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. Author manuscript; available in PMC: 2016 Jan 6.
Published in final edited form as: Clin Cancer Res. 2014 Apr 22;20(12):3064–3070. doi: 10.1158/1078-0432.CCR-13-3175

Molecular Pathways: Turning Proteasomal Protein Degradation into a Unique Treatment Approach

Sebastian Stintzing 1, Heinz-Josef Lenz 1
PMCID: PMC4703318  NIHMSID: NIHMS587579  PMID: 24756373

Abstract

Cancer treatment regimens have evolved from single cytotoxic substances affecting all proliferative tissues towards antibodies and kinase inhibitors targeting tumor specific pathways. Treatment efficacy and cancer survival has overall improved and side effects have become less frequent. The ubiquitin proteasome system (UPS) mediated proteasomal protein degradation is the most critical pathway to regulate the quantity of signal proteins involved in carcinogenesis and tumor progression. These processes are, as well as protein recycling, highly regulated and offer targets for biomarker and drug development.

Unspecific proteasome inhibitors such as bortezomib and carfilzomib have shown clinical efficacy and are approved for clinical use. Inhibitors of more substrate specific enzymes of degradation processes are developed and in early clinical trials. The novel compounds focus on the degradation of key regulatory proteins such as p53, p27Kip1 and β-catenin, and inhibitors specific for growth factor receptor kinases turnover are in pre-clinical testing.

BACKGROUND

Ubiquitin-proteasome-system (UPS)

The closely regulated ubiquitin-proteasome-system (UPS) clears the cell-plasma from damaged, misfolded and aged proteins. More than 80% of intracellular proteins are processed by the UPS (1), the remaining proteins are handled by the lysosome system. UPS is also involved in the inactivation of regulatory proteins by initiating the post-translational addition of multiple ubiquitin motifs which sorts intracellular proteins for degradation.

Ubiquitin is a small and highly conserved protein of 76 amino-acids. Poly-ubiquitination is facilitated by isopeptide bonds between the last amino-acid of ubiquitin (glycine) and one lysine (K) of another ubiquitin that functions as the substrate. Ubiquitin has seven lysine positions (K6, K11, K27, K29, K33, K48 and K63) with K48 and K63 being the most common positions where poly-ubiquitination occurs. The position of poly-ubiquitination determines whether a protein will be degraded (K48-linked) or will be activated (K63-linked) (2). Little is known about ubiquitination at the other lysine positions.

Ubiquitin-like Proteins

More than 20 ubiquitin-like proteins such as NEDD8 (neural precursor cell expressed, developmentally down-regulated 8), SUMO (small ubiquitin-related modifier) and ISG15 (interferon-induced 17 kDa protein) have been described which play important roles in posttranslational protein modification (3).

NEDD8 most importantly is modifying the ubiquitin dependent degradation process by interacting with cullin like E3 ligases (4). It activates cullin E3 ligases leading to a higher rate of poly-ubiquitination and therefore drives the degradation of proteins that are turned over by cullin E3 ligases (5).

SUMO, like ubiquitin, is facilitating lysine amino-acids within the substrate to bind to other proteins. SUMOylation therefore competes with ubiquitylation and can inhibit ubiquitin dependent proteolysis (6). It has been described in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease (7). SUMO modification of multiple substrates supports their physical interaction (SUMO glue) and thereby stimulates complex formation. This complex formation plays an important role in DNA repair mechanisms, ribosomal biogenesis and genome maintenance (8) and links SUMOylation to multiple diseases such as melanoma, renal cell carcinoma and cell stemness making it an interesting field for drug development (9).

ISG15 also modifies proteins by a lysine-glycin isopeptide bond and is involved in the inflammatory response to interferon-1. Its role in alternating lethality to virus infection has been investigated to a wide extent (10). As virus replication is for the most not affected and function differs between different viruses and host species, many questions remain unaddressed. It is ensured that ISG15 targets newly translated virus and host proteins under the influence of interferon-1 (11) and therefore is involved in the modulation of immune response o viral infections.

