| Abstract: | MLN4924, also known as pevonedistat, is the first-in-class inhibitor of NEDD8-activating enzyme, which blocks the entire neddylation modification of proteins. Previous preclinical studies and current clinical trials have been exclusively focused on its anticancer property. Unexpectedly, we show here, to our knowledge for the first time, that MLN4924, when applied at nanomolar concentrations, significantly stimulates in vitro tumor sphere formation and in vivo tumorigenesis and differentiation of human cancer cells and mouse embryonic stem cells. These stimulatory effects are attributable to (i) c-MYC accumulation via blocking its degradation and (ii) continued activation of EGFR (epidermal growth factor receptor) and its downstream pathways, including PI3K/AKT/mammalian target of rapamycin and RAS/RAF/MEK/ERK, via inducing EGFR dimerization. Finally, MLN4924 accelerates EGF-mediated skin wound healing in mouse and stimulates cell migration in an in vitro culture setting. Taking these data together, our study reveals that neddylation modification could regulate stem cell proliferation and differentiation and that a low dose of MLN4924 might have a therapeutic value for stem cell therapy and tissue regeneration.In cells, homeostasis is reached by a fine balance between the rates of protein synthesis and degradation. The process of protein degradation by the ubiquitin-proteasome system (UPS) involves two sequential steps: (i) ubiquitylation catalyzed by the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) and (ii) degradation catalyzed by 26S proteasome (1). Abnormal UPS activity abrogates the homeostasis and is associated with many human diseases, including cancer (2, 3). Bortezomib, the first Food and Drug Administration-approved proteasome inhibitor for the treatment of multiple myeloma and mantle cell lymphoma (4), possesses significant cytotoxicity against normal cells due to its universal inhibition of the UPS (5). This stimulated an intensive search for inhibitors targeting the enzymes upstream of the proteasome for improved specificity.Cullin-RING ligases (CRLs), consisting of scaffold cullins, adaptor proteins, substrate-recognizing receptors, and RING finger proteins, are the largest family of E3 ubiquitin ligases and are responsible for the ubiquitylation of about 20% of all cellular proteins (6, 7). The activity of CRLs requires neddylation of the cullin proteins (8, 9), a ubiquitylation-like process, which is sequentially catalyzed by three enzymes: a NEDD8-activating enzyme (NAE or E1), NEDD8-conjugating enzyme (E2), and NEDD8 ligase (E3). Abnormal elevation of CRL activity has been found in multiple human cancers, which makes them favorable targets for cancer treatment (10, 11).MLN4924 was descried in 2009 as the first-in-class NAE inhibitor exclusively in the application for cancer treatment by inactivation of CRLs via blockage of cullin neddylation (7). Since the initial report, more than 150 papers (by PubMed search) were published to show its preclinical anticancer activity, either acting alone or in combination with current chemotherapy and/or radiation (6, 11). One of the seven clinical trials of MLN4924 ({"type":"clinical-trial","attrs":{"text":"NCT00911066","term_id":"NCT00911066"}}NCT00911066) was published recently, concluding a modest effect of MLN4924 against acute myeloid leukemia (AML) (12).To further elucidate the role of blocking neddylation in cancer treatment, we thought to study the effect of MLN4924 on cancer stem cells (CSCs) or tumor-initiating cells (TICs), a small group of tumor cells with stem cell properties that have been claimed to be responsible for cancer initiation and relapse (13). Meanwhile, a recent study showed that pharmacological inactivation of SKP2 SCF (SKP1–Cullins–F-box proteins) E3 ubiquitin ligase (also known as CRL1) by a small molecule inhibitor, compound 25, could restrict CSC traits and cancer progression in prostate cancer (14).To our utter surprise, we found that at low drug concentrations (30–100 nM), MLN4924 stimulates in vitro tumor sphere (TS) formation and in vivo tumorigenesis of both cancer cells and embryonic stem cells. Mechanistic studies revealed the involvement of accumulation of c-MYC, and dimerization and activation of EGFR (epidermal growth factor receptor). We also found that MLN4924 could accelerate the process of EGF-induced skin wound healing in mouse and cell migration in an in vitro culture setting. Thus, MLN4924, when applied at low concentrations, may have potential utility in the field of stem cell therapy and tissue regeneration. |
