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Rationale: Androgen receptor splice variant 7 (AR-V7) is a leading cause of the development of castration-resistant prostate cancer (CRPC). However, the regulation and function of AR-V7 at levels of post-translational modifications in prostate cancer therapy remain poorly understood. Here, we conducted a library screen of natural products to identify potential small molecules responsible for AR-V7 protein degradation in human prostate cancer cell lines. Methods: A natural product library was used to screen the inhibitor of AR-V7. Co-IP and biomass spectrum assays were used to identify the AR-V7-interacting proteins, whereas western blot, confocal microscopy, RNA interfering, and gene transfection were used to validate these interactions. Cell viability, EDU staining, and colony formation assays were employed to detect cell growth and proliferation. Flowcytometry assays were used to detect the distribution of cell cycle. Mouse xenograft models were used to study the anti-CRPC effects in vivo. Results: This screen identified rutaecarpine, one of the major components of the Chinese medicine Evodia rutaecarpa, as a novel chemical that selectively induces AR-V7 protein degradation via K48-linked ubiquitination. Mechanically, this effect relies on rutaecarpine inducing the formation of a GRP78-AR-V7 protein complex, which further recruits the E3 ligase SIAH2 to directly promote the ubiquitination of AR-V7. Consequently, the genetic and pharmacological activation of the GRP78-dependent AR-V7 protein degradation restores the sensitivity of castration-resistant prostate cancer to anti-androgen therapy in cell culture and animal models. Conclusions: These findings not only provide a new approach for overcoming castration-resistance in prostate cancer therapy, but also increase our understanding about the interplay between molecular chaperones and ubiquitin ligase in shaping protein stability.

Background Androgen receptor (AR) is expressed in approximately 70% of breast tumors. Recent studies increasingly support AR as a potential therapeutic target of AR-positive breast cancer. We have previously reported that deubiquitinase USP14 stabilizes AR proteins by deubiquitination and USP14 inhibition results in inhibition of cell growth and tumor progression in AR-positive prostate cancer and breast cancer. The current study aims to explore the anticancer effect of a treatment combining AR antagonist enzalutamide with USP14 inhibition on breast cancer cells. Methods The combining effects of enzalutamide and USP14 inhibition on breast cancer cell proliferation and apoptosis and associated cell signaling were evaluated in vitro and in vivo. Results USP14 inhibition via administration of IU1 or USP14-specific siRNA/shRNA enhanced cell growth inhibition and apoptosis induction by enzalutamide in breast cancer cell lines in vitro and in vivo. Additionally, the combination of enzalutamide with USP14 inhibition/knockdown induced significant downregulation of AR proteins and suppression of AR-related signaling pathways, including Wnt/β-catenin and PI3K/AKT pathways. Moreover, AKT inhibition via MK2206 increased the antiproliferative and proapoptotic effects of enzalutamide+IU1 combined treatment. Conclusion Collectively, our data suggest that USP14 inhibition in combination with enzalutamide represents a potentially new therapeutic strategy for breast cancer. Electronic supplementary material The online version of this article (10.1186/s13046-019-1227-7) contains supplementary material, which is available to authorized users.

Auranofin (Aur) inhibits thioredoxin reductases and is also an inhibitor of 19S proteasome associated deubiquitinases, targeting USP14 and UCHL5. Androgen receptor is often over-expressed in prostate cancer (PCa) and is strongly linked to PCa growth and progression. Consequently, androgen deprivation therapy (ADT) that reduces androgen has been applied to treat androgen receptor-mediated PCa for decades. Nevertheless, most ADT treated patients experience relapse due to the development of the castration-resistant PCa. Numerous studies have shown that down-regulation of cellular androgen receptor level, including inhibiting its transcription and promoting its protein degradation, is lethal to PCa cells. Here we report that Aur arrested cell cycle progression and induced apoptosis of PCa cells. Co-inhibition of USP14 and UCHL5 with Aur facilitated the ubiquitination and degradation of androgen receptors in LNcap and 22RV1 PCa cells. Our results also show that Aur decreases the mRNA level of androgen receptors. In conclusion, our findings suggest that Aur is a promising agent for clinical translation to treat PCa.