Ubiquitin activation (E1)

The UPS can be separated into four different processes: Ubiquitin activation by E1-enzymes, ubiquitin conjugation by E2 enzymes, ubiquitin ligation by E3-enzymes and the proteolysis of the substrate in a 26S-proteasome (12). De-ubiquitinases (DUBs) can reverse this process by dissociating ubiquitin from the substrate and enable protein recycling.

Ubiquitin activation is ATP dependent and is achieved by one of the two known E1 human enzymes. UBE1 the principal ubiquitin activating protein in eukaryotes and the recently described UBE1L2 add an energy rich thioester bond to the C-terminal end of ubiquitin. Inhibitors of E1 enzymes are designed to interfere with this thioester bond.

Ubiquitin-conjugating enzymes (E2)

E2 enzymes are capable of transferring the activated ubiquitin onto an E3 enzyme-substrate complex. About 50 E2 enzymes have been identified. The central functional motif is an ubiquitin-conjugating catalytic (UBC) fold. The UBC exhibit a catalytic cysteine residue which together with the thioester bond of the activated ubiquitin forms a high-energetic conjugate. E2 enzymes define the position of ubiquitination (e.g. K48 vs. K63) and consequently determine the further destiny of the protein substrate (12). Characterized by the extensions to the UBC, four different classes of E2-enzymes have been defined: class I: no extension; class II: N-terminal extension, class III: C-terminal extension and class IV: extension on both ends (13).

Ubiquitin ligases (E3)

E3-ligase enzymes are highly substrate specific with more than a thousand enzymes estimated (14). The principle function of E3 ligases is to recruit specific proteins (substrates) and to interact with E2 enzymes to catalyze the covalent binding of ubiquitin. Three major classes of E3 enzymes have been defined according to the structure of the catalytic domain (15): (i) HECT (homologous to the E6AP carboxyl terminus), (ii) U-Box and (iii) RING (really interesting new gene) E3s. An important subgroup of RING E3s are cullin RING ligases (CRL) which ubiquitinate proteins with key roles in cell-cycle progression and signal transduction. Functional motif of many CRL is the SCF (Skp, Cullin, F-box containing) complex, making all three components interesting targets for drug development. F-box is a structural motif of about 50 amino acids that mediates protein–protein interactions (16). Skp (seventeen kilo Dalton protein) is a trimeric periplasmic chaperone that assists outer membrane proteins in their folding and insertion into membranes. Cullins are a family of proteins scaffolding the E3 ligase activity that are regulated by neddylation (4). The addition of NEDD8 to CRLs is driving the turnover of multiple regulatory proteins towards degradation such as growth factor receptor proteins (5).

26S Proteasome

Degradation within the UPS is processed by the 26S proteasome which consists of a 20S core particle and two regulatory 19S regulatory caps (17). Poly-ubiquitinated proteins are broken down by proteolysis. In cancer cell lines an augmented proteasome activity is a common phenomenon including degradation of proteins involved in tumor progression, apoptosis and cell cycle regulation.

CLINICAL–TRANSLATIONAL ADVANCES

Novel inhibitors of the UPS have been developed targeting key proteins of the major circuits of carcinogenesis as defined by Hanahan and Weinberg (18) (figure 1). Most of the compounds (table 1) are still in early clinical development (phase 1) and therefore examined for toxicity and tolerability. As single substance efficacy is not anticipated, studies testing drugs in further development (phase 1b/2) use combinations of standard of care chemotherapeutic substances such as antimetabolites (e.g. cytarabin, 5-fluorouracil) or mitotic inhibitors (e.g. taxol) depending on the underlying disease.

Figure 1.

Figure 1

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Inhibitors of the UPS targeting proteins involved in the intracellular circuits of carcinogenesis.

