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Inhibition of Mcl-1 enhances Pevonedistat-triggered apoptosis in osteosarcoma cells
Author’s Accepted Manuscript Inhibition of Mcl-1 enhances Pevonedistat-triggered apoptosis in osteosarcoma cells Yi Zhang, Chengcheng Shi, Li Yin, Wei Zhou, Haitao Wang, Jingjing Seng, Wencai Li www.elsevier.com/locate/yexcr
PII: DOI: Reference:
S0014-4827(17)30350-6 http://dx.doi.org/10.1016/j.yexcr.2017.06.019 YEXCR10642
To appear in: Experimental Cell Research Received date: 12 January 2017 Revised date: 21 May 2017 Accepted date: 23 June 2017 Cite this article as: Yi Zhang, Chengcheng Shi, Li Yin, Wei Zhou, Haitao Wang, Jingjing Seng and Wencai Li, Inhibition of Mcl-1 enhances Pevonedistattriggered apoptosis in osteosarcoma cells, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2017.06.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inhibition of Mcl-1 enhances Pevonedistat-triggered apoptosis in osteosarcoma cells Yi Zhang a,1, Chengcheng Shi b1, Li Yin a, Wei Zhou a, Haitao Wang a, Jingjing Seng c, Wencai, Li d a
Department of Orthopaedic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
b
Department of Pharmacy, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China c
Department of pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA d
Department of Pathology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
Corresponding author: Yi Zhang: No.1, East Jian She Road, Zhengzhou, 450052, Henan, China, Tel.: +86-371-66913032. Email: [email protected], Fax: +86-371-66295356
Running title: Mcl-1 attenuates Pevonedistat activity
1
These authors contributed equally to this work
Abstract
Neddylation inhibitor Pevonedistat (MLN4924) is a novel anticancer drug and has demonstrated broad-spectrum anticancer activity. Nevertheless, we found that Pevonedistat had only a modest apoptotic effect in osteosarcoma (OS) cells. Moreover, we noted that inhibition of neddylation by Pevonedistat led to accumulation of Mcl-1 protein in OS cells. Because Mcl-1 is an important anti-apoptotic protein and also because apoptosis has been shown to be a major cell death pathway, we hypothesized that Mcl-1 accumulation negatively impacted Pevonedistat-mediated anticancer activity in OS cells. In this regard, we employed genetic or pharmacological approaches to inhibit Mcl-1 expression and to examine the effect on Pevonedistat-induced apoptosis in OS cells. We found that inhibition of Mcl-1 expression by siRNA considerably enhanced Pevonedistat-triggered the activation of caspase-3, PARP cleavage and apoptosis, and also dramatically promoted the ability of Pevonedistat to inhibit colony formation of OS cells. Moreover, we observed that flavopiridol, a FDA approved drug, inhibited Mcl-1 expression and substantially enhanced Pevonedistat-mediated activation of apoptosis signaling and significantly augmented cell killing effect in OS cells. Altogether, our study shows that Mcl-1 is a critical resistance factor to Pevonedistat monotherapy, and suggests that Pevonedistat in combinations with flavopiridol may achieve better anticancer therapy.
Keywords: Pevonedistat; Mcl-1; Apoptosis Osteosarcoma
1. Introduction
Neddylation is a post-translational protein modification process through which ubiquitin-like protein NEDD8 is conjugated to protein substrates. This process is catalyzed by an enzymatic cascade comprising NEDD8-activating enzyme E1 (NAE1), NEDD8-conjugating enzyme E2, and substrate-specific NEDD8-E3 ligases.[1] The cullin protein family is the most established targets for neddylation. Neddylation allows activation of Cullin-RING E3 ubiquitin ligases (CRLs), which in turn regulate the turnover of proteins involved in many important cellular processes such as cell proliferation, DNA integrity, as well as apoptosis, via ubiquitin (Ub)/26S proteasome system (UPS).[1-3] Overactivation of neddylation pathway has been found in a variety of human cancers and may contribute to carcinogenesis and progression.[4-6] Therefore, neddylation is an attractive target for cancer therapy.
Pevonedistat (MLN4924) is a specific and selective inhibitor of neddylation.[7] By inducing accumulation of tumor suppressor proteins, Pevonedistat causes cell cycle arrest and DNA damage, triggers apoptosis, thus exhibiting broad-spectrum antitumor activity.[4, 8-10] We recently investigated the potential utility of Pevonedistat in osteosarcoma (OS) using in vitro and in vivo models and found that this drug was able to inhibit tumor growth in a mouse xenograft OS tumor model, suggesting that Pevonedistat holds promise for OS treatment.[11] Nevertheless, in the study, we observed that as compared to its extraordinary cytostatic effects, Pevonedistat exhibited only a modest apoptotic effect in OS cells. For instance, even at a very low concentration (0.04 µM), Pevonedistat effectively disrupted cell cycle progression in OS cells. Nonetheless, Pevonedistat triggered only modest activation of caspase-3 and only a small amount of cytochrome c release at a much higher concentration (1 µM). Moreover, we found that
Pevonedistat treatment led to a dramatic increase in the protein level of myeloid cell leukemia 1 (Mcl-1) in OS cells. Given that Mcl-1 is a critical anti-apoptotic member of Bcl-2 family proteins, we are interested in investigating whether Mcl-1 is a potential resistance factor to Pevonedistat monotherapy, and exploring approaches to improve Pevonedistat-based anticancer activity.
2. Materials and methods
2.1. Reagents and cell lines The cell lines Saos-2 and SJSA-1 obtained from China Center for Type Culture Collection (Wuhan, China) were maintained in PRMI1640 (HyClone/Thermo Fisher Scientific, Beijing, China) supplemented with 10% heat-inactivated fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd, Hangzhou, China) at 37°C in a humidified incubator containing 5% CO2. Pevonedistat and cyclin dependent kinase (CDK) inhibitor flavopiridol were obtained from Shanghai Selleck Chemicals (Shanghai, China) and both compounds were dissolved in Dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and stored at −20°C. 2.2. Cell death assay Cells were seeded in 96-well plates and treated with Pevonedistat or DMSO for the indicated times. Cell death was quantified by microscopic examination in trypan blue exclusion assays. 2.3. Western blot analysis Cells were lysed in RIPA buffer after the treatments. Equal amounts of lysate were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and evaluated by western blot analysis as described previously.[11] Rabbit anti-human PARP, caspase-3, Bcl-2, Bcl-xL and Cullin1 antibodies were purchased from Santa Cruz Biotechnology (Shanghai, China). Mouse
anti-human Mcl-1 was purchased from R&D System China (Shanghai, China). Mouse anti-beta actin, rabbit anti-FBXW7 and mouse anti-βTrCP were obtained from Sigma-Aldrich (Shanghai, China). Quantifications were performed using ImageJ 1.45 software. 2.4. Protein stability assay Protein stability assay was performed as described previously.[12] Briefly, cells were treated with DMSO or Pevonedistat for 6 h and then cycloheximide (5 μg/ml) was added to block protein synthesis. Collected protein samples were subjected to Western blot analysis using antiMcl-1 antibody. 2.5. Flow cytometry assay Apoptosis in treated cells was detected using an FITC Annexin-V Apoptosis Detection Kit (BD Biosciences-CN, Shanghai, China) according to the manufacturer's instructions. Briefly, the cells were digested with 0.25% trypsin, washed twice with cold phosphate-buffered saline (PBS), and then incubated with Annexin-V FITC for 15 min at room temperature in the dark. The population of apoptotic cells was analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, Shanghai, China). 2.6. Transfection of siRNA Mcl-1 siRNA was purchased from Santa Cruz Biotechnology (Beijing, China) for siMcl-1a (sc35877) and for non-targeting control siRNA (sc-12756), from Cell Signaling Technology (Shanghai, China) siMcl-1b (6315). Cul1 siRNA (L-004086) and FBW7 (L-004264) were purchased from GE (Shanghai, China). β-TrCP siRNA (sc-37178) was purchased from Santa Cruz Biotechnology (Beijing, China). Transfection of siRNA was conducted with RNAi Max Reagent (13778030, Invitrogen Trading Shanghai Co., Shanghai, China) according to the manufacturer's operating instructions.
