Therapeutically targeting the SUMOylation, Ubiquitination and Proteasome pathways as a novel anticancer strategy
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- Driscoll, J.J. & DeChowdhury, R. Targ Oncol (2010) 5: 281. doi:10.1007/s11523-010-0165-2
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The ubiquitin (Ub)+proteasome proteolytic pathway is responsible for the selective degradation of the majority of nuclear and cytosolic proteins. The proteasome is a high molecular weight multicatalytic protease that serves as the catalytic core of the complex Ub-dependent protein degradation pathway and is an exciting new target for the development of novel anticancer therapies. Inhibition of the proteasome, and consequently Ub-dependent proteolysis, with the small molecule pharmacologic agent bortezomib led to approval by the US Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM) that has subsequently been extended to other hematologic malignancies. Inhibition of the proteasome results in the intracellular accumulation of many ubiquitinated proteins that control essential cellular functions such as cellular growth and apoptosis. The accumulation of high molecular weight Ub~protein conjugates eventually triggers apoptosis, with tumor cells more susceptible to proteasome inhibition than non-malignant cells. The defined mechanism of action for proteasome inhibitors has not been completely characterized, not all patients respond to proteasome inhibitor-based therapy, and inevitably patients develop resistance to proteasome inhibitors. Further investigation of the Ub+proteasome system (UPS) is needed to develop more effective inhibitors, to develop agents that overcome bortezomib resistance and to avoid adverse effects such as neuropathy. Furthermore, there are newly uncovered pathways, e.g., the SUMOylation and NEDDylation pathways, which similarly attach Ub-like proteins (ULPs) to protein substrates. The functional consequence of these modifications is only beginning to emerge, but these pathways have been linked to tumorigenesis and may similarly provide therapeutic targets. The immunoproteasome is a specialized form of the proteasome that produces peptides that are presented at the cell surface as major histocompatibility complex (MHC) class I antigens. Proteasome inhibitors decrease the presentation of antigenic peptides to reduce tumor cell recognition by cytotoxic T cells (CTLs) but unexpectedly increase tumor cell recognition by natural killer (NK) cells. Inhibitors of the UPS are validated, cytotoxic agents that may be further exploited in immunotherapy since they modulate tumor cell recognition by effectors of the immune system. Targeting the UPS, SUMOylation and NEDDylation pathways offers great promise in the treatment of hematologic and solid malignancies.
The ubiquitin+proteasome system and cancer biology
Targeting of the UPS in multiple myeloma
Bortezomib is a novel cytotoxic agent that potently and specifically inhibits the proteolytic activity of the proteasome and, hence, inhibits the degradation of multi-ubiquitinated proteins [34, 35]. This agent is a modified dipeptidyl boronic acid derived from leucine and phenylalanine and is the first proteasome inhibitor to demonstrate antitumor activity. Bortezomib predominantly inhibits the chymotrypsin-like activity of the proteasome but inhibition of this activity alone is sufficient to block all proteasomal activity . Multiple myeloma (MM) is a B-cell malignancy of the plasma cell and represents the second most common hematological malignancy. The incidence and prevalence of MM are similar in the US and Europe with ~40,000 cases diagnosed annually. Bortezomib has shown cytotoxic activity against a variety of MM cell lines and MM patient plasma cells. Numerous mechanisms of action have been proposed for the effect of bortezomib on tumor cells. These include inhibition of nuclear factor-κB (NF-κB) activation, inhibition of myeloma cell adherence to the bone marrow stroma, decreased interleukin-6 signaling, reduced production of angiogenic factors, defective apoptotic regulation and the accumulation of unfolded, misfolded, or multi-ubiquitinated proteins. In addition, it has been proposed that the accumulation of Ub~conjugates by proteasome inhibitors decreases the intracellular pool of free Ub to promote cell death. It is noteworthy that bortezomib is effective in other hematologic malignancies such as lymphomas and leukemic cell lines. An attractive hypothesis is that proteasome inhibition prevents the clearance of misfolded proteins by the endoplasmic reticulum (ER)–associated degradation (ERAD) pathway and results in enhanced ER stress and cell death [37–39].