Background Hepatocellular carcinoma (HCC) remains a medical challenge due to its high proliferation and metastasis. Although deubiquitinating enzymes (DUBs) play a key role in regulating protein degradation, their pathological roles in HCC have not been fully elucidated. Methods By using biomass spectrometry, co-immunoprecipitation, western blotting and immunofluorescence assays, we identify ribosomal protein S16 (RPS16) as a key substrate of ubiquitin-specific peptidase 1 (USP1). The role of USP1-RPS16 axis in the progression of HCC was evaluated in cell cultures, in xenograft mouse models, and in clinical observations. Results We show that USP1 interacts with RPS16. The depletion of USP1 increases the level of K48-linked ubiquitinated-RPS16, leading to proteasome-dependent RPS16 degradation. In contrast, overexpression of USP1-WT instead of USP1-C90A (DUB inactivation mutant) reduces the level of K48-linked ubiquitinated RPS16, thereby stabilizing RPS16. Consequently, USP1 depletion mimics RPS16 deficiency with respect to the inhibition of growth and metastasis, whereas transfection-enforced re-expression of RPS16 restores oncogenic-like activity in USP1-deficient HCC cells. Importantly, the high expression of USP1 and RPS16 in liver tissue is a prognostic factor for poor survival of HCC patients. Conclusions These findings reveal a previously unrecognized role for the activation of USP1-RPS16 pathway in driving HCC, which may be further developed as a novel strategy for cancer treatment.

Melanoma, the most threatening cancer in the skin, has been considered to be driven by the carcinogenic RAF-MEK1/2-ERK1/2 signaling pathway. This signaling pathway is usually mainly dysregulated by mutations in BRAF or RAS in skin melanomas. Although inhibitors targeting mutant BRAF, such as vemurafenib, have improved the clinical outcome of melanoma patients with BRAF mutations, the efficiency of vemurafenib is limited in many patients. Here, we show that blood bilirubin in patients with BRAF-mutant melanoma treated with vemurafenib is negatively correlated with clinical outcomes. In vitro and animal experiments show that bilirubin can abrogate vemurafenib-induced growth suppression of BRAF-mutant melanoma cells. Moreover, bilirubin can remarkably rescue vemurafenib-induced apoptosis. Mechanically, the activation of ERK-MNK1 axis is required for bilirubin-induced reversal effects post vemurafenib treatment. Our findings not only demonstrate that bilirubin is an unfavorable for patients with BRAF-mutant melanoma who received vemurafenib treatment, but also uncover the underlying mechanism by which bilirubin restrains the anticancer effect of vemurafenib on BRAF-mutant melanoma cells.

Prostate cancer (PC) is the second most common cancer with limited treatment option in males. Although the reactivation of embryonic signals in adult cells is one of the characteristics of cancer, the underlying protein degradation mechanism remains elusive. Here, we show that the molecular chaperone GRP75 is a key player in PC cells by maintaining the protein stability of SIX1, a transcription factor for embryonic development. Mechanistically, GRP75 provides a platform to recruit the deubiquitinating enzyme USP1 to inhibit K48-linked polyubiquitination of SIX1. Structurally, the C-terminus of GRP75 (433-679 aa) contains a peptide binding domain, which is required for the formation of GRP75-USP1-SIX1 protein complex. Functionally, pharmacological or genetic inhibition of the GRP75-USP1-SIX1 protein complex suppresses tumor growth and overcomes the castration resistance of PC cells in vitro and in xenograft mouse models. Clinically, the protein expression of SIX1 in PC tumor tissues is positively correlated with the expression of GRP75 and USP1. These new findings not only enhance our understanding of the protein degradation mechanism, but also may provide a potential way to enhance the anti-cancer activity of androgen suppression therapy.