2.7. Transduction of lentivrial shRNA and colonies formation Lentivrial shMcl-1 (sc-43912) and control shRNA lentiviral (sc-108080) particles were obtained from Santa Cruz Biotechnology (Shanghai, China). Saos-2 cell line was transduced with the lentiviral particles and efficacy of transduction was examined by western blot. Cells (2,000 per well) transduced with shMcl-1 or control shRNA were treated by Pevonedistat as indicated for 14 days to allow cells forming colonies. Colonies were stained with 0.025% (m/v) and photographed. 2.8. Statistical analysis The results were presented as means ± SEM of three independent experiments. For statistical tests, Prism 5.0 (GraphPad Software, SanDiego, CA, USA) was used. p values less than 0.05 were considered statistically significant.
3. Results 3.1. Pevonedistat induces rapid Mcl-1 accumulation in OS cells. In this study, we first evaluated the effect of Pevonedistat on the neddylation status of cullin1 and on the protein expression level of a panel of Bcl-2 family members in OS cells. To this end, we treated OS Saos-2 and SJSA-1 cell lines with Pevonedistat (0.04, 0.2 and 1.0 μM) for 24 h and performed western blot assay. The results showed that Pevonedistat effectively inhibited the neddylated form of cullin1 in both cell lines. The results also showed that Pevonedistat treatment dramatically increased the protein level of Mcl-1 in both OS cells lines (Fig. 1a,b). These results confirmed our previous finding with another Mcl-1 antibody.[11] However, Pevonedistat had little impact on the expression levels of anti-apoptotic Bcl-2, Bcl-xl and pro-apoptotic Bax (Fig. 1a,b).
To investigate whether Pevonedistat treatment induces Mcl-1 accumulation in OS cells in a timedependent manner, OS cell lines were treated with 0.2 μM Pevonedistat for different period of time. Western blot results demonstrated that Pevonedistat induced Mcl-1 accumulation very rapidly in these cells. Mcl-1 accumulation started within 4 h after Pevonedistat treatment, became stronger over time and lasted at least for up to 24 h in the presence of the drug (Fig. 1c,d). These results suggest that Pevonedistat rapidly and dramatically induces Mcl-1 accumulation in OS cells. 3.2. Pevonedistat increases the protein stability of Mcl-1 in OS cells. To further identify the mechanism responsible for Pevonedistat-induced Mcl-1 accumulation, we examined the Mcl-1 expression at the transcript level by reverse transcription (RT)–qPCR. The mRNA level of Mcl-1 modestly increased at 4 h after Pevonedistat treatment, but slightly decreased at 14 and 24 h (Fig. 1e,f). These results suggest that Mcl-1 accumulation may not be through transcriptional upregulation of the gene. We next examined whether Pevonedistat had an impact on the stability of Mcl-1 protein. We treated both OS cell lines with Pevonedistat or DMSO for 6 h, then treated with 5 µg/mL Cycloheximide (CHX) for another 2, 4 and 6 h. The protein level of Mcl-1 was examined by western blot, and the intensity of bands was quantified by ImageJ software respectively (Fig. 2a-d). The results showed that CHX treatment for 2 h reduced Mcl-1 level by 72 and 65% in Sao2 and SJSA1 cell lines pretreated by DMSO, respectively. CHX treatment for 6 h reduced Mcl-1 level by more than 90% in both cell lines pretreated by DMSO. In contrast, CHX treatment for 2 h only reduced Mcl-1 level by 37 and 23% in Pevonedistat-pretreated Sao2 and SJSA1 cell lines, respectively. Moreover, after treatment with CHX for 6 h, there were still 27 and 36% of Mcl-1 remained in Pevonedistat-pretreated Sao2 and SJSA1 cell lines, respectively (Fig. 2a-d). These results show that Pevonedistat
treatment dramatically attenuates CHX-mediated Mcl-1 reduction in OS cells, and suggest that Mcl-1 accumulation caused by Pevonedistat is associated with the enhancement of protein stability. 3.3. Induction of Mcl-1 accumulation by Pevonedistat requires the presence of adaptor proteins Fbw7 or β-TrCP in OS cells. In order to investigate the mechanism underlying Mcl-1 accumulation in OS cells, we knocked down cullin1 by siRNAs in OS cells and found that inhibition of cullin1 markedly increased the level of Mcl-1 (Fig. 3a). These results confirm that Mcl-1 is a substrate of cullin1-associated CRL E3 ligase. Ubiquitination and proteasomal degradation of Mcl-1 by CRLs requires two F-box proteins, Fbw7 and beta-transducin repeats-containing protein (β-TrCP) as adaptor proteins to recruit Mcl1 to cullin1-dependent CRLs ubiquitin E3 ligases in certain types of cells. We next investigated the role of these two F-box proteins in the modulation of Mcl-1 by Pevonedistat with specific siRNA in Sao2 cell line. Western blotting analysis showed that siRNA transfection for 48 h effectively inhibited the expression of targeted genes. Of note, knockdown of either Fbw7 or βTrCP led to an increase of Mcl-1 level (Fig. 3a). These results indicate that both two F-box proteins were involved in Mcl-1 ubiquitination and degradation in OS cells. We next treated siRNA transfected-cells with Pevonedistat for another 6 h and analyzed the Mcl1 with western blotting analysis. The results showed that Pevonedistat treatment led to marked accumulation of Mcl-1 protein in control siRNA transfected cells, but only slightly or modestly increase the Mcl-1 level in β-TrCP siRNA and Fbw7 siRNA transfected cells (Fig. 3b,c). These results evidently suggest that induction of Mcl-1 accumulation by Pevonedistat requires the presence of these two adaptor proteins in this OS cell line.
3.4. Mcl-1 knockdown by siRNA enhanced Pevonedistat-induced apoptotic signaling in OS cells. It is well documented that Mcl-1 is an anti-apoptotic protein and protects tumor cells against apoptosis. Thus, it is highly possible that inhibition of Mcl-1 accumulation can enhance Pevonedistat-triggered apoptosis in OS cells. To address this issue, OS cells were transfected with Mcl-1 siRNA (siMcl-1a and siMcl-1b) and further were treated with Pevonedistat. Both siRNA very efficiently inhibited Mcl-1 expression. Furthermore, Pevonedistat treatment not only induced dramatic Mcl-1 accumulation in siCtl-transfected cells, but also could induce Mcl-1 accumulation in OS cells transfected with siMcl-1. However, Mcl-1 accumulation in siMcl-1transfected cells was negligible (Fig. 4a,b), suggesting that Mcl-1 siRNAs were extremely efficient in inhibition of Pevonedistat-mediated Mcl-1 accumulation in OS cells. Western blot also was performed to evaluate molecular biomarkers of apoptotic signaling. As shown in Fig. 4, Pevonedistat triggered much stronger caspase-3 activation and more PARP cleavage in cells transfected with siMcl-1 than in cells transfected with siCtl. Altogether, these results suggest that Mcl-1 negatively regulates Pevonedistat-mediated apoptotic in OS cells. 3.5. Knockdown of Mcl-1 sensitizes OS cells to Pevonedistat-mediated apoptotic cell death. To further determine the effect of Mcl-1 knockdown on Pevonedistat-mediated anti-OS activity, we examined apoptosis induction in the treated cells by Annexin-V FITC staining and flow cytometery assay in Saos-2 cell line. The results showed that Pevonedistat treatment for 24 h resulted in a significantly higher apoptosis proportion in Mcl-1 siRNA transfected cells (53.01 ±8.01 for siMcl-1a and 50.7 ± 7.5% for siMcl-1b) than in siCtl transfected cells (18.3 ± 3.5%, p < 0.01 versus both siMcl-1) (Fig. 5a,b).
