Proteasome deubiquitinases as novel targets for cancer therapy

The ubiquitin-proteasome system (UPS) is a conserved pathway regulating numerous biological processes including protein turnover, DNA repair, and intracellular trafficking. Tumor cells are dependent on a functioning UPS, making it an ideal target for the development of novel anti-cancer therapies. The development of bortezomib (Velcade^®) as a treatment for multiple myeloma and mantle cell lymphoma has verified this and suggests that targeting other components of the UPS may be a viable strategy for the treatment for cancer. We recently described a novel class of proteasome inhibitors that function by an alternative mechanism of action (D’Arcy et al., 2011). The small molecule b-AP15 blocks the deubiquitinase (DUB) activity of the 19S regulatory particle (19S RP) without inhibiting the proteolytic activities of the 20S core particle (20S CP). b-AP15 inhibits two proteasome-associated DUBs, USP14 and UCHL5, resulting in a rapid accumulation of high molecular weight ubiquitin conjugates and a functional proteasome shutdown. Interestingly, b-AP15 displays several differences to bortezomib including insensitivity to over-expression of the anti-apoptotic mediator Bcl-2 and anti-tumor activity in solid tumor models. In this review we will discuss the potential of proteasome deubiquitinase inhibitors as additions to the therapeutic arsenal against cancer.     

A Conserved Unfoldase Activity for the p97 AAA-ATPase in Proteasomal Degradation

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97^Ufd1/Npl4 complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.     

Proteasomal AAA-ATPases: structure and function

The 26S proteasome is a chambered protease in which the majority of selective cellular protein degradation takes place. Throughout evolution, access of protein substrates to chambered proteases is restricted and depends on AAA-ATPases. Mechanical force generated through cycles of ATP binding and hydrolysis is used to unfold substrates, open the gated proteolytic chamber and translocate the substrate into the active proteases within the cavity. Six distinct AAA-ATPases (Rpt1-6) at the ring base of the 19S regulatory particle of the proteasome are responsible for these three functions while interacting with the 20S catalytic chamber. Although high resolution structures of the eukaryotic 26S proteasome are not yet available, exciting recent studies shed light on the assembly of the hetero-hexameric Rpt ring and its consequent spatial arrangement, on the role of Rpt C-termini in opening the 20S 'gate', and on the contribution of each individual Rpt subunit to various cellular processes. These studies are illuminated by paradigms generated through studying PAN, the simpler homo-hexameric AAA-ATPase of the archaeal proteasome. The similarities between PAN and Rpts highlight the evolutionary conserved role of AAA-ATPase in protein degradation, whereas unique properties of divergent Rpts reflect the increased complexity and tighter regulation attributed to the eukaryotic proteasome.     

Proteasome inhibition by quercetin triggers macroautophagy and blocks mTOR activity

The bioflavonoid quercetin has long been known to exert anti-tumor effects, although the underlying mechanisms remain unknown. Investigation of the potential interference of this anti-oxidant with the efficacy of cell stress-inducing anti-cancer drugs revealed extensive intracellular vacuolation induced by quercetin in epithelial cancer cells that led to cell cycle arrest and ensuing apoptosis. Accumulation of biomarkers of autophagy, including fluorescent autophagy markers and acidotropic dyes characterized these vacuoles as phagolysosomes. Prior to the formation of autophagosomes, an immediate and pronounced inhibition of the autophagy-controlling mTOR activity in quercetin-treated cancer cells occurred, accompanied by a marked reduction in the phosphorylation of the mTOR substrates 4E-BP1 and p70S6 kinase. Assessment of cellular proteasome activity revealed an effective and immediate inhibition of the activity of the proteasome by quercetin in cancer cells. In addition to the formation of autophagosomes, accumulation of poly-ubiquitinated protein aggregates was observed. Thus, proteasome inhibition by quercetin can be regarded as a major cause of quercetin-induced cancer cell death. These results suggest potential new applications for quercetin in cancer science and identify quercetin as an easy-to-handle agent to study proteasome activity, mTOR signaling and autophagy in human cancer cells for cell biological purposes.     

Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.     