Background Our previous study in animal models revealed that bilirubin could induce Aβ formation and deposition. Bilirubin may be important in neurodegenerative dementia with Aβ deposition. Hence, lowering the concentration of the free bilirubin capable of crossing the blood brain-barrier may benefit the treatment of Alzheimer's disease (AD). Objectives The objectives of this study were to examine the change in the serum bilirubin and albumin concentrations of dementia patients with Aβ deposition, and to determine the effects of intravenous administration of albumin in the treatment of AD. Methods Bilirubin and albumin concentrations in dementia patients with Aβ deposition were examined. Cell viability and apoptosis were determined in dopaminergic neuron-like cells MN9D treated with bilirubin in the presence of diverse concentrations of serum. Human albumin at a dose of 10 g every 2 weeks for 24 weeks was administered intravenously to AD patients to examine the effect of albumin on AD symptoms. Results Significantly higher indirect bilirubin (IBIL) concentrations, lower albumin concentrations, and higher ratio of IBIL to albumin (IBIL/ALB) were observed in dementia patients with Aβ deposition, including AD, dementia with Lewy bodies, and general paresis of insane. In vitro assays showed that bilirubin-induced injury in cultured dopaminergic neuron-like cells negatively depends on the concentration of serum in the culture medium. General linear model with repeated measures analysis indicated a main effect of group on the change in albumin concentrations and Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory scale (ADCS-ADL) scores, and the main effect of time and group, and group-by-time interaction on the change of Clinical Dementia Rating Scale-Sum of Boxes (CDR-SB) scores. Analysis of the combined data of the entire 28 weeks of assessment period using the area under curve convincingly showed significantly improvements in the change of albumin concentrations, ADCS-ADL scores, and CDR-SB scores. Conclusion IBIL and the IBIL/ALB ratio are significantly higher in dementia patients with Aβ deposition, and intravenous administration of albumin is beneficial to AD treatment. Trial registration The intervention study was registered at http://www.chictr.org.cn (ChiCTR-IOR-17011539). Date of registration: June 1, 2017.

Estrogen receptor α (ERα) is expressed in ~67% of breast cancers and is critical to their proliferation and progression. The expression of ERα is regarded as a major prognostic marker, making it a meaningful target to treat breast cancer (BCa). However, hormone receptor-positive BCa was sometimes irresponsive or even resistant to classic anti-hormonal therapies (e.g., fulvestrant and tamoxifen). Hence, novel anti-endocrine therapies are urgent for ERα+ BCa. A phase II study suggested that bortezomib, an inhibitor blocking the activity of 20 S proteasomes, intervenes in cancer progression for anti-endocrine therapy in BCa. Here we report that proteasome-associated deubiquitinases (USP14 and UCHL5) inhibitors b-AP15 and platinum pyrithione (PtPT) induce growth inhibition in ERα+ BCa cells. Further studies show that these inhibitors induce cell cycle arrest and apoptosis associated with caspase activation, endoplasmic reticulum (ER) stress and the downregulation of ERα. Moreover, we suggest that b-AP15 and PtPT block ERα signaling via enhancing the ubiquitin-mediated degradation of ERα and inhibiting the transcription of ERα. Collectively, these findings demonstrate that proteasome-associated deubiquitinases inhibitors b-AP15 and PtPT may have the potential to treat BCa resistant to anti-hormonal therapy.

Background Ovarian carcinoma is one of the most aggressive gynecological malignant neoplasms and makes up 25–30% of all cancer cases of the female genital tract. Currently, resistance to traditional chemotherapy is a great challenge for patients with Epithelial ovarian cancer (EOC). Therefore, identifying novel agents for EOC treatment is essential and urgent. Method MTS assay was used to analyze the cell viability and proliferation of cancer cells. Flow cytometry was employed to analyze cell cycle distribution and cell apoptosis. Protein signaling pathways were detected by western blot and immunohistochemical staining. Nude mouse experiment was performed to test the in vivo effect of platinum pyrithione (PtPT). Results PtPT is a chemically well-characterized synthetic complex of platinum that potently inhibits proteasome-associated deubiquitinases USP14 and UCHL5 activity and shows selective cytotoxicity to multiple cancer cells without damaging DNA. We found that PtPT significantly accumulated ubquitinated-proteins and suppressed the proliferation of multiple EOC cells. Additionally, PtPT induced G2 phase arrest and apoptosis in both A2780 and SKOV3 cells. More importantly, animal experiments showed that PtPT dramatically suppressed the growth of EOC xenografts without obvious side effects. Conclusion These results suggest that through proteasome inhibition, PtPT significantly suppressed the proliferation of EOC in vitro and in vivo and could be developed as a novel agent for EOC treatment in the future.