Cell viability assay was performed to examine the effect of Mcl-1 inhibition on Pevonedistatmediated cell killing in both Saos-2 and SJSA-1 cell lines. In Saos-2 cell line, transfection with Mcl-1 siRNAs alone caused about 10% cell death, suggesting a role of Mcl-1 in sustaining cell survival of this cell line. Of note, Pevonedistat induced significantly more cell death in cells transfection with siMcl-1 than in cells transfected with siCtl (siCtl versus siMcl-1a or b, both p<0.01) (Fig. 5c). A substantial enhancement of Pevonedistat-mediated cell death by Mcl-1 knockdown was also observed in SJSA-1 cell line (siCtl versus siMcl-1a or b, both p<0.01) (Fig. 5d). Thus, inhibition of Mcl-1 accumulation could significantly promote Pevonedistat-mediated apoptotic cell death in OS cell lines. 3.6. Knockdown of Mcl-1 by shRNA enhances Pevonedistat-mediated colony formation. In order to determine the long-term effect of Mcl-1 knockdown on the Pevonedistat activity, Saos-2 cell line infected with lentivirus vectors expressing either a shRNA targeting Mcl-1 (shMcl-1) or a control shRNA (shCtl) were treated by Pevonedistat at 0.04 µM or DMSO as control treatment for 14 days to allow cells forming colonies. As shown in Fig.5e and 5f, cells infected with control shRNA or shMcl-1 lentiviruses both formed colonies. Although Pevonedistat treatment significantly reduced the colony numbers in cells with control shRNA, there were still a decent number of colonies formed after the treatment. In striking contrast, Pevonedistat treatment completely inhibited colony formation in cells bearing shMcl-1 lentivirus. These results suggest that Mcl-1 also inhibits the long-term anti-OS activity of Pevonedistat. 3.7. Flavopiridol enhances Pevonedistat-mediated apoptotic cell death. Flavopiridol is a first-generation CDK inhibitor which has been approved by FDA for the treatment of acute myeloid leukemia. [13, 14] This drug has been shown to inhibit Mcl-1 in multiple cancer cells. We here investigated whether flavopiridol could inhibit Mcl-1 and promote
Pevonedistat-mediated apoptotic cell death in OS cells. We treated the cells with 3 µM flavopiridol, 0.2 µM Pevonedistat or both for 24 h, and then examined the levels of Mcl-1, caspase-3 activation and PARP cleavage. We observed that flavopiridol distinctly reduced the protein level of Mcl-1 in both OS cell lines. Moreover, flavopiridol or Pevonedistat alone had little or modest inhibitory effect on the expression of full-length PARP, suggesting weak activation of apoptosis signaling. In striking contrast, treatment of Pevonedistat in combination with flavopiridol resulted in marked reduction of pro-PARP and induced massive accumulation of PARP cleavage and activated caspase-3 in both OS cell lines (Fig. 6a,b). Cell death analysis further showed that combination of the two drugs significantly enhanced cell killing effect as compared to single agent in both OS cell lines (Fig. 6c,d). These results suggest that combination with flavopiridol significantly enhances Pevonedistat-mediated apoptotic cell killing effect, likely through inhibiting Mcl-1 expression.
4. Discussion
Neddylation pathway plays an essential role in ubiquitination and proteasomal degradation of intracellular proteins, some of which are closely associated with cell survival, proliferation, apoptosis, DNA integrity, and many other crucial cellular processes.[6-9] Previous studies suggested that cancer cells usually are much more sensitive to inhibition of neddylation pathway than normal cells; therefore, inhibition of Neddylation is considered as a promising strategy of cancer treatment.[6-10] Indeed, the novel anticancer drug Pevonedistat has demonstrated promising anticancer activity via inhibition of neddylation pathway.[9-11] However, data from previous studies also showed that Pevonedistat only partially inhibited tumor growth, but could
not induce tumor regression.[15, 16] This suboptimal antitumor activity has inspired a number of groups to elucidate the mechanisms underlying Pevonedistat-resistance and explore approaches to overcome the resistance.[15, 16]
It has been reported that Pevonedistat elicited anticancer activity by inducing accumulation of tumor suppressive proteins, such as pro-apoptotic protein NOXA, cell cycle regulators p21, p27 and DNA replication licensing factor CDT1.[17-19] However, we and other groups observed that proteins with pro-survival function, such as cyclinE and Mcl-1, were also robustly increased by Pevonedistat in cancer cells.[11, 20-23] Thus, we hypothesized that the accumulation of these pro-survival proteins contributed to Pevonedistat-resistance. We focused our study on the role of Mcl-1 because Mcl-1 is an important anti-apoptotic protein and often plays a decisive role in the process of apoptosis.[24, 25] We have made several important observations in this study. Firstly, Pevonedistat treatment distinctly increases the protein level of Mcl-1 in OS cells and importantly, this increase occurs within 4 h, an early time compared to apoptosis induction that was observed after Pevonedistat treatment for 24 h. Secondly, inhibition of Mcl-1 by knockdown dramatically enhances Pevonedistat-triggered activation of caspase-3 and PARP cleavage, significantly promotes Pevonedistat-mediated cell death, and also boosts the ability of Pevonedistat to inhibit colony formation in OS cells. Importantly we found that flavopiridol, a CDK inhibitor with function of inhibiting Mcl-1 expression, also significantly augmented Pevonedistat-triggered apoptotic signaling in OS cells. Our finding thus demonstrates that Mcl-1 confers resistance of OS cells to Pevonedistat, and also indicate that combination with flavopiridol can be used to improve Pevonedistat-based anticancer therapy.
Mcl-1 is a unique member amongst the Bcl-2 family, with relatively short half-lives (1-3 h). The expression level of Mcl-1 can be modulated at both transcriptional and translational levels by numerous factors.[26] We analyzed the mechanism of Mcl-1 accumulation by Pevonedistat in OS cells. RT-qPCR analysis showed that Mcl-1 transcript did not increase significantly during Pevonedistat treatment, suggesting that transcriptional mechanisms are not involved in Mcl-1 accumulation. In contrast, western blot analysis showed that Pevonedistat dramatically delayed protein synthesis inhibitor cycloheximide-mediated Mcl-1 reduction. This result highly suggests that accumulation of Mcl-1 in OS cells is caused by Pevonedistat-mediated enhancement of Mcl1 stabilization. Mcl-1 has been unambiguously identified as a substrate of CRLs, and therefore inhibition of cullin neddylation blocks UPS-mediated degradation of Mcl-1 protein.[27] Additionally, the siRNA experiments shows that Pevonedistat-mediated Mcl-1 accumulation require the presence of Fbw7 and β-TrCP. Considering that Fbw7 and β-TrCP are not expressed in all cancer cells [28, 29], we assume that the expression of these two adaptor proteins might be useful for selecting patients for Pevonedistat in combination with Mcl-1-inhibiting agents.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors thank Dr. Lan Huang for her critical reading of this manuscript (The 1st Affiliated Hospital of Zhengzhou University).