Bortezomib was initially approved as third-line treatment for relapsed and refractory MM by the US Food and Drug Administration (FDA) under the accelerated approval program. The FDA evaluated the safety and effectiveness of bortezomib based on the SUMMIT trial of 202 patients with relapsed and refractory MM that had received at least two prior therapies and showed disease progression on their most recent therapy . Bortezomib was administered intravenously at 1.3 mg/m2/dose twice-weekly for 2 weeks, followed by a 10-day rest period (21-day treatment cycle) for a maximum of eight treatment cycles. Results in the 188 eligible and evaluable subjects included complete response (CR) in five patients, for a CR rate of 2.7%; partial responses (PR) occurred in 47 patients for a PR rate of 25%. Clinical remissions by Southwest Oncology Group (SWOG) criteria were observed in 17.6% of patients and median response time was 1 year. The CREST trial included 54 relapsed MM patient randomized to receive either 1.0 mg/m2 or 1.3 mg/m2 of bortezomib for up to 24 weeks (days 1, 4, 8 and 11 of a 21-day cycle, for up to eight cycles) . For patients in the 1.3-mg/m2 treatment group, the overall response (ORR) was defined as the combined total CR, PR and minimal response (MR), which was 69%. For patients in the 1.0-mg/m2 treatment group, the ORR was 59%. Next, the randomized phase III APEX study was performed and compared single-agent bortezomib to high-dose dexamethasone in 669 patients with relapsed MM who had received up to three prior therapies . The study demonstrated a significant OS advantage with bortezomib. The combined CR/PR rates were 38% for bortezomib and 18% for dexamethasone. Median times to progression in the bortezomib and dexamethasone groups were 6.22 months (189 days) and 3.49 months (106 days), respectively. The 1-year OS rate was 80% among patients taking bortezomib and 66% among patients taking dexamethasone. Grade 3 or 4 adverse events were reported in 75% of patients treated with bortezomib and in 60% of those treated with dexamethasone. Front-line approval was based on the phase III VISTA (VELCADE as Initial Standard Therapy in MM: Assessment with Melphalan and Prednisone) trial, which was a randomized, international, open-label trial that compared bortezomib in combination with the current standard-of-care vs. the standard-of-care alone in 682 previously untreated patients who were unsuitable for stem cell transplantation. VISTA demonstrated that patients receiving the standard-of-care plus bortezomib achieved a greater OS rate at 24 months (83%) compared to those who only received standard-of-care alone (70%). The CR rate in the bortezomib combination arm was 30% versus 4% in the standard-of-care arm alone.
While the proteasome has emerged as an important target for cancer therapy, there are noteworthy unwanted side effects such as neuropathy . Moreover, many patients do not respond to bortezomib and most patients that do respond eventually develop resistance. Therefore, there is an urgent need for additional next-generation proteasome inhibitors with improved efficacy and better toxicity profiles. A novel, irreversible, epoxomicin-related proteasome inhibitor, carfilzomib (Onyx Pharmaceuticals, Emeryville, CA, USA), has shown activity in MM models and specifically inhibits the chymotrypsin-like activity of both the proteasome and immunoproteasome. Carfilzomib induced a dose- and time-dependent inhibition of cell growth and apoptosis that was associated with an activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c and activation of both intrinsic and extrinsic caspase pathways. Carfilzomib has produced encouraging results in early-stage studies when administered as a single agent for relapsed and/or refractory MM, a promising safety profile and an absence of neuropathy. NPI-0052 (Nereus Pharmaceuticals, San Diego, CA, USA) is another novel proteasome inhibitor that was isolated from the marine actinomycete Salinispora tropica. It is a bicyclic β-lactone γ-lactam and differs structurally from other proteasome inhibitors in development that are peptide mimetics. NPI-0052 is also a potent inhibitor of the 20S proteasome and in vitro has demonstrated broad and potent activity. Similar to bortezomib, MLN9708 (Millennium: Takeda Oncology, Cambridge, MA) is a modified dipeptidyl boronic acid that is a potent, reversible and specific inhibitor of proteasomal peptidase activity. MLN9708 preferentially binds to and inhibits the enzymatic activity of the β5 within catalytic core of the 20S proteasome [44, 45]. MLN9708 hydrolyzes to MLN2238, the biologically active form, on exposure to aqueous solutions or plasma. It is currently in Phase I trials for the treatment of solid and hematologic malignancies.