// Jianyu Cai 1, * , Xiaohong Xia 1, * , Yuning Liao 1, * , Ningning Liu 1, 2 , Zhiqiang Guo 1 , Jinghong Chen 1 , Li Yang 1 , Huidan Long 1 , Qianqian Yang 1 , Xiaolan Zhang 1 , Lu Xiao 1 , Xuejun Wang 1, 3 , Hongbiao Huang 1 and Jinbao Liu 1 1 Protein Modification and Degradation Lab, SKLRD, School of Basic Medical Sciences, The Affiliated Cancer Hospital of Guangzhou Medical University, Guangdong 511436, China 2 Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510260, China 3 Division of Basic Biomedical Sciences, Sanford School of Medicine of The University of South Dakota, Vermillion, South Dakota 57069, USA * Cai J, Xia X and Liao Y contributed equally to this work Correspondence to: Hongbiao Huang, email: hhb800616@126.com Jinbao Liu, email: jliu@gzhmu.edu.cn Keywords: b-AP15, deubiquitinase inhibitor, prostate cancer, apoptosis Received: February 18, 2017 Accepted: May 22, 2017 Published: June 28, 2017 ABSTRACT Prostate cancer (PCa) remains a leading cause of cancer-related death in men. Especially, a subset of patients will eventually progress to the metastatic castrate-resistant prostate cancer (CRPC) which is currently incurable. Deubiquitinases (DUBs) associated with the 19S proteasome regulatory particle are increasingly emerging as significant therapeutic targets in numerous cancers. Recently, a novel small molecule b-AP15 is identified as an inhibitor of the USP14/UCHL5 (DUBs) of the 19S proteasome, resulting in cell growth inhibition and apoptosis in several human cancer cell lines. Here, we studied the therapeutic effect of b-AP15 in PCa, and our results indicate that (i) b-AP15 decreases viability, proliferation and triggers cytotoxicity to both androgen receptor-dependent and -independent PCa cells in vitro and in vivo , associated with caspase activation, inhibition of mitochondria function, increased reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress; (ii) pan-caspase inhibitor z-VAD-FMK and ROS scavenger N-acetyl-L-cysteine (NAC) efficiently block apoptosis but not proteasome inhibition induced by exposure of b-AP15; (iii) treatment with b-AP15 in androgen-dependent prostate cancer (ADPC) cells down-regulates the expression of androgen receptor (AR), which is degraded via the ubiquitin proteasome system. Hence, the potent anti-tumor effect of b-AP15 on both androgen receptor-dependent and -independent PCa cells identifies a new promising therapeutic strategy for prostate cancer.

Breast cancer is the most common malignancy in women around the world. Estrogen receptor α (ERα) is expressed in approximately 70% of breast tumors, and considered as one of most effective targets in breast cancer therapy. It has been reported that the degradation of ERα protein is mediated by ubiquitin-proteasome system. However, little is known about the regulation of ERα deubiquitination, a critical constituent of its degradation control. The current study first reports that there is a positive correlation between ERα and ubiquitin specific protease 7 (USP7) protein levels in human breast tumor tissues. Subsequent studies showed that USP7 physically interacted with the ERα, thereby mediating the deubiquitination and stabilization of ERα. In addition, USP7 inhibition or silencing led to growth inhibition and apoptosis of ERα-positive breast cancer cells both in vitro and in vivo. Furthermore, overexpression of ERα rescued the USP7 silencing-induced cell cycle arrest and apoptosis, supporting that ERα status is essential to the function of USP7 in breast carcinogenesis. Overall, this study suggests that targeting USP7-ERα complex could be a potential strategy to treat ERα-positive breast cancer.

Background Prostate cancer (PCa) remains a challenge worldwide. Due to the development of castration-resistance, traditional first-line androgen deprivation therapy (ADT) became powerlessness. Epidermal growth factor receptor (EGFR) is a well characterized therapeutic target to treat colorectal carcinoma and non-small cell lung cancer. Increasing studies have unraveled the significance of EGFR and its downstream signaling in the progression of castration-resistant PCa. Method MTS, colony formation and Edu staining assays were used to analyze the cell proliferation of PCa cells. Flow cytometry was used to analyze PCa cell cycle distribution and cell apoptosis. Western blot was used to measure the expression of key proteins associated with cell cycle progression, apoptosis and EGFR signaling pathways. Transfection of exogenous small interfering RNA (siRNA) or plasmid was used to intervene specific gene expression. Nude mouse model was employed to test the in vivo effect of Spautin-1. Results The current study reveals that Spautin-1, a known inhibitor of ubiquitin-specific peptidase 10 (USP10) and USP13, inhibits EGFR phosphorylation and the activation of its downstream signaling. Inhibition of EGFR signaling induced by Spautin-1 leads to cell cycle arrest and apoptosis of PCa in a USP10/USP13 independent manner. The application of Spautin-1 reduces the expression of glucose transporter 1 (Glut1) and dramatically induces cell death under glucose deprivation condition. In vivo experiments show a potent anti-tumor effect of Spautin-1 alone and in combination with Enzalutamide. Conclusion This study demonstrates the therapeutic potential of EGFR signaling inhibition by the use of Spautin-1 for PCa treatment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1165-4) contains supplementary material, which is available to authorized users.

