimmune hepatitis, however, the therapeutic mechanism remains to be investigated. USP4 is a deubiquitinating enzyme and plays an important role in tissue fibrosis and immune disease. Vialinin A is an extract from mushroom and is a specific USP4 inhibitor. However, there is lack of evidences that Vialinin A plays a role in autoimmune hepatitis. By employing S100-induced autoimmune hepatitis in mice and AML12 cell line, therapeutic mechanism of Vialinin A was examined. Inflammation was documented by liver histological staining and inflammatory cytokines. Fibrosis was demonstrated by Masson, Sirius red staining and fibrotic cytokines with western blot and real-time RT-PCR. In experimental animal, there were increases in inflammation and fibrosis as well as USP4, and which were reduced after treatment of Vialinin A. Vialinin A also reduced Rheb and phosphorylated mTOR. Moreover, in LPS-treated AML12 cells, LPS-induced USP4, inflammatory and fibrotic cytokines, phosphorylated mTOR and Rheb. Specific inhibitory siRNA of USP4 reduced USP4 level and the parameters mentioned above. In conclusion, USP4 was significantly elevated in autoimmune hepatitis mice and Vialinin A reduced USP4 level and attenuate inflammation and fibrosis in the liver. The mechanism may be related to regulation of Rheb/mTOR signalling.

Comprehensive analysis of ubiquitin-specific protease 1 reveals its importance in hepatocellular carcinoma.

OBJECTIVES: In this study, we comprehensively analysed the role of ubiquitin-specific protease 1(USP1) in hepatocellular carcinoma (HCC) using data from a set of public databases. MATERIALS AND METHODS: We analysed the mRNA expression of USP1 in HCC and subgroups of HCC using Oncomine and UALCAN. Survival analysis of USP1 in HCC was conducted with the Kaplan-Meier Plotter database. The mutations of USP1 in HCC were analysed using cBioPortal and the Catalogue of Somatic Mutations in Cancer database. Differential genes correlated with USP1 and WD repeat domain 48 (WDR48) were obtained using LinkedOmics. USP1 was knocked down with small interfering RNA (siRNA) or pharmacologically inhibited by ML-323 in MHCC97H or SK-Hep-1 cell lines for function analysis. RESULTS: High USP1 expression predicted unfavourable overall survival in HCC patients. USP1 showed positive correlations with the abundances of macrophages and neutrophils. We identified 98 differential genes that were positively correlated with both USP1 and WDR48. These genes were mainly involved in the cell cycle, aldosterone synthesis and secretion and oestrogen signalling pathways. Interactions between USP1 and WDR 48 were confirmed using co-immunoprecipitation. USP1 knockdown or ML-323 treatment reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E1. CONCLUSIONS: Overall, USP1 is a promising target for HCC treatment with good prognostic value. USP1 and WDR48 function together in regulating cancer cell proliferation via the cell cycle.

Inhibition of USP1, a new partner of Bcr-Abl, results in decrease of Bcr-Abl level in K562 cells.

AIM: To analyze interaction of ubiquitin specific peptidase 1 (USP1) with Bcr-Abl and to assess the relation between USP1 functional activity and Bcr-Abl expression in K562 chronic myeloid leukemia cells. MATERIALS AND METHODS: The interaction between USP1 and Bcr-Abl in K562 cells was analyzed by co-immunoprecipitation, Western blot analysis, and confocal microscopy. RESULTS: A direct interaction between Bcr-Abl oncoprotein and USP1 protein in K562 cells was established by co-immunoprecipitation. Immunofluorescence analysis and confocal microscopy revealed that Bcr-Abl/USP1 protein complex is formed in the cell nucleus. The inhibition of USP1 protein activity by ML323 reduced the level of Bcr-Abl oncoprotein in K562 cells. CONCLUSIONS: USP1 protein has been identified as a new protein partner of Bcr-Abl oncoprotein in chronic myeloid leukemia. The relationship between the functional activity of USP1 protein and the level of Bcr-Abl oncoprotein has been demonstrated, suggesting that the targeted inhibition of USP1 activity could be a challenging approach for reducing Bcr-Abl expression.

Knockdown of Ubiquitin-Specific Protease 53 Enhances the Radiosensitivity of Human Cervical Squamous Cell Carcinoma by Regulating DNA Damage-Binding Protein 2.

BACKGROUND: Cervical cancer ranks fourth in incidence and mortality among women. Ubiquitin-specific protein 53 binds to damage-specific DNA binding protein 2 and affects the biological properties of colon cancer. Damage-specific DNA binding protein is involved in nucleotide excision repair, which can repair DNA damage. However, the mechanism by which ubiquitin-specific protein 53 regulates the radiosensitivity of cervical cancer through damage-specific DNA binding protein remains unclear. METHODS: Tissue samples from 40 patients with cervical squamous cell carcinoma who received radiotherapy were examined by immunohistochemistry to detect the expression of ubiquitin-specific protein 53, and clinical data were collected for statistical analysis. The cell cycle was detected by flow cytometry in Siha cells transfected with Si-USP53 and exposed to 8 Gy irradiation. Cell viability was determined by the CCK8 method in cells transfected with Si-USP53 and exposed to 0, 2, 4, 6, 8, or 10 Gy. The expression of damage-specific DNA binding protein, cyclin-dependent kinase 1, and cell cycle checkpoint kinase 2 was detected in cells transfected with Si-USP53. RESULTS: The expression of ubiquitin-specific protein 53 in the tissues of patients with cervical squamous cell carcinoma was correlated with the sensitivity to radiotherapy. Knockdown of ubiquitin-specific protein 53 in Siha cells downregulated damage-specific DNA binding protein and caused G2/M cell cycle arrest and decreased the survival rate of cells in response to radiation. CONCLUSION: Ubiquitin-specific protein 53-induced cell cycle arrest and affected the radiotherapy sensitivity of tumors through damage-specific DNA binding protein.

