Blockage of neddylation modification stimulates tumor PNAS PLUS sphere formation in vitro and stem cell differentiation and wound healing in vivo Xiaochen Zhoua,b,1, Mingjia Tanb,1, Mukesh K. Nyatib, Yongchao Zhaoc,d, Gongxian Wanga,2, and Yi Sunb,c,e,2 aDepartment of Urology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, China; bDivision of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109; cInstitute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; dKey Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; and eCollaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310058, China Edited by Vishva M. Dixit, Genentech, San Francisco, CA, and approved March 10, 2016 (received for review November 13, 2015) MLN4924, also known as pevonedistat, is the first-in-class inhibitor acting alone or in combination with current chemotherapy of NEDD8-activating enzyme, which blocks the entire neddylation and/or radiation (6, 11). One of the seven clinical trials of MLN4924 modification of proteins. Previous preclinical studies and current (NCT00911066) was published recently, concluding a modest effect clinical trials have been exclusively focused on its anticancer property. of MLN4924 against acute myeloid leukemia (AML) (12). Unexpectedly, we show here, to our knowledge for the first time, To further elucidate the role of blocking neddylation in cancer that MLN4924, when applied at nanomolar concentrations, signif- treatment, we thought to study the effect of MLN4924 on cancer icantly stimulates in vitro tumor sphere formation and in vivo stem cells (CSCs) or tumor-initiating cells (TICs), a small group tumorigenesis and differentiation of human cancer cells and mouse of tumor cells with stem cell properties that have been claimed to embryonic stem cells. These stimulatory effects are attributable to be responsible for cancer initiation and relapse (13). Meanwhile, (i) c-MYC accumulation via blocking its degradation and (ii)contin- a recent study showed that pharmacological inactivation of SKP2 SCF (SKP1–Cullins–F-box proteins) E3 ubiquitin ligase (also ued activation of EGFR (epidermal growth factor receptor) and its known as CRL1) by a small molecule inhibitor, compound 25, downstream pathways, including PI3K/AKT/mammalian target of could restrict CSC traits and cancer progression in prostate rapamycin and RAS/RAF/MEK/ERK, via inducing EGFR dimerization. cancer (14). Finally, MLN4924 accelerates EGF-mediated skin wound healing in To our utter surprise, we found that at low drug concentra- mouse and stimulates cell migration in an in vitro culture setting. tions (30–100 nM), MLN4924 stimulates in vitro tumor sphere Taking these data together, our study reveals that neddylation (TS) formation and in vivo tumorigenesis of both cancer cells modification could regulate stem cell proliferation and differenti- and embryonic stem cells. Mechanistic studies revealed the ation and that a low dose of MLN4924 might have a therapeutic involvement of accumulation of c-MYC, and dimerization and value for stem cell therapy and tissue regeneration. activation of EGFR (epidermal growth factor receptor). We also found that MLN4924 could accelerate the process of EGF- MLN4924 | neddylation | EGFR | stem cell | wound healing induced skin wound healing in mouse and cell migration in an in vitro culture setting. Thus, MLN4924, when applied at low n cells, homeostasis is reached by a fine balance between the Irates of protein synthesis and degradation. The process of Significance protein degradation by the ubiquitin-proteasome system (UPS) involves two sequential steps: (i) ubiquitylation catalyzed by the MLN4924, a potent small-molecule inhibitor of NEDD8-activating ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme enzyme, blocks cullin-RING ligase activity through inhibiting (E2), and a ubiquitin ligase (E3) and (ii) degradation catalyzed cullin neddylation. MLN4924 is widely used in both preclinical by 26S proteasome (1). Abnormal UPS activity abrogates the and clinical settings for an anticancer application. We report here homeostasis and is associated with many human diseases, an unexpected finding: MLN4924 at nanomolar concentra- including cancer (2, 3). Bortezomib, the first Food and Drug tion stimulates stem cell proliferation, self-renewal, and differ- Administration-approved proteasome inhibitor for the treatment entiation in both tumor and normal stem cell models and of multiple myeloma and mantle cell lymphoma (4), possesses promotes skin wound healing in a mouse model and cell mi- significant cytotoxicity against normal cells due to its universal gration in vitro. Mechanistic studies revealed that MLN4924 inhibition of the UPS (5). This stimulated an intensive search for causes c-MYC accumulation and promotes EGFR (epidermal inhibitors targeting the enzymes upstream of the proteasome for growth factor receptor) dimerization to activate the EGFR sig- improved specificity. naling pathway. Our