The ubiquitin-proteasome system (UPS) plays a central role in various cellular processes through selectively degrading proteins involved in critical cellular functions. Targeting UPS has been validated as a novel strategy for treating human cancer, as inhibitors of the 20S proteasome catalytic activity are currently in clinical use for treatment of multiple myeloma and other cancers, and the deubiquitinase activity associated with the proteasome is also a valid target for anticancer agents. Recent studies suggested that zinc pyrithione, an FDA-approved antidandruff agent, may have antitumor activity, but the detailed molecular mechanisms remain unclear. Here we report that zinc pyrithione (ZnPT) targets the proteasome-associated DUBs (USP14 and UCHL5) and inhibits their activities, resulting in a rapid accumulation of protein-ubiquitin conjugates, but without inhibiting the proteolytic activities of 20S proteasomes. Furthermore, ZnPT exhibits cytotoxic effects against various cancer cell lines in vitro, selectively kills bone marrow cells from leukemia patients ex vivo, and efficiently inhibits the growth of lung adenocarcinoma cancer cell xenografts in nude mice. This study has identified zinc pyrithione, an FDA-approved pharmacological agent with potential antitumor properties as a proteasomal DUB inhibitor.

Cardiac hypertrophy, a compensatory response to various stimuli in the heart, independently predicts cardiovascular ailments and related deaths. Increasing evidence indicates ubiquitin-proteasome signaling contributes to cardiac hypertrophy regulation. Here, we identified ubiquitin-specific protease 14 (USP14), a 19S proteasome associated deubiquitinase (DUB), as a novel target for cardiac hypertrophy therapy via inhibition of the GSK-3β pathway. Indeed, USP14 expression was increased in an animal model of abdominal aorta constriction. In an angiotensin II (AngII) induced primary neonatal rat cardiomyocyte hypertrophy model, USP14 expression was increased in a time-dependent manner, and reduced USP14 deubiquitinase activity or USP14 knockdown resulted in lower expression levels of the myocardial hypertrophy specific marker β-MHC, and subsequent decreased GSK-3β phosphorylation. In conclusion, USP14 mediates the development of cardiac hypertrophy by promoting GSK-3β phosphorylation, suggesting that USP14 might represent a novel therapeutic target for cardiac hypertrophy treatment.

Bortezomib (Bor), a proteasome inhibitor, has marked therapeutic effects in multiple myeloma (MM), and its synergistic effects with other anticancer agents have been widely investigated. In the present study, endoplasmic reticulum (ER) stress was the target of the treatment strategy; anacardic acid (AA) and Bor induce ER stress, resulting in apoptosis of multiple myeloma cells. AA/Bor combination therapy exhibited overt cytotoxicity in MM cells, by synergistically reducing cell growth and promoting cell death. Notably, expression levels of the stress‑associated molecules binding protein, phosphorylated eukaryotic initiation factor 2α, activating transcription factor 4 (ATF4) and CCAAT‑enhancer binding protein homologous protein (CHOP) were increased following treatment. AA/Bor combination therapy‑induced U266 cell cytotoxicity was partially reversed by ATF4 gene silencing and slightly enhanced by CHOP knockdown. The results of the present study suggest that AA/Bor combination may be a potential therapeutic strategy for MM treatment.

Bcr-Abl is the primary cause as well as currently key therapeutic target of chronic myeloid leukemia (CML). SKP2, an E3 ligase, is a downstream factor of Bcr-Abl to motivate the cell cycle transition of CML and also found to bind and activate Bcr-Abl in reverse. Therefore, SKP2/Bcr-Abl pathway is an attractive target for CML treatment. This study aims to identify an inhibitor of the SKP2/Bcr-Abl pathway based on a large screening of the natural products. We demonstrate that Diosmetin, a kind of phytoestrogens, notably downregulates the expression of SKP2, Bcr-Abl phosphorylation, and moderately downregulates the Bcr-Abl level. Furthermore, Diosmetin displays a favorable anti-tumor activity in CML cells and xenograft models. Collectively, our study reveals a natural compound in the treatment of CML on the basis of SKP2/Bcr-Abl signaling pathway.