Funding
This work was supported by the Education Department of Henan Province (Grants No: 15A320086), The Science and Technology Department of Henan Province (Grants No: 162300410094), Young physicians funds of The 1st Affiliated Hospital of Zhengzhou University (2014006, 2015163).
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Figure legends
Figure 1. Pevonedistat induces accumulation of Mcl-1 in OS cells. OS cell lines Saos-2 (a) and SJSA-1 (b) were treated with 0.04, 0.2 and 1 μM Pevonedistat for 24 h. The expression levels of both neddylated- and basal- cullin1, Mcl-1, Bcl-2, Bcl-xL and Bax proteins were analyzed by western blot analysis. Beta actin was used as a loading control. Representative results are shown from three independent experiments. OS cell lines Saos-2 (c) and SJSA-1 (d) were treated with 0.2 μM Pevonedistat for 4, 14 and 24 h. The expression level of Mcl-1 was analyzed by western blot analysis. Beta actin was used as a loading control. (e,f) The relative level of Mcl-1 mRNA was analyzed by RT-qPCR analysis. Representative results were shown from three triplicate experiments.
Figure 2. Pevonedistat induces Mcl-1 accumulation by enhancing the stability of Mcl-1 protein in OS cells. OS cell lines Saos-2 (a) and SJSA-1 (b) were pretreated with DMSO or 0.2 μM Pevonedistat for 6 h, and then OS cells were treated with 5 ug/ml CHX for another 2, 4 and 6 h. The level of Mcl-1 was analyzed by western blot analysis. Beta actin was used as a loading control. Quantitative analysis of band intensity shown in (c,d) the using ImageJ software.
Figure 3. Induction of Mcl-1 accumulation by Pevonedistat requires the presence of adaptor proteins Fbw7 or β-TrCP in OS cells. Saos-2 cell line was transfected with siRNAs for 48 h. The expression of target genes was examined by western blot analysis. Beta actin was used as a loading control. Saos-2 cell line transfected with siCTL, siFbw7 (b) or siβTrCP (c) for 48 h was treated by Pevonedistat (Pevo) for another 6 h, the expression of Mcl-1 protein level was examined by western blotting analysis. Beta actin was used as a loading control.
Figure 4. Knockdown of Mcl-1 enhances Pevonedistat-triggered apoptosis signaling. OS cell lines Saos-2 (a) and SJSA-1 (b) were transfected with two Mcl-1 siRNAs, i.e. siM(a), siM(b) or control siRNA (siCtl). After transfection for 24 h, the cells were treated with Pevonedistat as indicated for another 24 h. The expression level of Mcl-1, neddylated- or basal- cullin1, and activated caspase-3 were analyzed by western blot analysis. Beta actin was used as a loading control. Representative results were shown from three separate experiments.
Figure 5. Knockdown of Mcl-1 enhances Pevonedistat-mediated anti-OS activity. (a,b) OS cell line Saos-2 was transfected with two siMcl-1s, i.e. siM(a), siM(b) or control siRNA (siCtl). The transfected Saos-2 cells were treated with 0.2 μM Pevonedistat for 24 h, and then stained with Annexin-V, apoptotic cells were analyzed by flow cytometry. (a) Graphs are representative of three independent experiments that have similar results. (b) The percentages of Annexin-V positive cells from three experiments were plotted using Prizm software. The asterisks (**) indicates a significant (p<0.01) increasing apoptosis compared to siCtl-transfected cells. Saos-2 (c) and SJSA-1 (d) cell lines were transfected with siRNA for 24 h, and then treated by DMSO (D) or Pevonedistat (P) for another 24 h. The percentages of dead cells were examined by trypan blue exclusion assay and plotted by using Prizm software. The asterisks (**) indicates a significant (p<0.01) increasing death cells in siMcl-1-transfected cells compared to siCtltransfected cells. (e,f) Saos-2 cell line infected with lentivirus vectors expressing either a shRNA targeting Mcl-1 (shMcl-1) or a control shRNA (shCtl) were treated by Pevonedistat at 0.04 µM for 14 days to allow cells forming colonies. (e) The colonies were stained and photographed, and results showed the representative of three experiments. (f) Average colonies numbers of three experiments were plotted. The asterisks (**) indicates a significant (p<0.01) decreasing colony numbers compared to DMSO treated group.
Figure 6. Flavopiridol enhances Pevonedistat-triggered apoptotic cell death in OS cells. Saos-2
and SJSA-1 cells lines were treated with 3 μM Flavopiridol (F) alone, 0.2 μM
Pevonedistat (P) alone or both for 24 h. DMSO (D) was used a treatment control. (a,b) The protein level of Mcl-1, PARP and activated caspase-3 were examined by western blot analysis. Beta actin was used as a loading control. (c,d) The cell viability was examined by trypan blue exclusion assay, and the percentages of dead cells from three experiments were plotted using Prizm software. The asterisks (**) indicates a significant (p<0.01) increasing death cells in combination group compared to either single agent-treated group.
Highlights
1. Pevonedistat is a neddylation inhibitor. 2. Pevonedistat induces accumulation of Mcl-1 in osteosarcoma (OS) cells. 3. Inhibition of Mcl-1 enhances Pevonedistat signaling in OS cells. 4. Flavopiridol enhances apoptotic effect of Pevonedistat in OS.
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PII: DOI: Reference:
S0014-4827(17)30350-6 http://dx.doi.org/10.1016/j.yexcr.2017.06.019 YEXCR10642
To appear in: Experimental Cell Research Received date: 12 January 2017 Revised date: 21 May 2017 Accepted date: 23 June 2017 Cite this article as: Yi Zhang, Chengcheng Shi, Li Yin, Wei Zhou, Haitao Wang, Jingjing Seng and Wencai Li, Inhibition of Mcl-1 enhances Pevonedistattriggered apoptosis in osteosarcoma cells, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2017.06.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inhibition of Mcl-1 enhances Pevonedistat-triggered apoptosis in osteosarcoma cells Yi Zhang a,1, Chengcheng Shi b1, Li Yin a, Wei Zhou a, Haitao Wang a, Jingjing Seng c, Wencai, Li d a
Department of Orthopaedic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
b
Department of Pharmacy, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China c
Department of pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA d
Department of Pathology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
Corresponding author: Yi Zhang: No.1, East Jian She Road, Zhengzhou, 450052, Henan, China, Tel.: +86-371-66913032. Email: [email protected], Fax: +86-371-66295356
Running title: Mcl-1 attenuates Pevonedistat activity
1
These authors contributed equally to this work
Abstract
Neddylation inhibitor Pevonedistat (MLN4924) is a novel anticancer drug and has demonstrated broad-spectrum anticancer activity. Nevertheless, we found that Pevonedistat had only a modest apoptotic effect in osteosarcoma (OS) cells. Moreover, we noted that inhibition of neddylation by Pevonedistat led to accumulation of Mcl-1 protein in OS cells. Because Mcl-1 is an important anti-apoptotic protein and also because apoptosis has been shown to be a major cell death pathway, we hypothesized that Mcl-1 accumulation negatively impacted Pevonedistat-mediated anticancer activity in OS cells. In this regard, we employed genetic or pharmacological approaches to inhibit Mcl-1 expression and to examine the effect on Pevonedistat-induced apoptosis in OS cells. We found that inhibition of Mcl-1 expression by siRNA considerably enhanced Pevonedistat-triggered the activation of caspase-3, PARP cleavage and apoptosis, and also dramatically promoted the ability of Pevonedistat to inhibit colony formation of OS cells. Moreover, we observed that flavopiridol, a FDA approved drug, inhibited Mcl-1 expression and substantially enhanced Pevonedistat-mediated activation of apoptosis signaling and significantly augmented cell killing effect in OS cells. Altogether, our study shows that Mcl-1 is a critical resistance factor to Pevonedistat monotherapy, and suggests that Pevonedistat in combinations with flavopiridol may achieve better anticancer therapy.
