Proteasome-Bound UCH37/UCHL5 Debranches Ubiquitin Chains to Promote Degradation.

The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables in providing tight control over many biological paradigms. There are clear roles for branched architectures in regulating proteasome-mediated degradation, but the proteins that selectively recognize and process these atypical chains are unknown. Here, using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, cleaves K48 branched chains. The activity and selectivity toward branched chains is markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using reconstituted proteasome complexes, we find that chain debranching promotes degradation of substrates modified with branched chains under multi-turnover conditions. These results are further supported by proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity impairs global protein turnover. Our work therefore defines UCH37 as a debranching deubiquitinase important for promoting proteasomal degradation.

UCHL5 promotes hepatocellular carcinoma progression by promoting glycolysis through activating Wnt/ß-catenin pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is highly malignant with a dismal prognosis, although the available therapies are insufficient. No efficient ubiquitinase has been identified as a therapeutic target for HCC despite the complicating role that of proteins ubiquitination plays in the malignant development of HCC. METHODS: The expression of ubiquitin carboxyl terminal hydrolase L5 (UCHL5) in HCC tumor tissue and adjacent normal tissue was determined using the cancer genome atlas (TCGA) database and was validated using real-time quantitative polymerase chain reaction (RT-qRCR), Western blot and immunohistochemistry (IHC), and the relation of UCHL5 with patient clinical prognosis was explored. The expression of UCHL5 was knocked down and validated, and the effect of UCHL5 on the biological course of HCC was explored using cellular assays. To clarify the molecular mechanism of action of UCHL5 affecting HCC, expression studies of Adenosine triphosphate adenosine triphosphate (ATP), extracellular acidification (ECAR), and glycolysis-related enzymes were performed. The effects of UCHL5 on ß-catenin ubiquitination and Wnt signaling pathways were explored in depth and validated using cellular functionalities. Validation was also performed in vivo. RESULTS: In the course of this investigation, we discovered that UCHL5 was strongly expressed in HCC at both cellular and tissue levels. The prognosis of patients with high UCHL5 expression is considerably worse than that of those with low UCHL5 expression. UCHL5 has been shown to increase the degree of glycolysis in HCC cells with the impact of stimulating the proliferation and metastasis of HCC cells in both in vivo and in vitro. UCHL5 downregulates its degree of ubiquitination by binding to ß-catenin, which activates the Wnt/ß-catenin pathway and accelerates HCC cell glycolysis. Thereby promoting the growth of the HCC. CONCLUSIONS: In summary, we have demonstrated for the first time that UCHL5 is a target of HCC and promotes the progression of hepatocellular carcinoma by promoting glycolysis through the activation of the Wnt/ß-catenin pathway. UCHL5 may thus serve as a novel prognostic marker and therapeutic target for the treatment of HCC.

Caveolin-2 in association with nuclear lamina controls adipocyte hypertrophy.

Here, we identify that Caveolin-2 (Cav-2), an integral membrane protein, controls adipocyte hypertrophy in association with nuclear lamina. In the hypertrophy stage of adipogenesis, pY19-Cav-2 association with lamin A/C facilitated the disengagement of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) from lamin A/C and repressed Cav-2 promoter at the nuclear periphery for epigenetic activation of Cav-2, and thereby promoted C/EBPα and PPARγ-induced adipocyte hypertrophy. Stable expression of Cav-2 was required and retained by phosphorylation, deubiquitination, and association with lamin A/C for the adipocyte hypertrophy. However, obese adipocytes exhibited augmented Cav-2 stability resulting from the up-regulation of lamin A/C over lamin B1, protein tyrosine phosphatase 1B (PTP1B), and nuclear deubiquitinating enzyme (DUB), Uchl5. Our findings show a novel epigenetic regulatory mechanism of adipocyte hypertrophy by Cav-2 at the nuclear periphery.

METTL14/YTHDF1 axis-modified UCHL5 aggravates atherosclerosis by activating the NLRP3 inflammasome.