Targeting the Ub-like SUMOylation and neddylation pathways
A number of pathways that mechanistically and enzymatically parallel the Ub pathway have recently been uncovered are now being actively investigated [46–49]. These pathways covalently attach small Ub-like proteins (ULPs) onto protein substrates, but each of these ULPs has an apparently distinct cellular function. Some of these ULPs, such as the small ubiquitin-like modifier (SUMO) and the related to Ub (RUB or NEDD) pathways, have been identified in all eukaryotes examined, whereas others, such as the ISG15 pathway, have a more restricted phylogenetic distribution. A small cellular protein of 12 kDa was isolated, shown to be 18% homologous to Ub and termed small ubiquitin-like modifier, or SUMO. SUMOylation is a post-translational modification that utilizes SUMO as the modifier group covalently attached to target substrates and is essential for viability, at least in budding yeast . SUMOylation is highly regulated in all eukaryotes and participates in diverse events such as nuclear transport, transcriptional regulation, chromosome segregation and cell-cycle control. SUMOylation helps in the protein transport from the cytoplasm to nucleus of cells, regulates transcriptional activities of proteins and mediates the binding of the protein to other proteins [50, 51]. Both monomeric and polymeric addition to substrates has been documented, but distinct biological functions have only recently been ascribed to these modifications. The SUMOylation pathway is required to target Ran GTPase-activating protein 1 (RANGAP1) to the nuclear pore complex. Although protein modification with SUMO has a role in a growing number of cellular pathways, it was previously thought that SUMO, unlike Ub, did not target proteins for proteasomal degradation. However, these views have been recently revised, as it has been demonstrated that SUMO can act as a signal for the recruitment of certain E3 Ub ligases, which leads to the ubiquitylation and degradation of the target [52–54].
Recently it was demonstrated that the SUMOylation pathway and its effectors were markedly enhanced in MM patients compared to normal PCs . Gene expression profiling indicated a relative induction of SUMOylation pathway genes in the pretreatment samples of MM patients. To further the development of prognostically relevant molecular subtypes, gene expression profiling from newly diagnosed patients subsequently treated with high-dose melphalan-based tandem transplants was used to address the role of the SUMOylation pathway in MM. A set of genes that encoded SUMOylation pathway components was enhanced in MM patients and correlated with patient outcome. Induction of the SUMOylation pathway conferred multiple properties on myeloma cells that promoted tumorigenesis. In contrast to the Ub-pathway where numerous (>30) conjugating enzymes exist with distinct substrate specificities, it appears that UBE2I encodes the sole SUMO-conjugating enzyme in mammalian cells. Thus, the fundamental role of UBE2I and the SUMOylation pathway in critical cellular processes, the correlation with treatment outcome, demonstrable elimination of functionality by a dominant-negative form that prevents homodimerization and an elevation of the sumo signature in all MM sub-branches collectively render and validate the UBE2I gene product as a highly attractive candidate for therapeutic intervention. As an essential E2-conjugating enzyme in SUMOylation, UBE2I plays a central role in SUMOylation-mediated cellular pathways and is most likely a tumor-promoting factor . UBE2I (also known as UBC9) is over expressed in several types of cancers to highlight the clinical significance. It was recently demonstrated that miRNAs such as miR-30e were able to specifically silence Ube2I/Ubc9 and provide new insight into Ube2I/Ubc9 regulation . Therefore, Ube2I/Ubc9 may serve as a potential biomarker for diagnosis or prognosis as well as a therapeutic target for cancer intervention. Mammalian RNF4 is a nuclear RING finger protein with E3 ligase activity and is an orthologue of Rfp1/Rfp2 in yeast. Rfp1 and Rfp2 lack E3 ligase activity but recruit Slx8, an active RING-finger Ub ligase, through a RING–RING interaction, to form a functional E3 [58–62]. RNF4 complements the growth and genomic stability defects of rfp1rfp2, slx8, and rfp1rfp2slx8 mutant cells. SUMOylated proteins accumulate in rfp1rfp2 double-null cells to suggest that Rfp/Slx8 also promotes Ub-dependent degradation of SUMOylated targets. Cells lacking Slx8-Rfp accumulated sumoylated proteins, displayed genomic instability and were hypersensitive to genotoxic stresses such as hydroxyurea, methylmethane sulfonate, camptothecin and UV radiation. The discovery of a family of E3 ligases that act as SUMO-Targeted Ub Ligases (STUbLs) is intriguing since STUbLs appear to be recruited to SUMOylated proteins to mediate their ubiquitination and degradation. STUbL dysfunction causes a specific accumulation of sumoylated protein species and correlated defects in DNA repair and genetic integrity. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1 to demonstrate the specificity of STUbLs as regulators of SUMOylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8-Rfp, underscoring the evolutionary functional conservation of STUbLs. Moreover, it has recently been demonstrated that RNF4 that recognizes poly-SUMOylated proteins for Ub+proteasomal degradation was more highly expressed in MM patients compared to normal donors. Additionally, RNF4 and other genes that express components of the SUMOylation, Ubiquitination and Proteasome system were also induced in MM patients that did not respond to bortezomib to suggest that RNF4 also protects cells against bortezomib-induced cell death. Poly-SUMOylated proteins were also shown to be ubiquitinated and associated with the 26S proteasome to imply a biologically interactive system.