Keywords: Pevonedistat; Mcl-1; Apoptosis Osteosarcoma
1. Introduction
Neddylation is a post-translational protein modification process through which ubiquitin-like protein NEDD8 is conjugated to protein substrates. This process is catalyzed by an enzymatic cascade comprising NEDD8-activating enzyme E1 (NAE1), NEDD8-conjugating enzyme E2, and substrate-specific NEDD8-E3 ligases.[1] The cullin protein family is the most established targets for neddylation. Neddylation allows activation of Cullin-RING E3 ubiquitin ligases (CRLs), which in turn regulate the turnover of proteins involved in many important cellular processes such as cell proliferation, DNA integrity, as well as apoptosis, via ubiquitin (Ub)/26S proteasome system (UPS).[1-3] Overactivation of neddylation pathway has been found in a variety of human cancers and may contribute to carcinogenesis and progression.[4-6] Therefore, neddylation is an attractive target for cancer therapy.
Pevonedistat (MLN4924) is a specific and selective inhibitor of neddylation.[7] By inducing accumulation of tumor suppressor proteins, Pevonedistat causes cell cycle arrest and DNA damage, triggers apoptosis, thus exhibiting broad-spectrum antitumor activity.[4, 8-10] We recently investigated the potential utility of Pevonedistat in osteosarcoma (OS) using in vitro and in vivo models and found that this drug was able to inhibit tumor growth in a mouse xenograft OS tumor model, suggesting that Pevonedistat holds promise for OS treatment.[11] Nevertheless, in the study, we observed that as compared to its extraordinary cytostatic effects, Pevonedistat exhibited only a modest apoptotic effect in OS cells. For instance, even at a very low concentration (0.04 µM), Pevonedistat effectively disrupted cell cycle progression in OS cells. Nonetheless, Pevonedistat triggered only modest activation of caspase-3 and only a small amount of cytochrome c release at a much higher concentration (1 µM). Moreover, we found that
Pevonedistat treatment led to a dramatic increase in the protein level of myeloid cell leukemia 1 (Mcl-1) in OS cells. Given that Mcl-1 is a critical anti-apoptotic member of Bcl-2 family proteins, we are interested in investigating whether Mcl-1 is a potential resistance factor to Pevonedistat monotherapy, and exploring approaches to improve Pevonedistat-based anticancer activity.
2. Materials and methods
2.1. Reagents and cell lines The cell lines Saos-2 and SJSA-1 obtained from China Center for Type Culture Collection (Wuhan, China) were maintained in PRMI1640 (HyClone/Thermo Fisher Scientific, Beijing, China) supplemented with 10% heat-inactivated fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd, Hangzhou, China) at 37°C in a humidified incubator containing 5% CO2. Pevonedistat and cyclin dependent kinase (CDK) inhibitor flavopiridol were obtained from Shanghai Selleck Chemicals (Shanghai, China) and both compounds were dissolved in Dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and stored at −20°C. 2.2. Cell death assay Cells were seeded in 96-well plates and treated with Pevonedistat or DMSO for the indicated times. Cell death was quantified by microscopic examination in trypan blue exclusion assays. 2.3. Western blot analysis Cells were lysed in RIPA buffer after the treatments. Equal amounts of lysate were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and evaluated by western blot analysis as described previously.[11] Rabbit anti-human PARP, caspase-3, Bcl-2, Bcl-xL and Cullin1 antibodies were purchased from Santa Cruz Biotechnology (Shanghai, China). Mouse
anti-human Mcl-1 was purchased from R&D System China (Shanghai, China). Mouse anti-beta actin, rabbit anti-FBXW7 and mouse anti-βTrCP were obtained from Sigma-Aldrich (Shanghai, China). Quantifications were performed using ImageJ 1.45 software. 2.4. Protein stability assay Protein stability assay was performed as described previously.[12] Briefly, cells were treated with DMSO or Pevonedistat for 6 h and then cycloheximide (5 μg/ml) was added to block protein synthesis. Collected protein samples were subjected to Western blot analysis using antiMcl-1 antibody. 2.5. Flow cytometry assay Apoptosis in treated cells was detected using an FITC Annexin-V Apoptosis Detection Kit (BD Biosciences-CN, Shanghai, China) according to the manufacturer's instructions. Briefly, the cells were digested with 0.25% trypsin, washed twice with cold phosphate-buffered saline (PBS), and then incubated with Annexin-V FITC for 15 min at room temperature in the dark. The population of apoptotic cells was analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, Shanghai, China). 2.6. Transfection of siRNA Mcl-1 siRNA was purchased from Santa Cruz Biotechnology (Beijing, China) for siMcl-1a (sc35877) and for non-targeting control siRNA (sc-12756), from Cell Signaling Technology (Shanghai, China) siMcl-1b (6315). Cul1 siRNA (L-004086) and FBW7 (L-004264) were purchased from GE (Shanghai, China). β-TrCP siRNA (sc-37178) was purchased from Santa Cruz Biotechnology (Beijing, China). Transfection of siRNA was conducted with RNAi Max Reagent (13778030, Invitrogen Trading Shanghai Co., Shanghai, China) according to the manufacturer's operating instructions.
2.7. Transduction of lentivrial shRNA and colonies formation Lentivrial shMcl-1 (sc-43912) and control shRNA lentiviral (sc-108080) particles were obtained from Santa Cruz Biotechnology (Shanghai, China). Saos-2 cell line was transduced with the lentiviral particles and efficacy of transduction was examined by western blot. Cells (2,000 per well) transduced with shMcl-1 or control shRNA were treated by Pevonedistat as indicated for 14 days to allow cells forming colonies. Colonies were stained with 0.025% (m/v) and photographed. 2.8. Statistical analysis The results were presented as means ± SEM of three independent experiments. For statistical tests, Prism 5.0 (GraphPad Software, SanDiego, CA, USA) was used. p values less than 0.05 were considered statistically significant.
3. Results 3.1. Pevonedistat induces rapid Mcl-1 accumulation in OS cells. In this study, we first evaluated the effect of Pevonedistat on the neddylation status of cullin1 and on the protein expression level of a panel of Bcl-2 family members in OS cells. To this end, we treated OS Saos-2 and SJSA-1 cell lines with Pevonedistat (0.04, 0.2 and 1.0 μM) for 24 h and performed western blot assay. The results showed that Pevonedistat effectively inhibited the neddylated form of cullin1 in both cell lines. The results also showed that Pevonedistat treatment dramatically increased the protein level of Mcl-1 in both OS cells lines (Fig. 1a,b). These results confirmed our previous finding with another Mcl-1 antibody.[11] However, Pevonedistat had little impact on the expression levels of anti-apoptotic Bcl-2, Bcl-xl and pro-apoptotic Bax (Fig. 1a,b).