BACKGROUND: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to VSMC proliferation and migration in atherosclerosis (AS). Nevertheless, the regulatory mechanism of VSMC phenotypic switching during AS progression is unclear. Here, the role and regulatory mechanism of UCHL5 in VSMC phenotypic switching during AS progression were investigated. METHODS: ApoE-/- mice were fed with high fat diet to establish AS model in vivo. VSMCs stimulated by ox-LDL were used as AS cellular model. VSMC proliferation and migration were examined by CCK8 assay and transwell assay, respectively. The levels of pro-inflammatory cytokines were assessed using ELISA. The interactions between METTL14/YTHDF1, UCHL5 and NLRP3 were analyzed using RIP and/or dual-luciferase reporter gene and/or Co-IP assays. NLRP3 ubiquitination was analyzed by ubiquitination analysis. RESULTS: UCHL5 was significantly upregulated in AS patients and ox-LDL-treated VSMCs. UCHL5 silencing ameliorated plaque formation and vascular remodeling in vivo and suppressed ox-LDL-induced VSMC proliferation, migration, inflammation and phenotypic switching in vitro. Moreover, METTL14 could increase UCHL5 mRNA m6A level and promoted UCHL5 expression by recruiting YTHDF1. Moreover, UCHL5 overexpression enhanced protein stability by deubiquitinating NLRP3. Rescue studies revealed that NLRP3 overexpression abrogated UCHL5 silencing-mediated biological effects in ox-LDL-treated VSMCs. CONCLUSION: UCHL5 modified by METTL14/YTHDF1 axis could facilitate the inflammation and vascular remodeling in atherosclerosis by activating the NLRP3 inflammasome.

Deubiquitinase UCHL5 stabilizes ELK3 to potentiate cancer stemness and tumor progression in pancreatic adenocarcinoma (PAAD).

Aberrant ubiquitin-proteasome system (UPS) contributes to tumorigeneisis or drug resistance of Pancreatic Adenocarcinoma (PAAD). Previous studies have implicated the deubiquitinase UCHL5 was abnormally expressed in multiple malignancies. However, little was reported about the specific roles of UCHL5 in PAAD. We aimed to identify the biological roles of UCHL5 in PAAD and demonstrate its prognostic significance. Differential analysis revealed that UCHL5 expressed highly in tumors versus normal tissues, like TCGA-PAAD, GSE28735, GSE15471 and collected samples. Patients with high UCHL5 expressions had worse survival outcomes relative to those with low UCHL5 levels. Experimental assays showed that UCHL5 overexpression could significantly enhance cell proliferation, colony formation and self-renewal capacities. UCHL5 could also promote PAAD migration in vitro and in vivo. Mechanistically, UCHL5 could directly deubiquitinate and stabilize ELK3 proteins. UCHL5 relied on accumulated ELK3 proteins to drive cell growth, stem-like properties and migration abilities. In addition, enrichment analysis based on RNA-seq data implicated that ELK3 mainly correlated with Notch1 signaling and ELK3 could notably elevate ELK3 mRNA levels. UCHL5 could thus promote self-renewal abilities of PAAD and targeting ELK3 could inhibit the stemness features. In contrast, UCHL5 deficiency could suppress PAAD stemness features, and ectopic expression of ELK3 could rescue this effect. Last of all, we utilized the UCHL5 inhibitor, b-AP15, to treat PAAD cells and found that b-AP15 could inhibit the growth of PAAD cells in a dose-dependent manner. Collectively, our study uncovered the underlying mechanisms of UCHL5/ELK3/Notch1 axis in PAAD progression and stemness maintaince, shedding light on individualized treatment and risk stratification for PAAD patients.

Targeting proteasomal deubiquitinases USP14 and UCHL5 with b-AP15 reduces 5-fluorouracil resistance in colorectal cancer cells.