Proteasomal insensitivity of apoptin in tumor cells

The small viral protein apoptin is capable of inducing apoptosis selectively in human tumor cells. In normal cells apoptin localizes in the cytoplasm where it forms aggregates, becomes epitope-shielded and eventually degraded. By inhibiting the proteasome activity with the chemical inhibitors bortezomib and Ada-Ahx(3)L(3)VS apoptin levels can be stabilized in normal cells similar to the tumor suppressor p53 protein. In contrast, proteasome inhibition in tumor cells did not affect the apoptin stability while it still stabilized p53 levels. Apparently, apoptin is degraded by proteasomal activity in normal human cells, a process that no longer takes place in tumor cells. This loss of proteasomal susceptibility appears to be specific for apoptin.     

Proteasomal activity in mammalian spermatozoa

The proteasome is a multicatalytic cellular complex, which possess three different enzymatic activities, trypsin-like, chymotrypsin-like, and peptidylglutamyl peptidase. Its function is to remove abnormal or aged proteins. Recently, it has been suggested the participation of the sperm proteasome during mammalian fertilization. In this study, we present evidence that indicates that sperm extracts from several mammalian species, including hamster, mice, rats, bovine, rabbits, and humans all possess proteasome activity. We characterized the three specific activities of the proteasome using specific synthetic substrates and specific proteasome inhibitors. The results indicates that the highest specific activity detected was in mouse sperm toward the trypsin substrates and it was 1,114% of the activity of human sperm toward the chymotrypsin substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC, which was considered as 100%). In all cases, the lowest activity was toward substrates for the peptidylglutamyl peptidase hydrolyzing activity, and it was lowest for rabbit sperm (1.7% of the activity of human sperm toward the chymotrypsin substrate SLLVY-AMC). In addition, specific proteasome inhibitors were able to block all proteasome activities almost 100%, with the exception of clasto-Lactacystin beta-lactone upon rat sperm. All sperm extracts tested evidenced bands of about 29-32 kDa by Western blots using a monoclonal antibody against proteasome subunits alpha 1, 2, 3, 5, 6, and 7. In conclusion, sperm from several mammals possess enzymatic activities that correspond to the proteasome. The proteasome from the different species hold similar but distinctive enzymatic characteristics.     

Proteasome activity in tumors of the female reproductive system

Although proteasomes (multiproteinase protein complexes) are known to play an important role in cancer pathogenesis, there is little information about their activity in human tumor tissues. The chymotrypsin-like activity of proteasomes in breast cancer (BC) and endometrial cancer (EC) tissues was studied. It was shown that the chymotrypsin-like total proteasome activity and the 20S and 26S proteasome pool activities were significantly higher in malignant than in normal tissues. An increase in the size of either BC or EC tumors did not affect the proteasome activity, whereas the propagation of a malignant process did. If compared with BC non-metastatic tumors, a reliable decrease in the total activity and the 26S proteasome activity was observed in BC tumors with regional lymph node metastases. In EC tissues, the total proteasome activity and the 20S and 26S proteasome pool activities increased when the depth of tumor myometrial invasion grew. These data demonstrated that the proteasome activity significantly varied in the process of carcinogenesis. Further proteasome studies could serve as the basis for the development of new criteria for prognosis of female reproductive system cancer and the search for effective antitumor agents in targeted therapy.     

Proteasome activity in tumors of female reproductive system

Proteasomes (multiproteinase protein complexes) are known to play an important role in cancer pathogenesis, however, few information about their activity in human tumor tissues is available so far. We studied chymotrypsin-like activity of proteasomes in tissues of breast cancer (BC) and endometrial cancer (EC). The chymotrypsin-like total proteasome activity and the 20S and 26S proteasome activity in malignant tissues were shown to be significantly higher in malignant tumors than in normal tissues. No increase in proteasome activity was registered with larger tumor size in both BC and EC, whereas proteasome activity was changed with respect to the extent of tumor involvement. In breast cancer tissues, significant reductions in the total and the 26S proteaome activities were observed in tumors with regional lymph node metastases as compared to tumors without metastases. In endometrial cancer tissues, the total proteasome activity and the 20S and 26S proteasome activities were increased as the depth of myometrial invasion. The data obtained indicate that the proteasome acyivity is significantly changed in the process of cancerogenesis and further study is needed to develop new additional prognostic criteria and effective anti-tumor agents in molecular-directed therapy.     

