Binding of USP4 to cortactin enhances cell migration in HCT116 human colon cancer cells.

Ubiquitin-specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating ß-catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on ß-catenin. Thus, we investigated an additional role of USP4 in cancer. In this study, we identified cortactin (CTTN), an actin-binding protein involved in the regulation of cytoskeleton dynamics and a potential prognostic marker for cancers, as a new cellular interacting partner of USP4 from proximal labeling of HCT116 cells. Additionally, the role of USP4 in CTTN activation and promotion of cell dynamics and migration was investigated in HCT116 cells. We confirmed that interacting of USP4 with CTTN increased cell movement. This finding was supported by the fact that USP4 overexpression in HCT116 cells with reduced expression of CTTN was insufficient to promote cell migration. Additionally, we observed that USP4 overexpression led to a significant increase in CTTN phosphorylation, which is a requisite mechanism for cell migration, by regulating Src/focal adhesion kinase (FAK) binding to CTTN and its activation. Our results suggest that USP4 plays a dual role in cancer progression, including stabilization of ß-catenin as a DUB and interaction with CTTN to promote cell dynamics by inducing CTTN phosphorylation. Therefore, this study demonstrates that USP4 is important for cancer progression and is a good target for treating or preventing cancer.

Ubiquitin-Specific Protease 21 Promotes Colorectal Cancer Metastasis by Acting as a Fra-1 Deubiquitinase.

Fos-related-antigen-1 (Fra-1), a member of the activator protein-1 (AP-1) transcription factor superfamily, has an essential role in cancer progress and metastasis and Fra-1 is considered a therapeutic target in metastatic cancer including metastatic colorectal cancer (mCRC). However, its regulation at protein level has not yet been clearly elucidated. We found that ubiquitin-specific protease 21 (USP21) increases Fra-1 stability by deubiquitinating Fra-1 and enhances the expression of Fra-1 target genes in colon cancer cells. We also showed that USP21 controlled Fra-1-dependent migration and invasion activities. The oncogenic property of USP21 was confirmed by a significant reduction in liver metastasis when USP21-knockdown cancer cells were injected intrasplenically into mice. Consistently, clinicopathological analysis of colorectal cancer patients revealed a correlation of USP21 expression with high-grade carcinoma and life span. These results demonstrate that USP21 enhances Fra-1 stability and AP-1 target gene expression by deubiquitinating Fra-1. Therefore, USP21 is considered an attractive therapeutic target in mCRC with high Fra-1 expression.

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.

Suppression of the Ubiquitin Pathway by Small Molecule Binding to Ubiquitin Enhances Doxorubicin Sensitivity of the Cancer Cells.

Development of inhibitors for ubiquitin pathway has been suggested as a promising strategy to treat several types of cancers, which has been showcased by recent success of a series of novel anticancer drugs based on inhibition of ubiquitin pathways. Although the druggability of enzymes in ubiquitin pathways has been demonstrated, ubiquitin itself, the main agent of the pathway, has not been targeted. Whereas conventional enzyme inhibitors are used to silence the ubiquitination or reverse it, they cannot disrupt the binding activity of ubiquitin. Herein, we report that the scaffolds of sulfonated aryl diazo compounds, particularly Congo red, could disrupt the binding activity of ubiquitin, resulting in the activity equivalent to inhibition of ubiquitination. NMR mapping assay demonstrated that the chemical directly binds to the recognition site for ubiquitin processing enzymes on the surface of ubiquitin, and thereby blocks the binding of ubiquitin to its cognate receptors. As a proof of concept for the druggability of the ubiquitin molecule, we demonstrated that Congo red acted as an intracellular inhibitor of ubiquitin recognition and binding, which led to inhibition of ubiquitination, and thereby, could be used as a sensitizer for conventional anticancer drugs, doxorubicin.

Ubiquitin-specific protease 4 (USP4) suppresses myoblast differentiation by down regulating MyoD activity in a catalytic-independent manner.

For myotube formation, proliferation and differentiation of myoblasts must be tightly regulated by various myogenic regulatory factors (MRFs) such as MyoD, myogenic factor 5 (Myf5), myogenin, and muscle-specific regulatory factor 4 (MRF4). However, it is not clear how the expression or activity of these MRFs is controlled during myogenesis. In this study, we identified ubiquitin-specific protease 4 (USP4), one of deubiquitinating enzymes, as a suppressor of MRFs by demonstrating that a knockdown of USP4 enhances myogenesis by controlling MyoD and the level of myogenesis marker proteins in C2C12 cells. However, it was revealed that the effect of USP4 on myogenesis is independent of its deubiquitinase activity because the catalytic-site mutant has the same inhibitory effects as the wild-type USP4 on myogenesis. We observed that the activity and protein levels of both HDAC1 and HDAC4 are decreased when myoblast differentiation is promoted by the USP4 knockdown. We also found that the role of USP4 in muscle differentiation is correlated with two major signaling pathways in myogenesis, AKT and the p38 mitogen-activated protein kinase pathways. According to these results, we propose that USP4 is a key player in myogenic differentiation; it controls myogenic regulatory factors in a catalytic-independent manner.