5-Fluorouracil (5-FU) is the first-line treatment for colorectal cancer (CRC) patients, but the development of acquired resistance to 5-FU remains a big challenge. Deubiquitinases play a key role in the protein degradation pathway, which is involved in cancer development and chemotherapy resistance. In this study, we investigated the effects of targeted inhibition of the proteasomal deubiquitinases USP14 and UCHL5 on the development of CRC and resistance to 5-FU. By analyzing GEO datasets, we found that the mRNA expression levels of USP14 and UCHL5 in CRC tissues were significantly increased, and negatively correlated with the survival of CRC patients. Knockdown of both USP14 and UCHL5 led to increased 5-FU sensitivity in 5-FU-resistant CRC cell lines (RKO-R and HCT-15R), whereas overexpression of USP14 and UCHL5 in 5-FU-sensitive CRC cells decreased 5-FU sensitivity. B-AP15, a specific inhibitor of USP14 and UCHL5, (1-5 µM) dose-dependently inhibited the viability of RKO, RKO-R, HCT-15, and HCT-15R cells. Furthermore, treatment with b-AP15 reduced the malignant phenotype of CRC cells including cell proliferation and migration, and induced cell death in both 5-FU-sensitive and 5-FU-resistant CRC cells by impairing proteasome function and increasing reactive oxygen species (ROS) production. In addition, b-AP15 inhibited the activation of NF-κB pathway, suppressing cell proliferation. In 5-FU-sensitive and 5-FU-resistant CRC xenografts nude mice, administration of b-AP15 (8 mg·kg-1·d-1, intraperitoneal injection) effectively suppressed the growth of both types of tumors. These results demonstrate that USP14 and UCHL5 play an important role in the development of CRC and resistance to 5-FU. Targeting USP14 and UCHL5 with b-AP15 may represent a promising therapeutic strategy for the treatment of CRC.

LncRNA CRNDE binds hnRNPA1 to facilitate carbon monoxide poisoning-induced delayed encephalopathy via inhibiting UCHL5-mediated SMO deubiquitination.

Delayed encephalopathy after acute carbon monoxide poisoning (DEACMP) is one of the most common complications following carbon monoxide intoxication. Long noncoding RNAs (lncRNAs) exert critical functions in numerous neurological disorders. We intended to investigate the role of CRNDE in DEACMP. The DEACMP model in rats and the oxygen-glucose deprivation/reoxygenation (OGD/R) model in PC-12 cells were established. Brain and cell injuries were assessed with H&E staining, Nissl staining, TUNEL and CCK8 assays, respectively. Related proteins and RNAs were quantified with western blot and qRT-PCR. The N6-methyladenosine (m6A) level was determined using MeRIP-qPCR and immunofluorescence. Loss and gain function studies were performed to investigate the biological function of CRNDE. The potential mechanisms between each factor were explored using RNA immunoprecipitation, RNA-pull down and co-immunoprecipitation. CRNDE was increased in the hippocampal tissues of DEACMP rats and in OGD/R-treated PC-12 cells, which was positively correlated to m6A modification. Knockdown of CRNDE reduced cell damage and elevated UCHL5 and SMO expressions in OGD/R-treated PC-12 cells. hnRNPA1 was upregulated in DEACMP. In addition, inhibiting hnRNPA1 prevented apoptosis in PC-12 cells subjected to OGD/R. hnRNPA1 bound to CRNDE and remained in the nucleus, which inhibited UCHL5 expression through the formation of CRNDE-hnRNPA1-mRNA complex. UCHL5 could inhibit SMO ubiquitination and suppress PC-12 cell apoptosis during OGD/R. CRNDE silencing blocked brain injury in DEACMP, while knocking down UCHL5 reversed these effects. CRNDE interacted with hnRNPA1 to facilitate DEACMP via inhibition of UCHL5-mediated SMO deubiquitination. CRNDE might be a latent therapeutic target for treating DEACMP.

The CDK4/6-UCHL5-BRD4 axis confers resistance to BET inhibitors in MLL-rearranged leukemia cells by suppressing BRD4 protein degradation.

Among acute leukemias, mixed-lineage leukemia-rearranged (MLL-r) leukemia is associated with poor prognosis. Bromodomain and extra-terminal inhibitors (BETi) are promising agents for treatment of hematological malignancies; however, the mechanisms underlying sensitivity to BETi and biomarkers to predict sensitivity are yet to be clarified. Here, we established OTX015-resistant MLL-r cell lines (OTX015-R cells) and used them to explore therapeutic targets in BETi-resistant MLL-r leukemia. OTX015-R cells exhibited resistance to various BETi, and levels of bromodomain-containing protein 4 (BRD4) and BRD4-regulated molecules, such as c-MYC and B-cell/CLL lymphoma-2 (BCL-2), were remarkably increased in OTX015-R cells relative to those in the parental cells; however, BRD4 mRNA transcript levels were not elevated. These results suggest that overexpression of BRD4 protein, through suppression of BRD4 degradation, may contribute to BETi-resistance. Notably, expression of ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5) was increased in OTX015-R cells. Further, a UCHL5 inhibitor, b-AP15, and UCHL5 knockdown had antitumor effects by degrading BRD4. In addition, sensitivity to OTX015 was partially recovered in OTX015-R cells pretreated with b-AP15. Furthermore, cyclin-dependent kinase 4/6 (CDK4/6) inhibition decreased UCHL5 expression, suppressed OTX015-R cell proliferation, and induced apoptosis. These results indicate that the CDK4/6-UCHL5-BRD4 axis confers resistance to BETi by suppressing BRD4 degradation. We propose that this pathway is a potential novel therapeutic target in BETi-resistant MLL-r leukemia with BRD4 overexpression.