Proteasomal regulation of caspase-8 in cancer cell apoptosis

Previous studies demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of caspase-8. The present study investigated the impact of proteasome inhibition on caspase-8 stability, ubiquitination, trafficking, and activation in cancer cells. Using caspase-8 deficient neuroblastoma (NB7) cells for reconstituting non-cleavable mutant forms of caspase-8, we demonstrated that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8, in response to proteasome inhibitor and GST-TRAIL. Moreover in the LNCaP human prostate cancer cells, caspase-8 polyubiquitination occurs after TRAIL stimulation and caspase-8 processing. Subcellular fractionation analysis revealed caspase-8 activity in both cytosol and plasma membrane fractions in both NB7 reconstituted caspase-8 cell lines, as well the LNCaP prostate cancer cells. The present results suggest that caspase-8 stabilization through proteasome inhibition leads to reactivation of the extrinsic pathway of apoptosis and identify E3 ligase mediating caspase-8 polyubiquitination, as a novel molecular target. Inhibition of this E3 ligase in combination with TRAIL towards restoring apoptosis signaling activation may have potential therapeutic significance in resistant tumors.     

Proteasomal degradation of retinoblastoma-related p130 during adipocyte differentiation.

Within 24 h of hormonally stimulated 3T3-L1 adipocyte differentiation, there are dramatic changes in the protein levels of p130 and p107, two members of the retinoblastoma tumor suppressor gene family. Designated the "p103:p107" switch, this alteration is characterized by a rapid and transient drop in p130 protein levels accompanied by a transient increase in both p107 mRNA and protein levels. Using protease inhibitors, the specific proteolytic pathway involved in degradation of p130 was examined. Treatment of cells with N-acetyl-leu-leu-norleucinal, an inhibitor that blocks proteolytic activity of type I calpain and the 26S proteasome, resulted in a complete block in the degradation of p130 protein, as well as adipocyte differentiation, suggesting that one of these pathways is involved in regulating p130 protein levels. Similar analysis with lactacystin, a specific inhibitor of the 26S proteasome, also resulted in a complete block in both differentiation and p130 degradation. Furthermore, both inhibitors blocked the increase in p107 protein levels normally observed on Day 1, suggesting that the p130:p107 switch is required for adipocyte differentiation and one of the early molecular events involved in activating the p130:p107 switch is the specific degradation of p130 by the 26S proteasome.     

Proteasomal ATPase-associated factor 1 negatively regulates proteasome activity by interacting with proteasomal ATPases

The 26S proteasome, composed of the 20S core and the 19S regulatory complex, plays a central role in ubiquitin-dependent proteolysis by catalyzing degradation of polyubiquitinated proteins. In a search for proteins involved in regulation of the proteasome, we affinity purified the 19S regulatory complex from HeLa cells and identified a novel protein of 43 kDa in size as an associated protein. Immunoprecipitation analyses suggested that this protein specifically interacted with the proteasomal ATPases. Hence the protein was named proteasomal ATPase-associated factor 1 (PAAF1). Immunoaffinity purification of PAAF1 confirmed its interaction with the 19S regulatory complex and further showed that the 19S regulatory complex bound with PAAF1 was not stably associated with the 20S core. Overexpression of PAAF1 in HeLa cells decreased the level of the 20S core associated with the 19S complex in a dose-dependent fashion, suggesting that PAAF1 binding to proteasomal ATPases inhibited the assembly of the 26S proteasome. Proteasomal degradation assays using reporters based on green fluorescent protein revealed that overexpression of PAAF1 inhibited the proteasome activity in vivo. Furthermore, the suppression of PAAF1 expression that is mediated by small inhibitory RNA enhanced the proteasome activity. These results suggest that PAAF1 functions as a negative regulator of the proteasome by controlling the assembly/disassembly of the proteasome.     

The Proteasome-associated Protein Ecm29 Inhibits Proteasomal ATPase Activity and in Vivo Protein Degradation by the Proteasome

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.     