FAF1 = Fas associated factor 1; SCF = Skp-Cullin-F-box, FBXW7 = F-box and WD repeat domain containing 7, p27Kip1 = cyclin-dependent kinase inhibitor 1B, c-Myc = Myc proto-oncogene protein, USP = ubiquitin-specific-processing protease, HAUSP = herpesvirus-associated ubiquitin-specific protease, Hdm2 = human double minute 2 homolog, p53 = tumor suppressor p53, Mcl-1 = induced myeloid leukemia cell differentiation protein, AP = apoptosis protein, XIAP = X-linked inhibitor of apoptosis protein, cIAP = cellular inhibitor of apoptosis protein, NF-κB = nuclear factor κB; IκB = inhibitor of nuclear factor κB, DUB = de-ubiquitinase, BRCA1 = breast cancer 1, early onset, HIF1α = hypoxia-inducible factor 1-alpha, FANC = Fanconi anemia (FA) pathway, VHL = von Hippel Lindau tumor suppressor, RAD6 = ubiquitin-conjugating enzyme E2 B

Table 1.

Modulators of the ubiquitin proteasome system (UPS) in clinical development for cancer treatment

Target Structure Name Clinical stage Remarks
Proteasome inhibitors
Bortezomib approved for PCM and MCL multiple clinical trials testing Bortezomib in combination with chemotherapeutic substances in PCM and MCL.
Carfilzomib approved for PCM after 2 prior therapies tested in a phase 1 study of advanced malignancies (including mCRC) with hepatic impairment (NCT01949545) and in combination with irinotecan in a phase 1/2 study of irinotecan sensitive advanced malignancies (including mCRC) (NCT01941316)
Oprozomib ONX 0912 phase 1 oral proteasome inhibitor under evaluation for the treatment of PCM and lymphomas and in a phase 1 study of advanced malignancies (including mCRC) (NCT01129349)
Marizomib NPI-0052 phase 1/2 tested in a phase 1 study for advanced solid malignancies (including mCRC) (NCT00629473)
Delanzomib CEP-18770 phase 1 tested in a phase 1 study for advanced solid malignancies (including mCRC) or non-Hodgkin’s lymphoma (NCT00572637)
Ixazomib MLN9708 phase 1/2 tested in a phase 1 study for advanced solid malignancies (including mCRC) (NCT00830869)
Nedd8 activating enzyme
NAE MLN4924 phase 1 currently tested in AML (NCT01814826), large B-cell lymphoma (NCT01415765) and advanced malignancies (NCT00677170) in combination with chemotherapeutic drugs
E3-ligases interacting Hdm2 –p53
RO5045337 phase 1 nutlin derivate tested in multiple phase 1 trials (NCT00559533), results not reported yet
RO5503781 phase 1 nutlin derivate currently tested in AML (NCT01773408), advanced malignancies except leukemia (NCT01462175)
DS-3032b phase 1 tested in a phase 1 study for advanced solid malignancies or lymphomas (NCT01877382)
SAR405838 phase 1 spiro-oxindole tested in phase 1 studies for advanced malignancies (NCT01636479) and in combination with pimasertib (NCT01985191)
JNJ-26854165 phase 1 successful phase 1 study with good tolerability and modest efficacy (21)
MK-8242 phase 1 tested in phase 1 studies for advanced solid malignancies (NCT01463696) and in combination with cytarabin in AML (NCT01451437)
CGM097 phase 1 tested in a phase 1 study for advanced solid malignancies (NCT01760525)
p28 phase 1 tested in pediatric patients with recurrent or progressive CNS tumors (NCT01975116)
E3-ligases interacting with apoptotic proteins
Birinapant TL32711 phase 2 testing the efficacy for ovarian, primary peritoneal or fallopian tube cancer (NCT01681368)
AEG35156 phase 1/2 preclinical studies showed activity but data of clinical phase 1/2 studies were disappointing
LCL161 phase 1/2 tested in solid tumors (NCT01098838), in combination with taxol in solid tumors (NCT01240655) and in breast cancer (NCT01617668)
AT-406 (Debio 1143) phase 1 encouraging data in mouse xenotransplant models of human ovarian cancer (56), tested in phase 1 design (NCT01078649)
GDC-0917 phase 1 (NCT01226277)
GDC-0152 phase 1 tested in solid tumors (NCT00977067)
HSG1029 phase 1 tested in solid tumors (NCT00708006) and lymphoid malignancies (NCT01013818)
CUDC-427 phase 1 tested in solid tumors and lymphomas (NCT01908413)
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PCM = plasma cell myeloma, MCL = mantle cell lymphoma, AML = acute myeloid leukemia, mCRC = metastatic colorectal cancer, NCT = National Clinical Trial number