Proteostasis in the Hedgehog signaling pathway.

The Hedgehog (Hh) signaling pathway is crucial for the development of vertebrate and invertebrate animals alike. Hh ligand binds its receptor Patched (Ptc), allowing the activation of the obligate signal transducer Smoothened (Smo). The levels and localizations of both Ptc and Smo are regulated by ubiquitination, and Smo is under additional regulation by phosphorylation and SUMOylation. Downstream of Smo, the Ci/Gli family of transcription factors regulates the transcriptional responses to Hh. Phosphorylation, ubiquitination and SUMOylation are important for the stability and localization of Ci/Gli proteins and Hh signaling output. Finally, Suppressor of Fused directly regulates Ci/Gli proteins and itself is under proteolytic regulation that is critical for normal Hh signaling.

Synthesis and evaluation of tiaprofenic acid-derived UCHL5 deubiquitinase inhibitors.

The ubiquitin-proteasome system (UPS) plays an important role in maintaining protein homeostasis by degrading intracellular proteins. In the proteasome, poly-ubiquitinated proteins are deubiquitinated by three deubiquitinases (DUBs) associated with 19S regulatory particle before degradation via 20S core particle. Ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) is one of three proteasome-associated DUBs that control the fate of ubiquitinated substrates implicated in cancer survival and progression. In this study, we have performed virtual screening of an FDA approved drug library with UCHL5 and discovered tiaprofenic acid (TA) as a potential binder. With molecular docking analysis and in-vitro DUB assay, we have designed, synthesized, and evaluated a series of TA derivatives for inhibition of UCHL5 activity. We demonstrate that one TA derivative, TAB2, acts as an inhibitor of UCHL5.

LncRNA DRAIC inhibits proliferation and metastasis of gastric cancer cells through interfering with NFRKB deubiquitination mediated by UCHL5.

BACKGROUND: Long non-coding RNA (lncRNA) as a widespread and pivotal epigenetic molecule participates in the occurrence and progression of malignant tumors. DRAIC, a kind of lncRNA whose coding gene location is on 15q23 chromatin, has been found to be weakly expressed in a variety of malignant tumors and acts as a suppressor, but its characteristics and role in gastric cancer (GC) remain to be elucidated. METHODS: Sixty-seven primary GC tissues and paired paracancerous normal tissues were collected. Bioinformatics is used to predict the interaction molecules of DRAIC. DRAIC and NFRKB were overexpressed or interfered exogenously in GC cells by lentivirus or transient transfection. Quantitative real-time PCR (qPCR) and western blotting were used to evaluate the expression of DRAIC, UCHL5 and NFRKB. The combinations of DRAIC and NFRKB or UCHL5 and NFRKB were verified by RNA-IP and Co-IP assays. Ubiquitination-IP and the treatment of MG132 and CHX were used to detect the ubiquitylation level of NFRKB. The CCK-8 and transwell invasion and migration assays measured the proliferation, migration and invasion of GC cells. RESULTS: DRAIC is down-regulated in GC tissues and cell lines while its potential interacting molecules UCHL5 and NFRKB are up-regulated, and DRAIC is positively correlated with NFRKB protein instead of mRNA. Lower DRAIC and higher UCHL5 and NFRKB indicated advanced progression of GC patients. DRAIC could increase NFRKB protein significantly instead of NFRKB mRNA and UCHL5, and bind to UCHL5. DRAIC combined with UCHL5 and attenuated binding of UCHL5 and NFRKB, meanwhile promoting the degradation of NFRKB via ubiquitination, and then inhibited the proliferation and metastasis of GC cells, which can be rescued by oeNFRKB. CONCLUSION: DRAIC suppresses GC proliferation and metastasis via interfering with the combination of UCHL5 and NFRKB and mediating ubiquitination degradation.

