Molecular mechanisms of bortezomib resistant adenocarcinoma cells

Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ∼30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response.     

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

The ubiquitin–proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.     

Dysfunction of the ubiquitin-proteasome system in multiple disease conditions: therapeutic approaches

The ubiquitin-proteasome system (UPS) is the major proteolytic pathway that degrades intracellular proteins in a regulated manner. Deregulation of the UPS has been implicated in the pathogenesis of many neurodegenerative disorders like Alzheimer's disease, Parkinson's diseases, Huntington disease, Prion-like lethal disorders, in the pathogenesis of several genetic diseases including cystic fibrosis, Angelman's syndrome and Liddle syndrome and in many cancers. Multiple lines of evidence have already proved that UPS has the potential to be an exciting novel therapeutic target for the treatment of these diseases. Here I review how aberrant functions of various genes have implicated UPS in many human disorders including neurodegeneration and cancers. I also discuss the finding that some proteasome inhibitors possess a therapeutic potential as drugs against many such diseases.     

Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.     

Ubiquitin–proteasome system and the role of its inhibitors in cancer therapy

Despite all the other cells that have the potential to prevent cancer development and metastasis through tumour suppressor proteins, cancer cells can upregulate the ubiquitin–proteasome system (UPS) by which they can degrade tumour suppressor proteins and avoid apoptosis. This system plays an extensive role in cell regulation organized in two steps. Each step has an important role in controlling cancer. This demonstrates the importance of understanding UPS inhibitors and improving these inhibitors to foster a new hope in cancer therapy. UPS inhibitors, as less invasive chemotherapy drugs, are increasingly used to alleviate symptoms of various cancers in malignant states. Despite their success in reducing the development of cancer with the lowest side effects, thus far, an appropriate inhibitor that can effectively inactivate this system with the least drug resistance has not yet been fully investigated. A fundamental understanding of the system is necessary to fully elucidate its role in causing/controlling cancer. In this review, we first comprehensively investigate this system, and then each step containing ubiquitination and protein degradation as well as their inhibitors are discussed. Ultimately, its advantages and disadvantages and some perspectives for improving the efficiency of these inhibitors are discussed.     

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.     

Proteasome inhibitors: Dozens of molecules and still counting

The discovery of the proteasome in the late 80’s as the core protease of what will be then called the ubiquitin–proteasome system, rapidly followed by the development of specific inhibitors of this enzyme, opened up a new era in biology in the 90’s. Indeed, the first proteasome inhibitors were instrumental for understanding that the proteasome is a key actor in most, if not all, cellular processes. The recognition of the central role of this complex in intracellular proteolysis in turn fuelled an intense quest for novel compounds with both increased selectivity towards the proteasome and better bioavailability that could be used in fundamental research or in the clinic. To date, a plethora of molecules that target the proteasome have been identified or designed. The success of the proteasome inhibitor bortezomib (Velcade^®) as a new drug for the treatment of Multiple Myeloma, and the ongoing clinical trials to evaluate the effect of several other proteasome inhibitors in various human pathologies, illustrate the interest for human health of these compounds.     

VLX1570 regulates the proliferation and apoptosis of human lung cancer cells through modulating ER stress and the AKT pathway

The ubiquitin‐proteasome system (UPS) possesses unique functions in tumorigenesis and has great potential for targeting tumours. The effectiveness of inhibitors targeting UPS in solid tumours remains to be studied. We screened a library of inhibitors targeting the ubiquitination system and found the highly potent, low‐concentration inhibitor molecule VLX1570 that specifically killed lung cancer cells. VLX1570 is an inhibitor of deubiquitinating enzyme activity and has also shown potential for preclinical cancer treatment. We found that VLX1570 significantly inhibited lung cancer cells proliferation and colony formation. VLX1570 induced a G2/M cell cycle arrest by downregulating CDK1 and CyclinB1. Moreover, VLX1570 significantly promoted the mitochondrial‐associated apoptosis. Mechanistically speaking, VLX1570 activated the PERK/IRE1/ATF6 pathway and induced ER stress in lung cancer cell lines. The inhibition of ER stress by tauroursodeoxycholic acid sodium or 4‐phenylbutyric acid enhanced VLX1570‐induced apoptosis. In addition, VLX1570 treatment led to the inactivation of Akt signalling and inhibited the proliferation of lung cancer cells by downregulating the Akt pathway. Moreover, combined treatment with VLX1570 and Afatinib or Gefitinib induced synergistic anti‐lung cancer activity. Our present study demonstrated a novel therapy using VLX1570, which inhibited the proliferation and increased apoptosis in human lung cancer.     