It has been well known that androgen receptor (AR) is critical to prostate cancer development and progression. It has also been documented that AR is expressed in more than 60% of breast tumors, which promotes the growth of estrogen receptor-negative (ER-)/AR-positive (AR+) breast cancer cells. Thus, AR might be a potential therapeutic target for AR-positive/ER-negative breast cancer patients. Previously we reported that in prostate cancer cells proteasome-associated deubiquitinase ubiquitin-specific protease 14 (USP14) stabilized AR protein level by removing its ubiquitin chain. In the current study, we studied the USP14-AR protein interaction and cell proliferation status after USP14 reduction or inhibition in breast cancer cells, and our results support the conclusion that targeting USP14 is a novel strategy for treating AR-responsive breast cancer. We found that inhibition of USP14 accelerated the K48-ubiquitination and proteasome-mediated degradation of AR protein. Additionally, both genetic and pharmacological inhibition of USP14 significantly suppressed cell proliferation in AR-responsive breast cancer cells by blocking G0/G1 to S phase transition and inducing apoptosis. Moreover, AR overexpression inhibited USP14 inhibition-induced events, suggesting that AR deubiquitination by USP14 is critical for breast cancer growth and USP14 inhibition is a possible strategy to treat AR-positive breast cancer.

EGF receptor tyrosine kinase inhibitors (EGFR-TKIs) have been widely used as a standard therapy in non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, most if not all of the patients who initially have responded to EGFR-TKIs later experience progression or deterioration of the disease while still on the treatment. Drug resistance becomes inevitable due to the emergence of the second-site EGFR T790M mutation within exon 20, MET and HER2 amplification, small cell histologic transformation and rare secondary BRAF mutations. The acquired drug resistance limits the efficacy of EGFR-TKIs in patients. Thalidomide is a widely used anti-angiogenic and immunomodulatory drug with anticancer effects. The current study was aimed to explore the combined effects of gefitinib and thalidomide on NSCLC. The combination of thalidomide and gefitinib induced antiproliferative and proapoptotic effects in HCC827, PC9, and PC9GR cells. The inhibition of EGFR phosphorylation and downstream signaling was more pronounced in the thalidomide and gefitinib co-treatment group as compared with the single agent treatment groups. Further study revealed that the inhibitors of AKT, MEK/ERK, and p38 increased the antiproliferative and proapoptotic effects of the combined treatment of thalidomide and gefitinib. However, JNK inhibition moderately abrogated cell apoptosis induced by the co-treatment. In conclusion, thalidomide and gefitinib exhibit synergistic effects on both TKI-sensitive and -resistant NSCLC cells by targeting the EGFR signaling pathways, suggesting that the combination strategy is promising for the treatment of NSCLC.

The molecular mechanism underlying bilirubin neurotoxicity remains obscure. Ubiquitin–proteasome system-mediated proteolysis is pivotal to virtually all cellular processes and cell survival. Here we report for the first time that bilirubin at a clinically relevant elevated level impairs proteasomal function via inhibiting both the 19S proteasome-associated deubiquitinases (USP14 and UCHL5) and the chymotrypsin-like (CT-like) peptidase activity of 20S proteasomes, thereby contributing to bilirubin neurotoxicity. This is supported by multiple lines of evidence. First, sera from patients with hyperbilirubinemia were able to inhibit the peptidase activity of purified 20S proteasome in vitro in a bilirubin concentration-dependent manner; meanwhile, the blood cells of these patients showed significantly increased levels of ubiquitinated proteins (Ub-prs), consistent with proteasome inhibition. Second, intracerebroventricular injection to adult rats or intraperitoneal injections to neonatal rats of bilirubin-induced neural accumulation of Ub-prs, concurrent with other neural pathology; and brain malfunction and pathology induced by neonatal exposure to hyperbilirubinemia were detectable in the rats during their adulthood. Third, in primary cultures of hippocampal neurons, bilirubin strikingly induced Ub-pr accumulation before the activation of cell death pathway becomes discernible. Finally, bilirubin in vitro directly inhibited both the deubiquitination activity of proteasome-associated USP14 and UCHL5 and the CT-like peptidase activity of purified 20S proteasomes, in a dose-dependent manner. Hence, this study has discovered that increased bilirubin at a clinically achievable level can act as a proteasome inhibitor via targeting the 19S proteasome-associated deubiquitinases (DUBs) and, perhaps to a less extent, the 20S proteasome, identifying a novel mechanism for bilirubin neurotoxicity.