USP1 inhibitor ML323 enhances osteogenic potential of human dental pulp stem cells.

LIM homeobox 8 (LHX8) is expressed during embryonic development of craniofacial tissues, including bone and teeth. In a previous study, the overexpression of LHX8 inhibited osteodifferentiation of human dental pulp stem cells (DPSCs). In this study, a cDNA microarray analysis was performed to reveal the molecular changes which occur in response to LHX8 overexpression in DPSCs and discover possible targets for an osteoinductive agent. There were 345 differentially expressed genes (DEGs) in response to osteoinductive signaling and 53 DEGs in response to LHX8 overexpression and osteoinductive signaling, respectively. Thirty-eight genes were common in both conditions, and among these, genes upregulated in LHX8 DPSCs but downregulated in osteodifferentiated DPSCs were chosen. Five of them had commercial inhibitors available. Among the tested inhibitors, ML323, which target DNA-binding protein inhibitor ID-1, promoted osteodifferentiation of DPSCs. In conclusion, inhibition of ID-1 led to increased osteogenesis of human DPSCs.

Targeting USP1-dependent KDM4A protein stability as a potential prostate cancer therapy.

The histone demethylase lysine-specific demethylase 4A (KDM4A) is reported to be overexpressed and plays a vital in multiple cancers through controlling gene expression by epigenetic regulation of H3K9 or H3K36 methylation marks. However, the biological role and mechanism of KDM4A in prostate cancer (PC) remain unclear. Herein, we reported KDM4A expression was upregulation in phosphatase and tensin homolog knockout mouse prostate tissue. Depletion of KDM4A in PC cells inhibited their proliferation and survival in vivo and vitro. Further studies reveal that USP1 is a deubiquitinase that regulates KDM4A K48-linked deubiquitin and stability. Interestingly, we found c-Myc was a key downstream effector of the USP1-KDM4A/androgen receptor axis in driving PC cell proliferation. Notably, upregulation of KDM4A expression with high USP1 expression was observed in most prostate tumors and inhibition of USP1 promotes PC cells response to therapeutic agent enzalutamide. Our studies propose USP1 could be an anticancer therapeutic target in PC.

A Selective Inhibitor of Ubiquitin-Specific Protease 4 Suppresses Colorectal Cancer Progression by Regulating ß-Catenin Signaling.

BACKGROUND/AIMS: Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of ß-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities. METHODS: By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of ß-catenin, as shown by immunoblotting, and it affected the target gene expression of ß-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model. RESULTS: We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased ß-catenin stability and reduced expression of ß-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume. CONCLUSION: The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.

Inhibition of USP4 attenuates pathological scarring by downregulation of the TGF­ß/Smad signaling pathway.

Pathological scarring is a result of the hypertrophy of scar tissue during tissue repair following trauma. The aim of the present study was to assess the effect of ubiquitin­specific protease 4 (USP4) silencing on pathological scarring, and to evaluate the mechanistic basis for the effect. An MTT assay was used to assess cell viability. Immunoprecipitation (IP) was used to determine ubiquitination levels of the TGF­ß receptor (TßR)I and Smad7. Tumor formation was assessed by injecting keloid fibroblasts. Hematoxylin and eosin staining was used to detect pathological changes in tumor tissue. Reverse transcription quantitative polymerase chain reaction and western blot analysis assays were used to evaluate the expression levels of TßRI and Smad7. Compared with the untreated control animals, cell viability and the expression of TßRI and Smad7 increased significantly in animals treated with TGF­ß. Short hairpin RNA for USP4 (shUSP4) decreased the cell viability of negative control cells, TGF­ß­induced cellular proliferation, and the expression of TßRI and Smad7. IP experiments indicated that the ubiquitination level of TßRI was decreased following USP4 silencing. There was no remarkable difference in the structure of scar tissue among the various animal groups at 14 days following treatment, while the necrotic area of the scar tissue in the shUSP4 and vialinin A (USP inhibitor)­treated animals increased significantly at the 28th and 42nd day compared with the control animals. At days 14, 28 and 42, the expression levels of TßRI and Smad7 in the shUSP4 and vialinin A­treated animals were significantly decreased compared with the control animals (P<0.05). In summary, interference with or inhibition of USP4 prevented the activity of the TGF­ß/Smad pathway signaling and inhibited the formation of pathological scars.

USP1 links platinum resistance to cancer cell dissemination by regulating Snail stability.

Resistance to platinum-based chemotherapy is a common event in patients with cancer, generally associated with tumor dissemination and metastasis. Whether platinum treatment per se activates molecular pathways linked to tumor spreading is not known. Here, we report that the ubiquitin-specific protease 1 (USP1) mediates ovarian cancer cell resistance to platinum, by regulating the stability of Snail, which, in turn, promotes tumor dissemination. At the molecular level, we observed that upon platinum treatment, USP1 is phosphorylated by ATM and ATR and binds to Snail. Then, USP1 de-ubiquitinates and stabilizes Snail expression, conferring resistance to platinum, increased stem cell-like features, and metastatic ability. Consistently, knockout or pharmacological inhibition of USP1 increased platinum sensitivity and decreased metastatic dissemination in a Snail-dependent manner. Our findings identify Snail as a USP1 target and open the way to a novel strategy to overcome platinum resistance and more successfully treat patients with ovarian cancer.