Development of inhibitors in the ubiquitination cascade

The ubiquitin proteasome system (UPS) is essential in regulating myriad aspects of protein functions. It is therefore a fundamentally important regulatory mechanism that impacts most if not all aspects of cellular processes. Indeed, malfunction of UPS components is implicated in human diseases such as neurodegenerative and immunological disorders and many cancers. The success of proteasome inhibitors in cancer therapy suggests that modulating enzymes in the ubiquitination cascade would be clinically important for therapeutic benefits. In this review, we summarize advances in developing inhibitors of a variety of UPS components. In particular, we highlight recent work done on the protein engineering of ubiquitin as modulators of the UPS, a novel approach that may shed light on innovative drug discovery in the future.     

Regulation of proteasome activity in health and disease

The ubiquitin–proteasome system (UPS) is the primary selective degradation system in the nuclei and cytoplasm of eukaryotic cells, required for the turnover of myriad soluble proteins. The hundreds of factors that comprise the UPS include an enzymatic cascade that tags proteins for degradation via the covalent attachment of a poly-ubiquitin chain, and a large multimeric enzyme that degrades ubiquitinated proteins, the proteasome. Protein degradation by the UPS regulates many pathways and is a crucial component of the cellular proteostasis network. Dysfunction of the ubiquitination machinery or the proteolytic activity of the proteasome is associated with numerous human diseases. In this review we discuss the contributions of the proteasome to human pathology, describe mechanisms that regulate the proteolytic capacity of the proteasome, and discuss strategies to modulate proteasome function as a therapeutic approach to ameliorate diseases associated with altered UPS function. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET—a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.Subject terms: Myeloma, Cancer therapeutic resistance     

The ubiquitin-proteasome system

The 2004 Nobel Prize in chemistry for the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first ‘tagged’ by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome. This article recounts the key observations that led to the discovery of ubiquitin-proteasome system (UPS). In addition, different aspects of proteasome biology are highlighted. Finally, some key roles of the UPS in different areas of biology and the use of inhibitors of this pathway as possible drug targets are discussed.     

The persisting challenge of selective and specific proteasome inhibition

Since the discovery of the proteasome and its structure elucidation intensive research programs in academic institutions and pharmaceutical industries led to identification of a wide spectrum of synthetic and natural small proteasomal inhibitors. Activity studies with these small molecules helped to deeply understand the complex biochemical organization and functioning of the proteasome. The new structural and biochemical insights placed the proteasome as an important anti‐cancer drug target, as revealed by the dipeptide boronate proteasome inhibitor, bortezomib, which is currently used for treatment of multiple myeloma. Serious side effects and partial cell resistance against bortezomib demand creation and discovery of new improved generations of more specific and potent proteasomal inhibitors. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Proteasome protease mediated regulation of cytokine induction and inflammation

We have previously demonstrated that proteasome serves as a central regulator of inflammation and macrophage function. Until recently, proteasomes have generally been considered to play a relatively passive role in the regulation of cellular activity, i.e., any ubiquitinated protein was considered to be in discriminatively targeted for degradation by the proteasome. We have demonstrated, however, by using specific proteasome protease inhibitors and knockout mice lacking specific components of immunoproteasomes, that proteasomes (containing X, Y, and Z protease subunits) and immunoproteasomes (containing LMP7, LMP2, and LMP10 protease subunits) have well-defined functions in cytokine induction and inflammation based on their individual protease activities. We have also shown that LPS-TLR mediated signaling in the murine RAW 264.7 macrophage cell line results in the replacement of macrophage immunoproteasomal subunits. Such modifications serve as pivotal regulators of LPS-induced inflammation. Our findings support the relatively novel concept that defects in structure/function of proteasome protease subunits caused by genetic disorders, aging, diet, or drugs may well have the potential to contribute to modulation of proteasome activity. Of particular relevance, we have identified quercetin and resveratrol, significant constituents present in berries and in red wine respectively, as two novel proteasome inhibitors that have been previously implicated as disease-modifying natural products. We posit that natural proteasome inhibitors/activators can potentially be used as therapeutic response modifiers to prevent/treat diseases through pathways involving the ubiquitin-proteasome pathway (UP-pathway), which likely functions as a master regulator involved in control of overall inflammatory responses. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Natural products are an important source for proteasome regulating agents