Androgen receptor (AR) is frequently over-expressed and plays a critical role in the growth and progression of human prostate cancer. The therapy attempting to target AR signalling was established in decades ago but the treatment of prostate cancer is far from being satisfactory. The assignable cause is that our understanding of the mechanism of AR regulation and re-activation remains incomplete. Increasing evidence suggests that deubiquitinases are involved in the regulation of cancer development and progression but the specific underlying mechanism often is not elucidated. In the current study, we have identified ubiquitin-specific protease 14 (USP14) as a novel regulator of AR, inhibiting the degradation of AR via deubiquitinating this oncoprotein in the androgen-responsive prostate cancer cells. We found that (i) USP14 could bind to AR, and additionally, both genetic and pharmacological inhibition of USP14 accelerated the ubiquitination and degradation of AR; (ii) downregulation or inhibition of USP14 suppressed cell proliferation and colony formation of LNcap cells and, conversely, overexpression of USP14 promoted the proliferation; and (iii) reduction or inhibition of USP14 induced G0/G1 phase arrest in LNcap prostate cancer cells. Hence, we conclude that USP14 promotes prostate cancer progression likely through stabilization of AR, suggesting that USP14 could be a promising therapeutic target for prostate cancer.

Based on the central role of the ubiquitin-proteasome system (UPS) in the degradation of cellular proteins, proteasome inhibition has been considered an attractive approach for anticancer therapy. Deubiquitinases (DUBs) remove ubiquitin conjugates from diverse substrates; therefore, they are essential regulators of the UPS. DUB inhibitors, especially the inhibitors of proteasomal DUBs are becoming a research hotspot in targeted cancer therapy. Previous studies have shown that metal complexes, such as copper and zinc complexes, can induce cancer cell apoptosis through inhibiting UPS function. Moreover, we have found that copper pyrithione inhibits both 19S proteasome-associated DUBs and 20S proteasome activity with a mechanism distinct from that of the classical 20S proteasome inhibitor bortezomib. In the present study, we reveal that (i) nickel pyrithione complex (NiPT) potently inhibits the UPS via targeting the 19S proteasome-associated DUBs (UCHL5 and USP14), without effecting on the 20S proteasome; (ii) NiPT selectively induces proteasome inhibition and apoptosis in cultured tumor cells and cancer cells from acute myeloid leukemia human patients; and (iii) NiPT inhibits proteasome function and tumor growth in nude mice. This study, for the first time, uncovers a nickel complex as an effective inhibitor of the 19S proteasomal DUBs and suggests a potentially new strategy for cancer treatment.

DNA is the well-known molecular target of current platinum-based anticancer drugs; consequently, their clinical use is severely restricted by their systemic toxicities and drug resistance originating from non-selective DNA damage. Various strategies have been developed to circumvent the shortcomings of platinum-based chemotherapy but the inherent problem remains unsolved. Here we report that platinum pyrithione (PtPT), a chemically well-characterized synthetic complex of platinum, inhibits proteasome function and thereby exhibits greater and more selective cytotoxicity to multiple cancer cells than cisplatin, without showing discernible DNA damage both in vitro and in vivo. Moreover, unlike the classical proteasome inhibitor bortezomib/Velcade which inhibits the proteasome via blocking the peptidase activity of 20S proteasomes, PtPT primarily deactivates 26S proteasome-associated deubiquitinases USP14 and UCHL5. Furthermore, PtPT can selectively induce cytotoxicity and proteasome inhibition in cancer cells from leukemia patients but not peripheral blood mononuclear cells from healthy humans. In nude mice, PtPT also remarkably inhibited tumor xenograft growth, without showing the adverse effects that were induced by cisplatin. Hence, we have discovered a new platinum-based anti-tumor agent PtPT which targets 26S proteasome-associated deubiquitinases rather than DNA in the cell and thereby exerts safer and more potent anti-tumor effects, identifying a highly translatable new platinum-based anti-cancer strategy.