Of all the ULPs, Rub1, also known as NEDD8, has a primary sequence most similar to that of Ub . NEDDylation has importance for cell cycle control, signal transmission, cell differentiation and DNA repair. The only known substrates are the Cullins, most or all of which are subunits of SCF (Skp1–Cullin–F-box protein) or SCF-related Ub ligases . The SCF-like family of E3s, which include a RING protein in their catalytic core, ubiquitinate a wide array of substrates. Cullin family proteins organize Ub ligase complexes to target numerous cellular proteins for polyubiquitinylation and subsequent proteasomal degradation. NEDD8-activating enzyme (NAE) transfers NEDD8 to a specific NEDD8-conjugating enzyme called ubc12, which then transfers NEDD8 to the Cullin subunit. MLN4924 (Millennium Pharmaceuticals, Cambridge, MA, USA) is a recently discovered potent, specific and reversible inhibitor of NAE. MLN4924 (Millennium: Takeda Oncology Cambridge MA) has been shown to inhibit cell growth across a wide range of tumors including breast, lung, and diffuse large B cell lymphomas .
Targeting the UPS to promote a tumor-specific immune response
Current treatment for advanced stage solid tumors and metastatic disease yield low response rates and generally do not provide significantly improved OS. Innovative treatment strategies beyond conventional therapies such as chemotherapy, surgery, and radiation are needed to overcome the high mortality and morbidity associated with solid tumors. Immunotherapy has emerged as an attractive approach for the treatment of cancers since it has the ability to specifically eradicate systemic tumors and control metastases without damaging normal cells. The UPS has been shown to be involved not only in antigen processing but also apoptosis, co-stimulation, adhesion and chemotaxis, and has generated interest for a role of proteasome inhibitors in immunotherapy. It is important to evaluate the immunotherapeutic effect of proteasome inhibitors in patients with coexisting malignancy-induced immuno-depression and the effect of prior chemotherapeutic treatments. The UPS has been shown to play a role in the processing of intracellular and viral proteins to generate small peptide products that then serve as major histocompatibility complex (MHC) class I restricted antigenic peptides . The immunoproteasome is therefore an essential component of the immune system and provides class I antigens for presentation to cytotoxic T cells (CTLs). Class I MHC components then bind the proteasome-generated peptide, and the peptide~MHC complex is escorted and presented at the cell surface for recognition by specific T cell receptors (TCRs) that reside on the CTLs. Certain subunits that comprise the proteasome are replaced by other γ-interferon-inducible proteasome subunits to generate a structurally and functionally distinct complex referred to as the immunoproteasome. The immunoproteasome appears to be the biologically relevant structure that produces antigenic peptides. CTLs and NK cells are the cytolytic effectors of the immune system and recognize targets using specific receptors through partially complementary mechanisms. CTLs recognize antigens present at the cell surface through their TCR, while NK target recognition is achieved by the absence of syngeneic MHC molecules through dominant NK-inhibiting receptors. Alternatively, NK cells may also utilize the presence of allogeneic MHC molecules or MHC-like molecules by NK-activating receptors to recognize targets. Targeted cells must express a cognate antigen~MHC complex to be recognized and susceptible to a CTL. In malignancy, MHC levels are down-regulated to render tumor cells less susceptible to CTL-induced lysis but may actually promote NK cell induced tumor lysis. Exposure of cells to bortezomib simultaneously results in divergent effects on NK and T cell function. Whereas bortezomib-treated tumors became sensitized to NK cell apoptosis, proteasome inhibition altered tumor-antigen presentation and paradoxically reduced tumor-specific T cell effector response.