BackgroundNatural medicines have a long history in the prevention and treatment of various diseases in East Asian region, especially in China. Modern research has proved that the pharmacological effects of numerous natural medicines involve the participation of ubiquitin proteasome system (UPS). UPS can degrade the unwanted and damaged proteins widely distributed in the nucleus and cytoplasm of various eukaryotes.PurposeThe objective of the present study was to review and discuss the regulatory effects of natural products and extracts on proteasome components, which may help to find new proteasome regulators for drug development and clinical applications.MethodsThe related information was compiled using the major scientific databases, such as CNKI, Elsevier, ScienceDirect, PubMed, SpringerLink, Wiley Online, and GeenMedical. The keywords “natural product” and “proteasome” were applied to extract the literature. Nature derived extracts, compounds and their derivatives involved in proteasome regulation were included, and the publications related to synthetic proteasome agents were excluded.ResultsThe pharmacological effects of more than 80 natural products and extracts derived from phytomedicines related to the proteasome regulation were reviewed. These natural products were classified according to their chemical properties. We also summarized some laws of action of natural products as proteasome regulators in the treatment of diseases, and listed the action characteristics of the typical natural products.ConclusionNatural products derived from nature can induce the degradation of damaged proteins through UPS or act as regulators to directly regulate the activity of proteasome. But few proteasome modulators are applied clinically. Summary of known rules for proteasome modulators will contribute to discover, modify and synthesize more proteasome modulators for clinical applications.     

Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.     

The ubiquitin–proteasome system in breast cancer

Ubiquitin–proteasome system (UPS) is a selective proteolytic system that is associated with the expression or function of target proteins and participates in various physiological and pathological processes of breast cancer. Inhibitors targeting the 26S proteasome in combination with other drugs have shown promising therapeutic effects in the clinical treatment of breast cancer. Moreover, several inhibitors/stimulators targeting other UPS components are also effective in preclinical studies, but have not yet been applied in the clinical treatment of breast cancer. Therefore, it is vital to comprehensively understand the functions of ubiquitination in breast cancer and to identify potential tumor promoters or tumor suppressors among UPS family members, with the aim of developing more effective and specific inhibitors/stimulators targeting specific components of this system.     

Targeting cullin-RING ligases for cancer treatment: rationales,advances and therapeutic implications

New therapeutic intervention strategies for the treatment of human malignancies are always desired. Approval of bortezomib as a front-line treatment for multiple myeloma highlighted the significance of ubiquitin–proteasome system (UPS) as a promising therapeutic target. However, due to the broad impact of proteasome inhibition, deleterious side effects have been reported with bortezomib treatment. Cullin RING ligases (CRLs)-mediated ubiquitin conjugation process is responsible for the ubiquitin conjugation of 20 % cellular proteins that are designated for degradation through the UPS, most of them are critical proteins involved in cell cycle progression, signaling transduction and apoptosis. Studies have depicted the upstream NEDDylation pathway that controls the CRL activity by regulating the conjugation of an ubiquitin-like-protein NEDD8 to the cullin protein in the complex. A specific pharmaceutical inhibitor of NEDD8 activating enzyme (NAE; E1) MLN4924 was recently developed and has been promoted to Phase I clinical trials for the treatment of several human malignancies. This article summarizes the most recent understanding about the process of NEDD8 conjugation, its relevance for cancer therapy and molecular mechanisms responsible for the potent anti-tumor activity of MLN4924.