study raises a concern in anticancer appli- Cullin-RING ligases (CRLs), consisting of scaffold cullins, adaptor cation of MLN4924, but at the same time provides an opportunity proteins, substrate-recognizing receptors, and RING finger proteins, for future development of MLN4924 as an agent for stem cell are the largest family of E3 ubiquitin ligases and are responsible for therapy and tissue regeneration. the ubiquitylation of about 20% of all cellular proteins (6, 7). The activity of CRLs requires neddylation of the cullin proteins (8, 9), a Author contributions: X.Z., M.K.N., G.W., and Y.S. designed research; X.Z., M.T., and Y.Z. ubiquitylation-like process, which is sequentially catalyzed by three performed research; X.Z. and Y.S. contributed new reagents/analytic tools; X.Z., M.K.N., CELL BIOLOGY enzymes: a NEDD8-activating enzyme (NAE or E1), NEDD8- and Y.S. analyzed data; and X.Z. and Y.S. wrote the paper. conjugating enzyme (E2), and NEDD8 ligase (E3). Abnormal el- The authors declare no conflict of interest. evation of CRL activity has been found in multiple human cancers, This article is a PNAS Direct Submission. which makes them favorable targets for cancer treatment (10, 11). Freely available online through the PNAS open access option. MLN4924 was descried in 2009 as the first-in-class NAE in- 1X.Z. and M.T. contributed equally to this work. hibitor exclusively in the application for cancer treatment by 2To whom correspondence may be addressed. Email: [email protected] or wanggx- inactivation of CRLs via blockage of cullin neddylation (7). Since [email protected]. the initial report, more than 150 papers (by PubMed search) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. were published to show its preclinical anticancer activity, either 1073/pnas.1522367113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1522367113 PNAS | Published online May 9, 2016 | E2935–E2944 Downloaded by guest on September 27, 2021 concentrations, may have potential utility in the field of stem cell therapy and tissue regeneration. Results Bipolar Effects of MLN4924 on Proliferation of Cancer Cells. Sup- pression of cancer cell growth by MLN4924 has been well- documented in the literature (for a review, see ref. 11). Indeed, we found a dose-dependent growth inhibition in multiple human cancer cell lines, including H1299 (lung cancer), MCF7 and SUM159 (breast cancer), and HCT116 (colorectal cancer), when cultured in the media containing 10% (vol/vol) or 1% serum (Fig. S1 A and B). Unexpectedly, when cultured in serum-free medium, we observed various degrees of dose-dependent growth stimulation at low MLN4924 concentrations (30–300 nM) in H1299 [maximum effective concentration (ECmax), 0.1 μM, P < 0.0001 (Fig. S1A)], HCT116 (ECmax,0.3μM, P < 0.0001) (Fig. S1B), MCF7 (ECmax,0.1μM, P = 0.0001) (Fig. S1B), and SUM159 (ECmax,0.1μM, P = 0.0098) (Fig. S1B) cells, followed by growth suppression at higher concentrations. We confirmed via trypan blue exclusion assay that the number of living cells was indeed signif- icantly increased following treatment with a low dose of MLN4924 under serum-free conditions (Fig. S1C)(P < 0.0001). Collectively, we uncovered the bipolar effects of MLN4924 on cell proliferation, both stimulatory and inhibitory, on cancer cells depending on se- rum and drug concentrations. We then focused our study on the growth-stimulating effect of MLN4924. MLN4924 Stimulates TS Formation in Multiple Cancer Cell Lines. The observation that MLN4924 at low concentrations stimulated cell growth under serum-starved conditions triggered us to test whether MLN4924 would promote TS formation, an assay widely used to study the stem cell potential in a serum-free suspension culture (15). We first tested H1299 nonsmall cell lung cancer Fig. 1. MLN4924 stimulates TS formation and in vivo tumorigenesis. (A–C) H1299 (NSCLC) cells that formed typically spherical TSs in standard TS formation assay. H1299 cells were plated at clonal density in the TS me- conditioned TS medium (16). We found a significant dose- dium in 24-well ULA plates and treated with the indicated concentration dependent stimulation by MLN4924 on TS formation with an of MLN4924 for up to 16 d. Representative pictures were taken (A) and SSSs ECmax of 100 nM, followed by a dose-dependent inhibition of TS were documented at 4-d intervals. Fold-changes in SSSs were calculated by formation, with 1 μM MLN4924 completely disrupting TS for- arbitrarily setting the SSS of vehicle treated wells as 1 and were plotted mation (Fig. 1A). To quantify, we used our recently developed against dose (B) and time (C). (D) Ki67 staining of H1299 TS. H1299 TS cul- Standardized Sphere Score (SSS) (16) as the assessment pa- tured in DMSO (vehicle control) and 0.1 μM MLN4924 were harvested and rameter and found that 0.1 μM MLN4924 induced a greater than sectioned for Ki67 staining. (E) Effect of MLN4924 on TS formation in the 30-fold increase in SSS in H1299 cells (Fig.