Novel methods to utilize proteasome inhibitors are currently being investigated to overcome tumor resistance to the desired apoptotic effect of immune effector cells and as adjuncts to targeted anticancer therapies. Evidence from animal models suggests that proteasome inhibitors have immunosuppressive properties and impair monocyte-derived dendritic cells (DCs) . Specifically, bortezomib exposure reduced the phagocytic capacity of DCs, decreased phenotypic maturation in response to lipopolysaccharide (LPS), CD40L, and TNF-α, and reduced cytokine production and immunostimulatory capacity. DC functions are profoundly affected by bortezomib, possibly through disruption of the NF-κB or other essential signaling pathways and point to a potential modulatory role of proteasome inhibitors in Toll-like receptor signaling and the immune response . DNA vaccine strategies may be enhanced by using proteasome inhibitors to modulate MHC class I antigen processing by the UPS and subsequent presentation in DCs . Therefore, it was hypothesized that the combination of a DNA vaccine that encoded calreticulin (CRT) linked to HPV-16 E7 in combination with bortezomib may lead to enhanced antitumor effects against E7-expressing tumors. Potent E7-specific CD8+ T cell immune responses and significant effects against TC-1 tumors were detected. Mechanistically, it would appear that bortezomib does not increase MHC class I antigen production but rather may upregulate death receptors (DR5).
A number of anticancer agents such as bortezomib have been shown to sensitize tumors to death receptor signaling pathways used by both NK and T cells and to thus selectively induce tumor cell death . Human leukocyte antigen class I molecules expressed by tumor cells are fundamental in controlling the NK cell-mediated immune response . Bortezomib was shown to reduce class I in MM cell lines and in MM patient cells. This down-regulation sensitized MM cells to allogeneic and autologous NK cell-mediated lysis and was consistent with the notion that the degree of HLA class I down-regulation may be physiologically relevant. It those studies, bortezomib did not alter interaction with TRAIL, NKG2D and NCRs on NK cells. Bortezomib also sensitized primary MM cells to cytolysis by NK cells. Normally, MM cells display self-molecules that inactivate autologous NK cells. This finding may explain the relative lack of autologous NK cell activity after adoptive transfer observed in the solid tumor setting . Moreover, bortezomib overcomes KIR-L inhibition of NK cells by MM and may facilitate autologous adoptive NK cell therapy. Similar studies demonstrated that bortezomib sensitized tumors to death receptor signaling pathways used by both NK and T cells to induce tumor apoptosis . Human and murine tumors exposed to bortezomib upregulated TRAIL death receptors and displayed increased caspase-8 activity with enhanced NK cell killing through TRAIL, Fas, and perforin granzyme. These studies demonstrated that bortezomib-treated tumors were sensitized to NK cell-induced apoptosis but paradoxically acquired resistance to tumor-specific CTLs.
Sustained regression of metastatic solid cancers has been achieved through immunotherapeutic strategies primarily in immune-sensitive cancers. However, these strategies have response rates that range from 5–10% in melanoma and renal cell carcinoma patients. The antitumor activity of stimulated tumor antigen-specific T cells is limited by local factors within the tumor microenvironment. Modulation of the tumor-surrounding milieu may be necessary to overcome resistance to immunotherapy. The Ub+proteasome, SUMOylation and NEDDylation pathways are ideal candidates to target as immunopotentiators to overcome oncogenic mechanisms that promote a proapoptotic state. Further understanding the mechanisms of tumor cell escape from immune surveillance may allow rational combinatorial approaches of novel therapies such as proteasome inhibitors to target tumor-induced immunosuppressive or antiapoptotic steps and reverse resistance to effectors of the immune system.
Conflict of interest statement
No conflict of interest.