To investigate whether Pevonedistat treatment induces Mcl-1 accumulation in OS cells in a timedependent manner, OS cell lines were treated with 0.2 μM Pevonedistat for different period of time. Western blot results demonstrated that Pevonedistat induced Mcl-1 accumulation very rapidly in these cells. Mcl-1 accumulation started within 4 h after Pevonedistat treatment, became stronger over time and lasted at least for up to 24 h in the presence of the drug (Fig. 1c,d). These results suggest that Pevonedistat rapidly and dramatically induces Mcl-1 accumulation in OS cells. 3.2. Pevonedistat increases the protein stability of Mcl-1 in OS cells. To further identify the mechanism responsible for Pevonedistat-induced Mcl-1 accumulation, we examined the Mcl-1 expression at the transcript level by reverse transcription (RT)–qPCR. The mRNA level of Mcl-1 modestly increased at 4 h after Pevonedistat treatment, but slightly decreased at 14 and 24 h (Fig. 1e,f). These results suggest that Mcl-1 accumulation may not be through transcriptional upregulation of the gene. We next examined whether Pevonedistat had an impact on the stability of Mcl-1 protein. We treated both OS cell lines with Pevonedistat or DMSO for 6 h, then treated with 5 µg/mL Cycloheximide (CHX) for another 2, 4 and 6 h. The protein level of Mcl-1 was examined by western blot, and the intensity of bands was quantified by ImageJ software respectively (Fig. 2a-d). The results showed that CHX treatment for 2 h reduced Mcl-1 level by 72 and 65% in Sao2 and SJSA1 cell lines pretreated by DMSO, respectively. CHX treatment for 6 h reduced Mcl-1 level by more than 90% in both cell lines pretreated by DMSO. In contrast, CHX treatment for 2 h only reduced Mcl-1 level by 37 and 23% in Pevonedistat-pretreated Sao2 and SJSA1 cell lines, respectively. Moreover, after treatment with CHX for 6 h, there were still 27 and 36% of Mcl-1 remained in Pevonedistat-pretreated Sao2 and SJSA1 cell lines, respectively (Fig. 2a-d). These results show that Pevonedistat
treatment dramatically attenuates CHX-mediated Mcl-1 reduction in OS cells, and suggest that Mcl-1 accumulation caused by Pevonedistat is associated with the enhancement of protein stability. 3.3. Induction of Mcl-1 accumulation by Pevonedistat requires the presence of adaptor proteins Fbw7 or β-TrCP in OS cells. In order to investigate the mechanism underlying Mcl-1 accumulation in OS cells, we knocked down cullin1 by siRNAs in OS cells and found that inhibition of cullin1 markedly increased the level of Mcl-1 (Fig. 3a). These results confirm that Mcl-1 is a substrate of cullin1-associated CRL E3 ligase. Ubiquitination and proteasomal degradation of Mcl-1 by CRLs requires two F-box proteins, Fbw7 and beta-transducin repeats-containing protein (β-TrCP) as adaptor proteins to recruit Mcl1 to cullin1-dependent CRLs ubiquitin E3 ligases in certain types of cells. We next investigated the role of these two F-box proteins in the modulation of Mcl-1 by Pevonedistat with specific siRNA in Sao2 cell line. Western blotting analysis showed that siRNA transfection for 48 h effectively inhibited the expression of targeted genes. Of note, knockdown of either Fbw7 or βTrCP led to an increase of Mcl-1 level (Fig. 3a). These results indicate that both two F-box proteins were involved in Mcl-1 ubiquitination and degradation in OS cells. We next treated siRNA transfected-cells with Pevonedistat for another 6 h and analyzed the Mcl1 with western blotting analysis. The results showed that Pevonedistat treatment led to marked accumulation of Mcl-1 protein in control siRNA transfected cells, but only slightly or modestly increase the Mcl-1 level in β-TrCP siRNA and Fbw7 siRNA transfected cells (Fig. 3b,c). These results evidently suggest that induction of Mcl-1 accumulation by Pevonedistat requires the presence of these two adaptor proteins in this OS cell line.
3.4. Mcl-1 knockdown by siRNA enhanced Pevonedistat-induced apoptotic signaling in OS cells. It is well documented that Mcl-1 is an anti-apoptotic protein and protects tumor cells against apoptosis. Thus, it is highly possible that inhibition of Mcl-1 accumulation can enhance Pevonedistat-triggered apoptosis in OS cells. To address this issue, OS cells were transfected with Mcl-1 siRNA (siMcl-1a and siMcl-1b) and further were treated with Pevonedistat. Both siRNA very efficiently inhibited Mcl-1 expression. Furthermore, Pevonedistat treatment not only induced dramatic Mcl-1 accumulation in siCtl-transfected cells, but also could induce Mcl-1 accumulation in OS cells transfected with siMcl-1. However, Mcl-1 accumulation in siMcl-1transfected cells was negligible (Fig. 4a,b), suggesting that Mcl-1 siRNAs were extremely efficient in inhibition of Pevonedistat-mediated Mcl-1 accumulation in OS cells. Western blot also was performed to evaluate molecular biomarkers of apoptotic signaling. As shown in Fig. 4, Pevonedistat triggered much stronger caspase-3 activation and more PARP cleavage in cells transfected with siMcl-1 than in cells transfected with siCtl. Altogether, these results suggest that Mcl-1 negatively regulates Pevonedistat-mediated apoptotic in OS cells. 3.5. Knockdown of Mcl-1 sensitizes OS cells to Pevonedistat-mediated apoptotic cell death. To further determine the effect of Mcl-1 knockdown on Pevonedistat-mediated anti-OS activity, we examined apoptosis induction in the treated cells by Annexin-V FITC staining and flow cytometery assay in Saos-2 cell line. The results showed that Pevonedistat treatment for 24 h resulted in a significantly higher apoptosis proportion in Mcl-1 siRNA transfected cells (53.01 ±8.01 for siMcl-1a and 50.7 ± 7.5% for siMcl-1b) than in siCtl transfected cells (18.3 ± 3.5%, p < 0.01 versus both siMcl-1) (Fig. 5a,b).
Cell viability assay was performed to examine the effect of Mcl-1 inhibition on Pevonedistatmediated cell killing in both Saos-2 and SJSA-1 cell lines. In Saos-2 cell line, transfection with Mcl-1 siRNAs alone caused about 10% cell death, suggesting a role of Mcl-1 in sustaining cell survival of this cell line. Of note, Pevonedistat induced significantly more cell death in cells transfection with siMcl-1 than in cells transfected with siCtl (siCtl versus siMcl-1a or b, both p<0.01) (Fig. 5c). A substantial enhancement of Pevonedistat-mediated cell death by Mcl-1 knockdown was also observed in SJSA-1 cell line (siCtl versus siMcl-1a or b, both p<0.01) (Fig. 5d). Thus, inhibition of Mcl-1 accumulation could significantly promote Pevonedistat-mediated apoptotic cell death in OS cell lines. 3.6. Knockdown of Mcl-1 by shRNA enhances Pevonedistat-mediated colony formation. In order to determine the long-term effect of Mcl-1 knockdown on the Pevonedistat activity, Saos-2 cell line infected with lentivirus vectors expressing either a shRNA targeting Mcl-1 (shMcl-1) or a control shRNA (shCtl) were treated by Pevonedistat at 0.04 µM or DMSO as control treatment for 14 days to allow cells forming colonies. As shown in Fig.5e and 5f, cells infected with control shRNA or shMcl-1 lentiviruses both formed colonies. Although Pevonedistat treatment significantly reduced the colony numbers in cells with control shRNA, there were still a decent number of colonies formed after the treatment. In striking contrast, Pevonedistat treatment completely inhibited colony formation in cells bearing shMcl-1 lentivirus. These results suggest that Mcl-1 also inhibits the long-term anti-OS activity of Pevonedistat. 3.7. Flavopiridol enhances Pevonedistat-mediated apoptotic cell death. Flavopiridol is a first-generation CDK inhibitor which has been approved by FDA for the treatment of acute myeloid leukemia. [13, 14] This drug has been shown to inhibit Mcl-1 in multiple cancer cells. We here investigated whether flavopiridol could inhibit Mcl-1 and promote
Pevonedistat-mediated apoptotic cell death in OS cells. We treated the cells with 3 µM flavopiridol, 0.2 µM Pevonedistat or both for 24 h, and then examined the levels of Mcl-1, caspase-3 activation and PARP cleavage. We observed that flavopiridol distinctly reduced the protein level of Mcl-1 in both OS cell lines. Moreover, flavopiridol or Pevonedistat alone had little or modest inhibitory effect on the expression of full-length PARP, suggesting weak activation of apoptosis signaling. In striking contrast, treatment of Pevonedistat in combination with flavopiridol resulted in marked reduction of pro-PARP and induced massive accumulation of PARP cleavage and activated caspase-3 in both OS cell lines (Fig. 6a,b). Cell death analysis further showed that combination of the two drugs significantly enhanced cell killing effect as compared to single agent in both OS cell lines (Fig. 6c,d). These results suggest that combination with flavopiridol significantly enhances Pevonedistat-mediated apoptotic cell killing effect, likely through inhibiting Mcl-1 expression.