Proteasomal degradation of tau protein

Filamentous inclusions composed of the microtubule-associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non-transfected SH-SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi-directional, proceeding from both N- and C-termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C-terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin-independent pathways for the proteasomal degradation of unfolded proteins.     

1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity

Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, l-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs.     

Cadmium pyrithione suppresses tumor growth in vitro and in vivo through inhibition of proteasomal deubiquitinase

The ubiquitin–proteasome system (UPS) is indispensable to the protein quality control in eukaryotic cells. Due to the remarkable clinical success of using proteasome inhibitors for clinical treatment of multiple myeloma, it is anticipated that targeting the UPS upstream of the proteasome step be an effective strategy for cancer therapy. Deubiquitinases (DUB) are proteases that remove ubiquitin from target proteins and therefore regulate multiple cellular processes including some signaling pathways altered in cancer cells. Thus, targeting DUB is a promising strategy for cancer drug discovery. Previously, we have reported that metal complexes, such as copper and gold complexes, can disrupt the UPS via suppressing the activity of 19S proteasome-associated DUBs and/or of the 20S proteasomes, thereby inducing cancer cell death. In this study, we found that cadmium pyrithione (CdPT) treatment led to remarkable accumulation of ubiquitinated proteins in cultured cancer cells and primary leukemia cells. CdPT potently inhibited the activity of proteasomal DUBs (USP14 and UCHL5), but slightly inhibited 20S proteasome activity. The anti-cancer activity of CdPT was associated with triggering apoptosis via caspase activation. Moreover, treatment with CdPT inhibited proteasome function and repressed tumor growth in animal xenograft models. Our results show that cadmium-containing complex CdPT may function as a novel proteasomal DUB inhibitor and suggest appealing prospects for cancer treatment.     

Scaled-down purification protocol to access proteomic analysis of 20S proteasome from human tissue samples: comparison of normal and tumor colorectal cells

The proteasome is a proteolytic complex that constitutes the main pathway for degradation of intracellular proteins in eukaryotic cells. It regulates many physiological processes and its dysfunction can lead to several pathologies like cancer. To study the 20S proteasome structure/activity relationship in cells that derive from human biopsy samples, we optimized an immuno-purification protocol for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the cytoplasmic 20S proteasome of adjacent normal and tumor colorectal cells arising from tissue samples of several patients. Proteomic analyses based on two-dimensional gel electrophoresis (2DE) and mass spectrometry showed that the subunit composition of 20S proteasomes from these normal and tumor cells were not significantly different. The proteasome activity was also assessed in the cytoplasmic extracts and was similar or higher in tumor colorectal than in the corresponding normal cells. The scaled-down 20S proteasome purification protocol developed here can be applied to any human clinical tissue samples and is compatible with further proteomic analyses.     

26S proteasome activity is down-regulated in lung cancer stem-like cells propagated in vitro

Cancer stem cells (CSCs) are a small subset of cancer cells capable of self-renewal and tumor maintenance. Eradicating cancer stem cells, the root of tumor origin and recurrence, has emerged as one promising approach to improve lung cancer survival. Cancer stem cells are reported to reside in the side population (SP) of cultured lung cancer cells. We report here the coexistence of a distinct population of non-SP (NSP) cells that have equivalent self-renewal capacity compared to SP cells in a lung tumor sphere assay. Compared with the corresponding cells in monolayer cultures, lung tumor spheres, formed from human non-small cell lung carcinoma cell lines A549 or H1299, showed marked morphologic differences and increased expression of the stem cell markers CD133 and OCT3/4. Lung tumor spheres also exhibited increased tumorigenic potential as only 10,000 lung tumor sphere cells were required to produce xenografts tumors in nude mice, whereas the same number of monolayer cells failed to induce tumors. We also demonstrate that lung tumor spheres showed decreased 26S proteasome activity compared to monolayer. By using the ZsGreen-cODC (C-terminal sequence that directs degradation of Ornithine Decarboxylase) reporter assay in NSCLC cell lines, only less than 1% monolayer cultures were ZsGreen positive indicating low 26S proteasome, whereas lung tumor sphere showed increased numbers of ZsGreen-positive cells, suggesting the enrichment of CSCs in sphere cultures.