An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors.

The ubiquitin pathway plays a critical role in regulating diverse biological processes, and its dysregulation is associated with various diseases. Therefore, it is important to have a tool that can control the ubiquitin pathway in order to improve understanding of this pathway and to develop therapeutics against relevant diseases. We found that Chicago Sky Blue 6B binds directly to the ß-groove, a major interacting surface of ubiquitin. Hence, it could successfully inhibit the enzymatic activity of ubiquitin processing enzymes and the binding of ubiquitin to the CXCR4, a cell surface ubiquitin receptor. Furthermore, we demonstrated that this ubiquitin binding chemical could effectively suppress the ubiquitin induced cancer cell migration by blocking ubiquitin-CXCR4 interaction. Current results suggest that ubiquitin binding molecules can be developed as inhibitors of ubiquitin-protein interactions, which will have the value not only in unveiling the biological role of ubiquitin but also in treating related diseases.

Ubiquitin-specific protease 4 controls metastatic potential through ß-catenin stabilization in brain metastatic lung adenocarcinoma.

Brain metastasis is the most common type of intracranial cancer and is the main cause of cancer-associated mortality. Brain metastasis mainly originates from lung cancer. Using a previously established in vitro brain metastatic model, we found that brain metastatic PC14PE6/LvBr4 cells exhibited higher expression of ß-catenin and increased migratory activity than parental PC14PE6 cells. Knockdown of ß-catenin dramatically suppressed the motility and invasiveness of PC14PE6/LvBr4 cells, indicating ß-catenin is involved in controlling metastatic potential. Since ß-catenin protein was increased without a significant change in its mRNA levels, the mechanism underlying increased ß-catenin stability was investigated. We found that ubiquitin-specific protease 4 (USP4), recently identified as a ß-catenin-specific deubiquitinylating enzyme, was highly expressed in PC14PE6/LvBr4 cells and involved in the increased stability of ß-catenin protein. Similar to ß-catenin knockdown, USP4-silenced PC14PE6/LvBr4 cells showed decreased migratory and invasive abilities. Moreover, knockdown of both USP4 and ß-catenin inhibited clonogenicity and induced mesenchymal-epithelial transition by downregulating ZEB1 in PC14PE6/LvBr4 cells. Using bioluminescence imaging, we found that knockdown of USP4 suppressed brain metastasis in vivo and significantly increased overall survival and brain metastasis-free survival. Taken together, our results indicate that USP4 is a promising therapeutic target for brain metastasis in patients with lung adenocarcinoma.

Ubiquitin specific protease 4 positively regulates the WNT/ß-catenin signaling in colorectal cancer.

ß-catenin is a key signal transducer in the canonical WNT pathway and is negatively regulated by ubiquitin-dependent proteolysis. Through screening of various deubiquitinating enzymes (DUBs), we identified ubiquitin specific protease 4 (USP4) as a candidate for ß-catenin-specific DUB. The effects of USP4 overexpression or knockdown suggested that USP4 positively controls the stability of ß-catenin and enhances ß-catenin-regulated transcription. Domain mapping results revealed that the C-terminal catalytic domain is responsible for ß-catenin binding and nuclear transport. Examination of colon cancer tissues from patients revealed a correlation between elevated expression levels of USP4 and ß-catenin. Consistent with this correlation, USP4 knockdown in HCT116, a colon cancer cell line, reduced invasion and migration activity. These observations indicate that USP4 acts as a positive regulator of the WNT/ß-catenin pathway by deubiquitination and facilitates nuclear localization of ß-catenin. Therefore, we propose that USP4 is a potential target for anti-cancer therapeutics.

Poncirin promotes osteoblast differentiation but inhibits adipocyte differentiation in mesenchymal stem cells.