4. Discussion
Neddylation pathway plays an essential role in ubiquitination and proteasomal degradation of intracellular proteins, some of which are closely associated with cell survival, proliferation, apoptosis, DNA integrity, and many other crucial cellular processes.[6-9] Previous studies suggested that cancer cells usually are much more sensitive to inhibition of neddylation pathway than normal cells; therefore, inhibition of Neddylation is considered as a promising strategy of cancer treatment.[6-10] Indeed, the novel anticancer drug Pevonedistat has demonstrated promising anticancer activity via inhibition of neddylation pathway.[9-11] However, data from previous studies also showed that Pevonedistat only partially inhibited tumor growth, but could
not induce tumor regression.[15, 16] This suboptimal antitumor activity has inspired a number of groups to elucidate the mechanisms underlying Pevonedistat-resistance and explore approaches to overcome the resistance.[15, 16]
It has been reported that Pevonedistat elicited anticancer activity by inducing accumulation of tumor suppressive proteins, such as pro-apoptotic protein NOXA, cell cycle regulators p21, p27 and DNA replication licensing factor CDT1.[17-19] However, we and other groups observed that proteins with pro-survival function, such as cyclinE and Mcl-1, were also robustly increased by Pevonedistat in cancer cells.[11, 20-23] Thus, we hypothesized that the accumulation of these pro-survival proteins contributed to Pevonedistat-resistance. We focused our study on the role of Mcl-1 because Mcl-1 is an important anti-apoptotic protein and often plays a decisive role in the process of apoptosis.[24, 25] We have made several important observations in this study. Firstly, Pevonedistat treatment distinctly increases the protein level of Mcl-1 in OS cells and importantly, this increase occurs within 4 h, an early time compared to apoptosis induction that was observed after Pevonedistat treatment for 24 h. Secondly, inhibition of Mcl-1 by knockdown dramatically enhances Pevonedistat-triggered activation of caspase-3 and PARP cleavage, significantly promotes Pevonedistat-mediated cell death, and also boosts the ability of Pevonedistat to inhibit colony formation in OS cells. Importantly we found that flavopiridol, a CDK inhibitor with function of inhibiting Mcl-1 expression, also significantly augmented Pevonedistat-triggered apoptotic signaling in OS cells. Our finding thus demonstrates that Mcl-1 confers resistance of OS cells to Pevonedistat, and also indicate that combination with flavopiridol can be used to improve Pevonedistat-based anticancer therapy.
Mcl-1 is a unique member amongst the Bcl-2 family, with relatively short half-lives (1-3 h). The expression level of Mcl-1 can be modulated at both transcriptional and translational levels by numerous factors.[26] We analyzed the mechanism of Mcl-1 accumulation by Pevonedistat in OS cells. RT-qPCR analysis showed that Mcl-1 transcript did not increase significantly during Pevonedistat treatment, suggesting that transcriptional mechanisms are not involved in Mcl-1 accumulation. In contrast, western blot analysis showed that Pevonedistat dramatically delayed protein synthesis inhibitor cycloheximide-mediated Mcl-1 reduction. This result highly suggests that accumulation of Mcl-1 in OS cells is caused by Pevonedistat-mediated enhancement of Mcl1 stabilization. Mcl-1 has been unambiguously identified as a substrate of CRLs, and therefore inhibition of cullin neddylation blocks UPS-mediated degradation of Mcl-1 protein.[27] Additionally, the siRNA experiments shows that Pevonedistat-mediated Mcl-1 accumulation require the presence of Fbw7 and β-TrCP. Considering that Fbw7 and β-TrCP are not expressed in all cancer cells [28, 29], we assume that the expression of these two adaptor proteins might be useful for selecting patients for Pevonedistat in combination with Mcl-1-inhibiting agents.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors thank Dr. Lan Huang for her critical reading of this manuscript (The 1st Affiliated Hospital of Zhengzhou University).
Funding
This work was supported by the Education Department of Henan Province (Grants No: 15A320086), The Science and Technology Department of Henan Province (Grants No: 162300410094), Young physicians funds of The 1st Affiliated Hospital of Zhengzhou University (2014006, 2015163).
References
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Figure legends
Figure 1. Pevonedistat induces accumulation of Mcl-1 in OS cells. OS cell lines Saos-2 (a) and SJSA-1 (b) were treated with 0.04, 0.2 and 1 μM Pevonedistat for 24 h. The expression levels of both neddylated- and basal- cullin1, Mcl-1, Bcl-2, Bcl-xL and Bax proteins were analyzed by western blot analysis. Beta actin was used as a loading control. Representative results are shown from three independent experiments. OS cell lines Saos-2 (c) and SJSA-1 (d) were treated with 0.2 μM Pevonedistat for 4, 14 and 24 h. The expression level of Mcl-1 was analyzed by western blot analysis. Beta actin was used as a loading control. (e,f) The relative level of Mcl-1 mRNA was analyzed by RT-qPCR analysis. Representative results were shown from three triplicate experiments.
Figure 2. Pevonedistat induces Mcl-1 accumulation by enhancing the stability of Mcl-1 protein in OS cells. OS cell lines Saos-2 (a) and SJSA-1 (b) were pretreated with DMSO or 0.2 μM Pevonedistat for 6 h, and then OS cells were treated with 5 ug/ml CHX for another 2, 4 and 6 h. The level of Mcl-1 was analyzed by western blot analysis. Beta actin was used as a loading control. Quantitative analysis of band intensity shown in (c,d) the using ImageJ software.
Figure 3. Induction of Mcl-1 accumulation by Pevonedistat requires the presence of adaptor proteins Fbw7 or β-TrCP in OS cells. Saos-2 cell line was transfected with siRNAs for 48 h. The expression of target genes was examined by western blot analysis. Beta actin was used as a loading control. Saos-2 cell line transfected with siCTL, siFbw7 (b) or siβTrCP (c) for 48 h was treated by Pevonedistat (Pevo) for another 6 h, the expression of Mcl-1 protein level was examined by western blotting analysis. Beta actin was used as a loading control.