Cell Cycle Regulation

Accelerated degradation of regulatory cell-cycle proteins causing lower intracellular expression of tumor suppressors such as p53 has been demonstrated in a variety of neoplasias. E3 ligases reducing intracellular p53 levels are associated with carcinogenesis and prognosis. Examples are (i) E6AP ubiquitin-protein ligase (E6-AP) which is activated by human papilloma virus (HPV) (13) and leads to HPV associated carcinomas and (ii) RING finger and CHY zinc finger domain-containing protein 1 (aka Pirh2), which is overexpressed in hepatocellular carcinoma, head and neck cancers, lung cancer and prostate cancer correlates with poor overall survival in hepatocellular carcinoma (HCC) (19).

Giving the central role of p53 in DNA damage repair and cell cycle regulation, the turnover of p53 has been extensively studied and several potential regulatory proteins of p53 degradation have been identified. Higher levels of Hdm2 which is the most important E3 ligase for p53 were identified in leukemias, lymphomas and solid tumors (12) making it a valuable target for drug development. Substances interacting with the Hdm2-p53 binding site have been shown to increase p53 levels in p53 non-mutant cell lines and consequently led to cell cycle arrest and apoptosis (20). Among those, nutlins were the first class discovered and two members (RO5045337 and RO5503781) made it to phase 1 trials but results have not been reported. Recently, clinical data on serdemetan (JNJ-26854165) a tryptamine compound belonging to the second class of Hdm2 antagonists have been reported (21). Serdemetan has shown to increase p53 levels radio-sensitizing tumors in xenograft models (22). It was well tolerated in a phase 1 trial. Spiro-oxindoles are the third class of small inhibiting molecules interacting with Hdm2 (23) with MI-773 (SAR405838) currently being tested in two clinical trials (NCT01636479, NCT01985191). Other compounds interacting with Hdm2 are being developed and are in early clinical testing (see table 1). Their specific mechanisms of action are not reported.

Targeting de-ubiquitinases (DUBs) plays an important role in the regulation of p53 levels. Both, the DUB HAUSP (herpesvirus-associated ubiquitin-specific protease) and USP10 (ubiquitin specific protease 10), targeting poly-ubiquitinated p53, have been shown to restore p53 levels even when Hdm2 is overexpressed (24, 25).

It has also been shown that the inhibition USP7 and USP2a which are de-ubiquitinases of Hdm2, increases Hdm2 proteolysis and stabilizes p53 levels in multiple myeloma cells (26, 27) enabling the initiation of apoptosis. The inhibition of USP7 with HBX 41,108, a DUB to Hdm2 has shown to induce p53 dependent apoptosis with an IC(50) in sub-micro-molar concentrations. The same principle was demonstrated with other compounds such as P5091 (26) and P22077 (28) in preclinical models, however no de-ubiquitinase inhibitor has entered clinical development.

Other potential targets in cell cycle regulation such as cyclin E, C-Myc and p27Kip1 are known to be degraded by F-box containing SCF E3 ligases.

Mutations leading to a loss function in the F-box FBXW7 have been identified in solid and hematological malignancies (29) leading to increased proliferation. The reported frequency of FBXW7 loss of function mutations in T-ALL is approximately 20% (30) but are also frequently found in cholangiocarcinoma (~35%), colorectal (~10%), ovarian, and endometrial cell lines (29, 30). Modulating of FBXW7 degradation is promising for novel drug development as a variety of tumors (15).

Degradation of p27Kip1 is mediated by a specific SCF Skp2 which plays a role in cellular senescence, cancer progression, and metastasis (31). No specific Skp2 inhibitor has been reported in clinical development.