Computational and biochemical studies of isothiocyanates as inhibitors of proteasomal cysteine deubiquitinases in human cancer cells.

Isothiocyanates (ITCs) are natural chemoprotective products found abundantly in cruciferous vegetables. However, the cancer-relevant targets and molecular mechanisms of ITCs remain unclear. We hypothesize that ITCs, as electrophiles, can interact with the catalytic triads (CYS, HIS, and ASP) of the proteasomal cysteine deubiquitinases USP14 and UCHL5, ultimately inhibiting their activities. In the current study, we exploited this possibility by performing both computational docking and biochemical validation assays using human breast and prostate cancer cell models. Docking results suggest that benzyl isothiocyanate, phenethyl isothiocyanate, and DL-sulforaphane are more potent inhibitors of UCHL5 than USP14, and these ITCs could interact with the catalytic triads of UCHL5 and USP14. Indeed, ubiquitin vinyl sulfone assay confirmed the inhibitory activity of each ITC on the ubiquitin-binding activity of UCHL5 and USP14. We also found that inhibition of USP-14 and UCHL5 activities by the ITCs caused increased levels of USP14 and UCHL5 proteins, but not the third 19S-deubiquitinating enzyme (DUB), POH1/RPN11, suggesting feedback loop activation and further supporting that ITCs are inhibitors of proteasomal cysteine DUBs. Associated with DUB inhibition by ITCs, ubiquitinated proteins were significantly increased, accompanied with induction of apoptosis, inhibition of proliferation and suppression of cell invasion. Our findings of ITCs as proteasomal cysteine DUB inhibitors should provide insightful information for designing, discovering and developing potent, specific 19S-DUB inhibitors for cancer therapies.

Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation.

BACKGROUND/AIMS: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. METHODS: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. RESULTS: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. CONCLUSIONS: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.

Molecular Characterization and Expression Profiles of Sp-uchl3 and Sp-uchl5 during Gonad Development of Scylla paramamosain.

Ubiquitin C-terminal hydrolases (UCHLs) are a subset of deubiquitinating enzymes, and are involved in numerous physiological processes. However, the role of UCHLs during gonad development has not been studied in crustaceans. In this study, we have first cloned and analyzed expression profiling of Sp-uchl3 and Sp-uchl5 genes from mud crab Scylla paramamosain. The full-length cDNA of Sp-uchl3 is of 1804 bp. Its expression level in the ovary was significantly higher than in other tissues (p < 0.01), and during gonadal development, its expression in both O1 and O5 stages was significantly higher than in the other three stages of ovaries (p < 0.05), while in T3 it was higher than in the former two stages of testes (p < 0.05). Meanwhile, the full-length cDNA of Sp-UCHL5 is 1217 bp. The expression level in the ovary was significantly higher than in other tissues (p < 0.01). Its expression in ovaries was higher than in testes during gonadal development (p < 0.05). The expression level in the O5 stage was the highest, followed by the O3 stage in ovarian development, and with no significant difference in the testis development (p > 0.05). These results provide basic data showing the role of Sp-UCHL3 and Sp-UCHL5 in the gonad development of the crab.

[Influence of UCHL5 on proliferation and apoptosis of SW527 breast cancer cells].

Objective: To investigate the effect of UCHL5 on proliferation and apoptosis of breast cancer cells. Methods: SW527 cells were infected with lentiviral vector carrying short hairpin RNA to delete the expression of UCHL5. 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to examine cell proliferation, and subcutaneous transplantation experiments were performed to detect tumor growth. Cell apoptosis was detected using Annexin V/ Propidium iodide (PI) double staining. The correlation between UCHL5 expression and the expressions of proliferation and apoptosis associated genes was analyzed using TCGA breast invasive carcinoma data set. The relationship between UCHL5 expression and breast cancer patients'survival was analyzed using Kaplan-Meier Plotter online tool. Results: After knockdown of UCHL5, A values of SW527 cells on day 2 and day 4 were 0.822±0.017 and 1.045±0.023, respectively, which were significantly lower than 0.976±0.016 and 1.284±0.025 of control cells on day 2 and day 4 (P<0.001). In vivo xenografted mouse model, the volume in UCHL5-suppressed group was (166.90±75.05) mm(3,) significantly smaller than (329.80±35.84) mm(3) in control group (P=0.029). Flow cytometry analysis showed the apoptotic rate of SW527 cells was (8.60±1.13)% after knockdown of UCHL5, significantly higher than (2.95±0.07)% of control group (P=0.020). TCGA database analysis showed that the expression of UCHL5 was positively correlated with the expressions of genes related to cell proliferation, in paralled with the increased expression of UCHL5, the expression of the pro-apoptosis associated genes was decreased. Kaplan-Meier Plotter analysis demonstrated that the overall survival and relapse-free survival of breast cancer patients with high expression of UCHL5 were much shorter (all P<0.001). Conclusions: Down-regulation of UCHL5 inhibits the proliferation and tumor formation and promotes apoptosis of SW527 cells. High expression of UCHL5 may predict poor prognosis of breast cancer patients.