Figure 4. Knockdown of Mcl-1 enhances Pevonedistat-triggered apoptosis signaling. OS cell lines Saos-2 (a) and SJSA-1 (b) were transfected with two Mcl-1 siRNAs, i.e. siM(a), siM(b) or control siRNA (siCtl). After transfection for 24 h, the cells were treated with Pevonedistat as indicated for another 24 h. The expression level of Mcl-1, neddylated- or basal- cullin1, and activated caspase-3 were analyzed by western blot analysis. Beta actin was used as a loading control. Representative results were shown from three separate experiments.
Figure 5. Knockdown of Mcl-1 enhances Pevonedistat-mediated anti-OS activity. (a,b) OS cell line Saos-2 was transfected with two siMcl-1s, i.e. siM(a), siM(b) or control siRNA (siCtl). The transfected Saos-2 cells were treated with 0.2 μM Pevonedistat for 24 h, and then stained with Annexin-V, apoptotic cells were analyzed by flow cytometry. (a) Graphs are representative of three independent experiments that have similar results. (b) The percentages of Annexin-V positive cells from three experiments were plotted using Prizm software. The asterisks (**) indicates a significant (p<0.01) increasing apoptosis compared to siCtl-transfected cells. Saos-2 (c) and SJSA-1 (d) cell lines were transfected with siRNA for 24 h, and then treated by DMSO (D) or Pevonedistat (P) for another 24 h. The percentages of dead cells were examined by trypan blue exclusion assay and plotted by using Prizm software. The asterisks (**) indicates a significant (p<0.01) increasing death cells in siMcl-1-transfected cells compared to siCtltransfected cells. (e,f) Saos-2 cell line infected with lentivirus vectors expressing either a shRNA targeting Mcl-1 (shMcl-1) or a control shRNA (shCtl) were treated by Pevonedistat at 0.04 µM for 14 days to allow cells forming colonies. (e) The colonies were stained and photographed, and results showed the representative of three experiments. (f) Average colonies numbers of three experiments were plotted. The asterisks (**) indicates a significant (p<0.01) decreasing colony numbers compared to DMSO treated group.
Figure 6. Flavopiridol enhances Pevonedistat-triggered apoptotic cell death in OS cells. Saos-2
and SJSA-1 cells lines were treated with 3 μM Flavopiridol (F) alone, 0.2 μM
Pevonedistat (P) alone or both for 24 h. DMSO (D) was used a treatment control. (a,b) The protein level of Mcl-1, PARP and activated caspase-3 were examined by western blot analysis. Beta actin was used as a loading control. (c,d) The cell viability was examined by trypan blue exclusion assay, and the percentages of dead cells from three experiments were plotted using Prizm software. The asterisks (**) indicates a significant (p<0.01) increasing death cells in combination group compared to either single agent-treated group.
Highlights
1. Pevonedistat is a neddylation inhibitor. 2. Pevonedistat induces accumulation of Mcl-1 in osteosarcoma (OS) cells. 3. Inhibition of Mcl-1 enhances Pevonedistat signaling in OS cells. 4. Flavopiridol enhances apoptotic effect of Pevonedistat in OS.
e o f M c l- 1 m R N A
1 .2 1 .1 1 .0 0 .9 0 .8 0 .7 0 .6 0 .5 0 .4 0 .3 0 .2 0 .1 0 .0
Mcl-1
Beta actin
c
Bcl-xl Bax
Bcl-2
N-Cullin1 Cullin1 Mcl-1
Beta actin
a
R e la tiv e e x p re s s io n
Figure 1 DMSO D
0
4
14
14
P e v o ( h r)
4
Pevo (hr)
Saos-2
0.04 0.2
Pevo (µM)
24
24
1
f
d
b
R e la tiv e e x p re s s io n
0
DMSO o f M c l- 1 m R N A
1 .2 1 .1 1 .0 0 .9 0 .8 0 .7 0 .6 0 .5 0 .4 0 .3 0 .2 0 .1 0 .0
4
D
4
14
14
24
1
P e v o ( h r)
Pevo (hr)
SJSA-1
0.04 0.2
Pevo (µM)
24
40KD
42KD
23KD
27KD
25KD
40KD
85KD
42KD
Mcl-1
Beta actin
Mcl-1
Beta actin
b
Mcl-1
Beta actin
Mcl-1
Beta actin
a 4
Saos-2
2
0
4
SJSA-1
2
6
CHX time (hr)
0
CHX time (hr) 6
Pevo
DMSO
Pevo
DMSO
d
c
re la tiv e M c l-1 le v e l
Figure 2
2
2
4
0 .0
0 .2
4 tim e (h r)
6
Pevo 0 .6 0 .4
DMSO
0 .0
0 .2
tim e (h r)
6
Pevo
0 .6 0 .4
DMSO
0 .8
1 .0
0 .8
1 .0
re la tiv e M c l-1 le v e l
90KD 62KD 62KD 40KD
βTrCP
Fbw7
Mcl-1
CTL
Cullin1
Cul1
42KD
a βTrCP
Beta actin
siRNA
Figure 3
Fbw7
siCtl
40KD
Mcl-1
0.2
40KD
42KD
42KD
0
siβTrCP
0 0.2
siFbw7
Beta actin
0.2
siCtl
0 0.2
Pevo (µM) 0
c
Mcl-1
Beta actin
Pevo (µM)
b
Figure 4
1
20KD 120KD 90KD
fl-PARP
cl-PARP
42KD
cl-Casp3
1
85KD
0.2
N-Cul1 Cul1
0
40KD
SJSA-1
0.2
0
0.2
Mcl-1
Beta actin
Pevo (µM)
siM(a)
siCtl 1
90KD
cl-PARP
0
120KD
fl-PARP
siM(b)
20KD
cl-Casp3
Saos-2
85KD
N-Cul1 Cul1
b
40KD
0 0.04 0.2
siM(b)
Mcl-1
0 0.04 0.2
siM(a)
42KD
0.04 0.2
siCtl
Beta actin
Pevo (µM) 0
a
Saos-2
e
c
Saos-2
0
20
40
60
80
100
P
P
s iM ( a )
D
DMSO
s iC t l
D
**
P
Pevo
s iM ( b )
D
**
53%
siM(a) (Pevo)
8%
siM(a) (DMSO)
S a o s -2
22%
siCtl (Pevo)
7%
siCtl (DMSO)
f
d
51%
siM(b) (Pevo)
9%
siM(b) (DMSO)
0
50
100
150
200
0
20
40
60
80
100
0
20
40
60
80
100
% d e a d c e lls
( n o r m a r ilz e d to c o n tr o l)
# c o lo n ie s
a
P
P
P
D
P
**
P
P
D
P
**
s iM ( b )
D
**
s h M c l- 1
P
s iM ( b )
D
M c l-1
a c tin
s iM ( a )
**
s h C tl
D
s iC t l
D
D
**
s iM ( a )
S J S A -1
s iC t l
D
b
s h M c l-1
Figure 5
% d e a d c e lls
shCtrl shMcl-1
s h C tl
% A n n e x in - V ( + ) c e lls
c
-
Pevo F la v
0
20
40
60
80
100
cl-Casp3
fl-PARP cl-PARP
Beta actin Mcl-1
a
% d e a d c e ll
Figure 6
D
-
+
P
**
Saos-2
+
-
**
F
+
+
C
d
b
% d e a d c e ll
0
20
40
60
80
100
F
F la v
Pevo
D
+
-
SJSA-1
-
-
P
**
C
-
+
**
+
+
20KD 18KD
120KD 90KD
40KD
42KD