Viability pathways

The down-regulation of apoptosis and upregulation of survival pathways are characteristic for cancer development and progression. Inhibitors of apoptosis proteins (IAP) are E3 ligases regulating caspase activity which is required for apoptosis. In a number of hematological diseases including AML, ALL, CLL and lymphomas IAPs are overexpressed (32) making them a promising target for drug development.

A detailed review of IAP inhibitors in cancer has been recently published (33) and multiple IAP inhibitors are currently tested in phase 1 and 2 trials (table 1). AEG 35156 is the only substance with clinical outcome data. Unfortunately, due to a lack of efficacy in two phase 2 trials (34, 35), further development was terminated.

Nuclear factor-κB (NF-κB) is a transcriptional factor involved in inflammatory processes, cell proliferation and cell survival. Active NF-κB is released after degradation of IκB (inhibitor of κB). Canonical release through TNFα signaling and non-canonical activation through NIK (NF-κB inducing kinase) are both dependent on cellular IAPs (cIAP) (33). Brinapant is an antagonist inhibiting cIAP1 and cIAP2 and was shown to restore TNFα dependent apoptosis in breast and melanoma cancer cells (36, 37). Brinapant is currently tested in phase 2 trials in ovarian, fallopian tube and primary peritoneal cancer.

Another target for novel drug development interacting with viability circuits is USP9x which de-ubiquitinases the induced myeloid leukemia cell differentiation protein Mcl-1. USP9x is overexpressed in lymphomas, CML and PCM (plasma cell myeloma) where higher Mcl-1 levels cause a block in apoptosis (38). In preclinical models the USP9x inhibitor WP1130 has shown to increase pro-apoptotic proteins and to decrease anti-apoptotic proteins (39) and therefore increase tumor cell sensitivity to multiple chemotherapeutic agents (40). Further clinical testing will depend on promising data generated in pre-clinical models.

Regulation of Cell Motility

A key regulatory protein that is commonly associated with a large number of cancers and hematological tumors is β-catenin. Depending on its intracellular localization it has distinct functions in cell-proliferation and cell motility. Usually bound to cytoskeleton proteins, cell plasma levels are critical for the function as a transcription factor. The F-Box E3 ligase SCF β-TrCP (41) and Siah-1–SIP–Skp1 (42) degrade cell plasma β-catenin reducing wnt-β-catenin signaling. FAF1 (FAS-associated factor 1) has shown to increase β-catenin degradation by activating the β-TrCP F-Box complex (43). K63 ubiquitination of β-catenin by the help of the E2 ligase RAD6 (Ubiquitin-conjugating enzyme E2 B) increase cell plasma β-catenin. K63 poly-ubiquitinated β-catenin is not available for K48 ubiquitination (44) but is functional active. RAD6 has been shown to be overexpressed in breast cancer (45) and inhibitors are in preclinical development (46).

Growth factor dependent pathways

Growth factor pathway dependent carcinogenesis, proliferation and metastasis are activated through extracellular ligands, trans-membrane receptors or by activating mutations within the intracellular part of the pathways. The amount of receptors and ligands expressed on the cell surface is dependent on recycling and degradation processes.

Solid tumors are in the need of growth factors to migrate, proliferate and create their own vessel system through angiogenesis. These signaling pathways are often regulated through growth factor receptor kinases such as ErbB family members or VEGFR (vascular endothelial growth factor receptor) family. After ligand binding, the growth factor receptor is internalized, the tyrosine activity is shut down and the protein is degraded with the help of c-Cbl, an SCF (Skp1-Cullin-F-box) ligase that is activated by neddylation (47, 48). Only inhibitors of neddylation (ML4232) are in clinical development (4).

The von-Hippel-Lindau (VHL) syndrome is caused by a mutation in the E3-ligease (VHL) reducing degradation of HIF1α and leading to increased signaling of pro-angiogenic cytokines (49) which further supports the key role of degradation in the regulation of growth factor signaling.

