Distinct outcomes of CRL–Nedd8 pathway inhibition reveal cancer cell plasticity

Rulina, Anastasia V; Mittler, Frédérique; Obeïd, Patricia; Gerbaud, Sophie; Guyon, Laurent; Sulpice, Eric; Kermarrec, Frédérique; Assard, Nicole; Dolega, Monika E.; Gidrol, Xavier; Balakirev, Maxim Y.

doi:10.1038/cddis.2016.395

Cited by 14 publications

(31 citation statements)

References 49 publications

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“…To establish drug potency and re-examine the cell cycle effects of neddylation inhibition we treated asynchronous HeLa cells with 0.3 µM MLN4924 for 24 to 48 h and used the ES-FUCCI (Fluorescent, Ubiquitination-based Cell Cycle Indicator [33]) reporter system [34] to monitor the levels of the CRL substrate CDT1 degraded in G1/S (mCherry-CDT1), and the APC-CDH1 substrate Geminin (Citrine-Geminin) degraded in G2/M ( Figure 1). We observed a significant increase in CDT1 expression in MLN4924-treated cells over time as measured by the integrated fluorescent intensity of mCherry-CDT1 per cell (p < 0.0001), a result consistent with previous findings that MLN4924 treatment above 0.25 µM can strongly block neddylation and stabilize CDT1 [29,35]. MLN4924 treatment for 48 h also increased Citrine-Geminin expression, which is not a CRL substrate, indicating that cells not initially arrested in G1/S began to accumulate in G2.…”

Section: Inhibition Of Neddylation By Mln4924 Leads To Cdt1 Accumulatsupporting

confidence: 91%

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

2019

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The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis.

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Section: Inhibition Of Neddylation By Mln4924 Leads To Cdt1 Accumulatsupporting

confidence: 91%

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

2019

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“…Another argument speaking in favor of the model presented in our work is that low Cand1 expression in primary prostate carcinoma tissue could be linked to better patient survival in four out of five investigated study cohorts. Besides the findings of Rulina that knockdown of Cand1 induces apoptosis in PCa, our findings of reduced cell proliferation due to Cand1 knockdown are also in line with a recent study in liver cancer where Che et al showed that Cand1 was overexpressed in cancerous issues compared to the corresponding adjacent benign ones and that high expression of Cand1 was associated with poor OS [35,40].…”

Section: Discussionsupporting

confidence: 91%

“…The deregulation of CRL components such as of substrate-receptors or Cand1 have been described in various cancer entities, however there is conflicting evidence concerning the impact of Cand1 in prostatic malignancies. For example, Rulina et al previously showed that the knockdown of Cand1 induces apoptosis in PCa cell lines, while Murata et al provided evidence that Cand1 downregulation enhances cell growth in LNCaP PCa cells [35,36]. Therefore, the present study elucidated Cand1 in PCa using not only a preclinical model, but also patient samples to better clarify its clinical impact.…”

Section: Discussionmentioning

confidence: 78%

“…Besides impaired cell viability and proliferation, we were able to show that downregulation of Cand1 resulted in significantly increased apoptosis, reflected by increased caspase 3/7 activity and elevated levels of cPARP in LNCaP and PC3 cells. Consistent with this finding, Rulina et al demonstrated that Cand1 knockdown induces apoptosis in PCa cell lines [35]. Chua and colleagues hypothesized that Cand1 differentially regulates CRLs depending on their cellular localization, as they found Cand1 in human embryonic kidney cells (HEK293) to be predominantly present in the cytoplasm [41].…”

Section: Discussionmentioning

confidence: 83%

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The Impact of Cand1 in Prostate Cancer

Eigentler

Tymoszuk

Zwick

et al. 2020

Cancers

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Evidence has accumulated asserting the importance of cullin-RING (really interesting new gene) ubiquitin ligases (CRLs) and their regulator Cullin-associated neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) dissociated protein 1 (Cand1) in various cancer entities. However, the role of Cand1 in prostate cancer (PCa) has not been intensively investigated so far. Thus, in the present study, we aimed to assess the relevance of Cand1 in the clinical and preclinical setting. Immunohistochemical analyses of radical prostatectomy specimens of PCa patients showed that Cand1 protein levels are elevated in PCa compared to benign areas. In addition, high Cand1 levels were associated with higher Gleason Scores, as well as higher tumor recurrence and decreased overall survival. In line with clinical findings, in vitro experiments in different PCa cell lines revealed that knockdown of Cand1 reduced cell viability and proliferation and increased apoptosis, therefore underlining its role in tumor progression. We also found that the cyclin-dependent kinase inhibitor p21 is significantly upregulated upon downregulation of Cand1. Using bioinformatic tools, we detected genes encoding for proteins linked to mRNA turnover, protein polyubiquitination, and proteasomal degradation to be significantly upregulated in Cand1high tumors. Next generation sequencing of PCa cell lines resistant to the anti-androgen enzalutamide revealed that Cand1 is mutated in enzalutamide-resistant cells, however, with little functional and clinically relevant impact in the process of resistance development. To summarize the present study, we found that high Cand1 levels correlate with PCa aggressiveness.

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“…MLN is currently being evaluated in clinical trials for the treatment of hematologic malignancies and solid tumors ( 88 , 89 ). Different cancer cells have different sensitivities to MLN, and as we have shown recently ( 77 ), MLN treatment has complex outcomes that vary from cell cycle arrest and protective dormancy to senescence and apoptosis. Here, we show that the complexity of the effects of MLN as well as of other cancer therapeutics with different mechanisms of action (MOAs) can be addressed using the 3D spheroid method.…”

Section: Introductionmentioning

confidence: 95%

High-Content Monitoring of Drug Effects in a 3D Spheroid Model

et al. 2017

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A recent decline in the discovery of novel medications challenges the widespread use of 2D monolayer cell assays in the drug discovery process. As a result, the need for more appropriate cellular models of human physiology and disease has renewed the interest in spheroid 3D culture as a pertinent model for drug screening. However, despite technological progress that has significantly simplified spheroid production and analysis, the seeming complexity of the 3D approach has delayed its adoption in many laboratories. The present report demonstrates that the use of a spheroid model may be straightforward and can provide information that is not directly available with a standard 2D approach. We describe a cost-efficient method that allows for the production of an array of uniform spheroids, their staining with vital dyes, real-time monitoring of drug effects, and an ATP-endpoint assay, all in the same 96-well U-bottom plate. To demonstrate the method performance, we analyzed the effect of the preclinical anticancer drug MLN4924 on spheroids formed by VCaP and LNCaP prostate cancer cells. The drug has different outcomes in these cell lines, varying from cell cycle arrest and protective dormancy to senescence and apoptosis. We demonstrate that by using high-content analysis of spheroid arrays, the effect of the drug can be described as a series of EC50 values that clearly dissect the cytostatic and cytotoxic drug actions. The method was further evaluated using four standard cancer chemotherapeutics with different mechanisms of action, and the effect of each drug is described as a unique multi-EC50 diagram. Once fully validated in a wider range of conditions, this method could be particularly valuable for phenotype-based drug discovery.

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Distinct outcomes of CRL–Nedd8 pathway inhibition reveal cancer cell plasticity

Cited by 14 publications

References 49 publications

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

The Impact of Cand1 in Prostate Cancer

High-Content Monitoring of Drug Effects in a 3D Spheroid Model

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