Poncirin, flavanone glycoside, isolated from the fruit of Poncirus trifoliata, has anti-bacterial and anti-inflammatory activities. In this study, the effects of poncirin on the differentiation of mesenchymal stem cells were investigated. The C3H10T1/2 mesenchymal stem cells and primary bone marrow mesenchymal stem cells were studied. In the C3H10T1/2 cells, poncirin prevented adipocyte differentiation, as demonstrated by inhibition of cytoplasm lipid droplet accumulation and peroxisome proliferator-activating receptor-γ (PPAR-γ) and CCAAT-enhancer-binding protein-ß (C/EBP-ß) mRNA expression. By contrast, poncirin enhanced the expression of the key osteogenic transcription factors, runt-related transcription factor 2 (Runx2) and transcriptional coactivator with PDZ-binding motif (TAZ). Poncirin also enhanced expression of the osteogenic marker genes including alkaline phosphatase (ALP) and osteocalcin (OC). Poncirin increased mineral nodule formation in primary bone marrow mesenchymal stem cells. These results suggest that poncirin prevents adipogenesis and enhances osteoblast differentiation in mesenchymal stem cells.

In vitro and in vivo inhibition of glucocorticoid-induced osteoporosis by the hexane extract of Poncirus trifoliata.

This study was performed to discover a novel herbal therapeutic for effective glucocorticoid-induced osteoporosis (GIO) treatment and further to clarify its molecular mechanism of action. Ethanol or methanol extracts of 68 edible Korean native plants were screened to find effective natural plant sources for the treatment of GIO, and Poncirus trifoliata (L.) (Rutaceae, PT) was selected as a final candidate because of its high inhibitory activity plus its novelty. The hexane extract of PT (PT-H) inhibited apoptotic cell death in dexamethasone-induced osteoblastic cell lines, C3H10T1/2 and MC3T3-E1. In vivo mouse results indicated that PT-H not only had an inhibitory effect on the bone loss caused by glucocorticoid, but also promoted bone formation. The molecular mechanisms behind the effect of PT-H on GIO were further clarified by screening of differentially expressed genes (DEGs) between dexamethasone (Dex)-induced osteoblastic cells with or without PT-H treatment. Finally, it was found that the expression level of AnxA6 in Dex-induced osteoblastic cells and prednisolone (PD)-treated GIO-model mice was significantly decreased by PT-H treatment. These findings suggest that PT-H has a strong in vitro and in vivo inhibitory effect on GIO, and decreased expression of AnxA6 may play a key role in this inhibition.

Glycogen synthase kinase-3beta regulates etoposide-induced apoptosis via Bcl-2 mediated caspase-3 activation in C3H10T1/2 cells.

Glycogen synthase kinase-3beta (GSK3beta) controls the survival of osteoblasts during bone development through Wnt canonical signaling. GSK3beta is a key factor for osteoblastogenesis, but relatively less is known regarding its role in osteoblast apoptosis. Genotoxic stress induced by etoposide promoted apoptotic signaling by GSK3beta activation in C3H10T1/2 cells, a mouse mesenchymal cell line. Etoposide led to the time-dependent activation of GSK3beta and caspase-3, which resulted in PARP cleavage. LiCl (a specific inhibitor) and siRNA (gene knock-down) of GSK3beta prevented the effects of etoposide on apoptosis. Staurosporine also induced apoptosis in C3H10T1/2 cells, but LiCl could not rescue. Bcl-2 was decreased in the cells by exposure to etoposide. LiCl completely recovered Bcl-2 expression as shown by both the mRNA and the protein expression levels. In conclusion, etoposide-induced apoptosis in C3H10T1/2 cells is mediated by GSK3beta, which leads to caspase-3 activation via decrease in Bcl-2 expression.

Glucocorticoid induces apoptosis of osteoblast cells through the activation of glycogen synthase kinase 3beta.

Glucocorticoids (GCs), which play an important role in the normal regulation of bone remodeling, are widely used as anti-inflammatory and chemotherapeutic agents. However, continued exposure to GCs results in osteoporosis, which is partially due to apoptosis of osteoblasts and osteocytes. To understand the mechanism of how GCs induce cell death in osteoblasts, we examined apoptotic effects of dexamethasone (Dex), GC, on MC3T3-E1 osteoblast cells. Results revealed that Dex-induced apoptosis was inhibited by a GC receptor antagonist, mifepristone, and a general caspase inhibitor, Z-VAD-fmk, indicating that Dex induces apoptosis of MC3T3-E1 cells through the pathways involved in GC receptor and caspase. Glycogen synthase kinase 3beta (GSK3beta) is known to participate in apoptosis signaling in MC3T3-E1 cells. Dex activated both GSK3beta and p38-mitogen-activated protein kinase (MAPK). The inhibition of GSK3beta by inhibitor (LiCl) or small interference RNA (siRNA) decreased apoptosis. In contrast, the inhibition of p38-MAPK by inhibitor (SB203580) or siRNA did not decrease, but increase apoptosis. These results suggest that Dex-mediated apoptosis of osteoblasts is facilitated by GSK3beta, but prevented by p38-MAPK.