DNA repair mechanisms

There are no inhibitors of the UPS associated DNA repair mechanisms available, however alterations in the BRCA and FANC E3-ligases are a good example for the clinical significance of degradation for diagnosis, prognosis, prevention and treatment strategies. Reduced DNA repair mechanisms lead to accumulation of DNA damage promoting carcinogenesis. One of the best known hereditary cancer syndromes is caused by mutations within the BRCA1 gene which is an E3 ligase involved in DNA repair emphasizing the importance of alterations in the UPS in carcinogenesis and tumor progression. Loss of function mutations in FANCs E3 ligases that are involved in the ubiquitination of the FANCI-FANCD2 which is also associated with DNA repair are causing FA (50) and are associated with childhood T-ALL and testicular seminoma (51).

Inhibitors of neddylation and the 26S proteasome

Inhibition of the 26S proteasome

The inhibition of the 26S proteasome causes an accumulation of intracellular proteins that leads to an inhibition of NF-κB activity and angiogenesis, alters degradation of cell cycle and apoptotic proteins and effects endoplasmatic reticulum stress (1, 52). The reversible inhibitor Bortezomib (53) and the irreversible inhibitor Carfilzomib (54) have been approved for treatment of plasma cell lymphoma (PCM). Bortezomib is also approved for the clinical use of mantel-cell lymphoma (MCL) after disease progression after one prior therapy and is currently tested in clinical trials in combination with chemotherapeutic agents in AML and ALL. More potent and less toxic proteasome inhibitors (table 1) are under evaluation in clinical trials for patients with plasma cell myeloma (PCM) and advanced solid tumors.

Inhibiting ubiquitin and NEDD8 activating enzymes

Inhibitors of E1 enzymes have been identified using high throughput screening for substances targeting p53 and p27. PYR-41 was the first UAE inhibitor to be tested in pre-clinical models and was able to inhibit p53 degradation and down-regulate cytokine induced NF-κB signaling (12). In addition to p53 levels, the structurally related substance PYZD-4409 was able to stabilize cyclin D3 levels and was able to induce cell death by induction of endoplasmatic reticulum stress (55). The ubiquitin activating enzyme inhibitors have been shown activity in preclinical models but no clinical trials are ongoing, so far. Many E3 ligases are cullin-RING-ubiquitin ligases which are activated by neddylation. Neddylation accelerates K48-linked-poly-ubiquitination of multiple regulatory proteins such as p53, p21, p27Kip1, growth-factor receptor tyrosine kinases, apoptosis proteins. One inhibitor of NEDD8 activation (MLN4924) is in early clinical development.

Conclusion

Degradation plays a key regulatory role in all major cell circuits representing the hallmarks of cancer. The challenge to translate these novel compounds successfully into the clinic is to identify the tumor tissue-specific degradation processes to personalize therapy with specific inhibitors.

Targeting the UPS for cancer treatment is a unique approach which has been proven to be effective as the proteasome inhibitors bortezomib and carfilzomib are established in the treatment of plasma cell myeloma and mantle cell lymphoma. Inhibitors of specific UPS enzymes of key regulatory proteins of carcinogenesis and tumor progression such as apoptosis proteins and p53 are currently tested in phase 1 and phase 2 trials in a variety of cancers including leukemia and solid tumors.

Increased molecular understanding of the regulation of protein degradation in cancer and liquid tumors will be essential for development of more specific and more effective and less toxic compounds. Identification of tumor specific UPS enzymes in cancers and liquid tumors will be critical for selection of patients who benefit the most from specific UPS inhibitors

Acknowledgments

H.-J. Lenz is supported by the National Institutes of Health (P30CA014089) and the Nancy Bernstein Research Fund. S. Stintzing is supported by a postdoctoral fellowship from the German Cancer Aid (Mildred-Scheel Foundation).

Footnotes

Disclosure of Potential Conflicts of Interest

H.-J. Lenz reports receiving speakers bureau honoraria from and is a consultant/advisory board member for Bristol-Myers Squibb and Merck. No potential conflicts of interest were disclosed by the other author.

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