UCHL5 expression associates with improved survival in lymph-node-positive rectal cancer.

Colorectal cancer is among the three most common cancer types for both genders, with a rising global incidence. To date, prognostic evaluation is difficult and largely dependent on early detection and successful surgery. UCHL5/Uch37 is an integral part of the protein homeostasis network as one of the three deubiquitinating enzymes associated with the 26S proteasome. Here, we have investigated in colorectal cancer the possible association of UCHL5 tumor expression and patient survival. UCHL5 tumor expression was evaluated by immunohistochemistry in 779 surgically treated colorectal cancer patients from Helsinki University Hospital, Finland, with assessment of clinicopathological parameters and the effect of UCHL5 expression on patient survival. High and undetectable UCHL5 expression both correlated with increased overall disease-specific survival in the subgroup of patients with lymph-node-positive (Dukes C/stage III) rectal cancer. Within this subgroup of 105 stage-III rectal cancer patients, none of the 7 with high UCHL5 expression died of colorectal cancer within 10 years after surgery ( p = 0.012). A similar, though less prominent, survival trend occurred throughout the whole patient cohort. In conclusion, UCHL5 is a promising novel prognostic marker in lymph-node-positive rectal cancer. Our results also advance the currently limited knowledge of biomarkers in colorectal cancer treatment.

Nuclear ubiquitin C-terminal hydrolase L5 expression associates with increased patient survival in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma is a lethal disease with an overall 5-year survival of less than 5%. Prognosis among surgically treated patients is difficult and identification of new biomarkers is essential for accurate prediction of patient outcome. As part of one of the major cellular protein degradation systems, the proteasome plays a fundamental role in both physiological and pathophysiological conditions including cancer. The proteasome-associated deubiquitinating enzyme ubiquitin C-terminal hydrolase L5 (UCHL5)/Uch37 is a modulator of proteasome activity with cancer prognostic marker potential. Cytoplasmic and nuclear immunoexpression of UCHL5 was evaluated in 154 surgical specimens from pancreatic ductal adenocarcinoma patients treated at Helsinki University Hospital, Finland, in 2000-2011. UCHL5 expression in relation to clinicopathological parameters and the association between UCHL5 In this study, positive expression and patient survival were assessed. Positive nuclear UCHL5 expression was associated with increased patient survival ( p = 0.005). A survival benefit was also detectable in these subgroups of patients: over 65 years ( p < 0.001), at tumor stages IIB to III ( p = 0.007), or with lymph-node positivity ( p = 0.006). In stages IIB to III disease, patients with positive nuclear UCHL5 expression showed a twofold increase in 5-year cancer-specific survival compared to those with negative expression. Multivariate analysis identified positive nuclear UCHL5 expression as an independent prognostic factor ( p = 0.012). In conclusion, UCHL5 expression could function as a prognostic marker in pancreatic ductal adenocarcinoma, particularly at disease stages IIB to III. As UCHL5 is one of the few markers predicting increased survival, our results may be of clinical relevance.

Expression of possible targets for new proteasome inhibitors in diffuse large B-cell lymphoma.

OBJECTIVES: Investigating expression of possible targets for proteasome inhibitors in patients with diffuse large B-cell lymphoma (DLBCL) and correlating the findings to clinical parameters and outcome. METHODS: Tumour material from 92 patients with DLBCL treated with either R-CHOP like (n = 69) or CHOP like (n = 23) regimens were stained for possible targets of proteasome inhibitors. RESULTS: The primary target molecule of bortezomib, proteasome subunit beta, type 5 (PSMB5), was not detected in the tumour cells in any of the cases but showed an abundant expression in cells in the microenvironment. However, the deubiquitinases (DUBs) of the proteasome, the ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) and the ubiquitin specific peptidase 14 (USP14), were detected in the cytoplasm of the tumour cells in 77% and 74% of the cases, respectively. The adhesion regulating molecule 1 (ADRM1) was detected in 98% of the cases. There was no correlation between the expression of any of the studied markers and clinical outcome or GC/non-GC phenotype. CONCLUSIONS: We suggest that UCHL5 and/or USP14 should be further evaluated as new targets for proteasome inhibitors in DLBCL. The lack of expression of PSMB5 on the tumour cells might provide an explanation of the relatively poor results of bortezomib in DLBCL.

Regulation of E2 promoter binding factor 1 (E2F1) transcriptional activity through a deubiquitinating enzyme, UCH37.

E2F1 is tightly controlled by multiple mechanisms, but whether ubiquitination regulates its transcriptional activity remains unknown. Here we identify UCH37 as the first, to our knowledge, deubiquitinating enzyme for E2F1. UCH37 does not deubiquitinate UbK48 chains or affect E2F1 protein stability. Instead, UCH37, but not a catalytically dead mutant, decreases the Lys-63-linked ubiquitination of E2F1 and activates its transcriptional activity. UCH37 depletion reduces the gene expression of both proliferative and pro-apoptotic E2F1 target genes. UCH37 depletion also decreases both cell proliferation and apoptosis induction in functional assays. Interestingly, UCH37 expression is induced by E2F1, and its level rises in G1/S transition and S phase, suggesting a positive feedback loop between UCH37 and E2F1. UCH37 protein and mRNA levels are also induced after DNA damage. UCH37 localizes to the promoters of E2F1 pro-apoptotic target genes such as caspase 3, caspase 7, PARP1, and Apaf-1 and activates their expression after DNA damage. Moreover, the expression of E2F1 proliferative and pro-apoptotic genes is correlated with the levels of UCH37 in many primary tumors. These results uncover a novel mechanism for E2F1 transcriptional activation through removal of its Lys-63-linked ubiquitination by UCH37.

Piperlongumine overcomes imatinib resistance by inducing proteasome inhibition in chronic myelogenous leukemia cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper longum L., an herbal medicine used in India and other Asian countries, is prescribed routinely for a range of diseases, including tumor. Piperlongumine, a natural product isolated from Piper longum L., has received widespread attention due to its various pharmacological activities, such as anti-inflammatory, antimicrobial, and antitumor effects. AIM OF THE STUDY: Chronic myelogenous leukemia (CML) is a hematopoietic disease caused by Bcr-Abl fusion gene, with an incidence of 15% in adult leukemias. Targeting Bcr-Abl by imatinib provides a successful treatment approach for CML. However, imatinib resistance is an inevitable issue for CML treatment. In particular, T315I mutant is the most stubborn of the Bcr-Abl point mutants associated with imatinib resistance. Therefore, it is urgent to find an alternative approach to conquer imatinib resistance. This study investigated the role of a natural product piperlongumine in overcoming imatinib resistance in CML. MATERIALS AND METHODS: Cell viability and apoptosis were evaluated by MTS assay and Annexin V/propidium iodide counterstaining assay, respectively. Levels of intracellular signaling proteins were assessed by Western blots. Mitochondrial membrane potential was reflected by the fluorescence intensity of rhodamine-123. The function of proteasome was detected using 20S proteasomal activity assay, proteasomal deubiquitinase activity assay, and deubiquitinase active-site-directed labeling. The antitumor effects of piperlongumine were assessed with mice xenografts. RESULTS: We demonstrate that (i) Piperlongumine inhibits proteasome function by targeting 20S proteasomal peptidases and 19S proteasomal deubiquitinases (USP14 and UCHL5) in Bcr-Abl-WT and Bcr-Abl-T315I CML cells; (ii) Piperlongumine inhibits the cell viability of CML cell lines and primary CML cells; (iii) Proteasome inhibition by piperlongumine leads to cell apoptosis and downregulation of Bcr-Abl; (iv) Piperlongumine suppresses the tumor growth of CML xenografts. CONCLUSIONS: These results support that blockade of proteasome activity by piperlongumine provides a new therapeutic strategy for treating imatinib-resistant CML.