The p21-Dependent Radiosensitization of Human Breast
Cancer Cells by MLN4924, an Investigational Inhibitor of
NEDD8 Activating Enzyme
Dong Yang1.¤, Mingjia Tan1., Gongxian Wang2, Yi Sun1*
1Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan, United States of America, 2Department of
Urology, The First Affiliated Hospital of Nanchang University, Nanchang, China
Radiotherapy is a treatment choice for local control of breast cancer. However, intrinsic radioresistance of cancer cells limits
therapeutic efficacy. We have recently validated that SCF (SKP1, Cullins, and F-box protein) E3 ubiquitin ligase is an
attractive radiosensitizing target. Here we tested our hypothesis that MLN4924, a newly discovered investigational small
molecule inhibitor of NAE (NEDD8 Activating Enzyme) that inactivates SCF E3 ligase, could act as a novel radiosensitizing
agent in breast cancer cells. Indeed, we found that MLN4924 effectively inhibited cullin neddylation, and sensitized breast
cancer cells to radiation with a sensitivity enhancement ratio (SER) of 1.75 for SK-BR-3 cells and 1.32 for MCF7 cells,
respectively. Mechanistically, MLN4924 significantly enhanced radiation-induced G2/M arrest in SK-BR-3 cells, but not in
MCF7 cells at early time point, and enhanced radiation-induced apoptosis in both lines at later time point. However,
blockage of apoptosis by Z-VAD failed to abrogate MLN4924 radiosensitization, suggesting that apoptosis was not causally
related. We further showed that MLN4924 failed to enhance radiation-induced DNA damage response, but did cause minor
delay in DNA damage repair. Among a number of tested SCF E3 substrates known to regulate growth arrest, apoptosis and
DNA damage response, p21 was the only one showing an enhanced accumulation in MLN4924-radiation combination
group, as compared to the single treatment groups. Importantly, p21 knockdown via siRNA partialy inhibited MLN4924-
induced G2/M arrest and radiosensitization, indicating a causal role played by p21. Our study suggested that MLN4924
could be further developed as a novel class of radiosensitizer for the treatment of breast cancer.
Citation: Yang D, Tan M, Wang G, Sun Y (2012) The p21-Dependent Radiosensitization of Human Breast Cancer Cells by MLN4924, an Investigational Inhibitor of
NEDD8 Activating Enzyme. PLoS ONE 7(3): e34079. doi:10.1371/journal.pone.0034079
Editor: Wafik S. El-Deiry, Penn State Hershey Cancer Institute, United States of America
Received January 11, 2012; Accepted February 21, 2012; Published March 22, 2012
Copyright: ? 2012 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by the NCI grants, CA118762 and CA156744 to YS and the Nanchang University Scholarship Fund in China to DY. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Department of Orthopaedics, The First Affiliated Hospital of Nanchang University, Nanchang, China
. These authors contributed equally to this work.
The SCF E3 ubiquitin ligases consist of Skp1, cullins, F-box
proteins, and a RING protein, RBX or ROC. By promoting the
ubiquitination and degradation of various key regulatory proteins,
SCF E3 ligases control several important biological processes
including cell cycle progression, signal transduction and DNA
replication [1,2]. While the substrate specificity of SCF E3s is
determined by the F box proteins that bind to Skp1 and Cullins
through its F-box domain and to substrates through its WD40 or
leucine rich region domains [3,4], the activity of SCF E3 ubiquitin
ligases requires 1) the RING protein RBX or ROC that binds to E2
and facilitates ubiquitin transfer from E2 to substrates  and 2)
cullin neddylation, which disrupts inhibitory binding by CAND1
[6,7,8,9]. Accumulated evidence strongly suggests that abnormal
regulation of SCF E3 ubiquitin ligases contributes to uncontrolled
proliferation, genomic instability, and cancer , and SCF E3
ligases appear to be an attractive anticancer targets [10,11,12].
Although no specific small molecule inhibitor of E3 ubiquitin
ligases has been successfully discovered and developed [13,14],
MLN4924, a potent small molecule inhibitor of Nedd8 activating
enzyme (NAE) has demonstrated an inhibitory activity against
SCF E3 ligases via inhibiting cullin neddylation . Mechanis-
tically, MLN4924 binds to NAE to create a covalent Nedd8-
MLN4924 adduct that blocks NAE enzymatic activity . By
inactivating SCF E3 ubiquitin ligases, MLN4924 caused the
accumulation of a number of SCF E3 substrates to induce
apoptosis [15,17,18] and senescence [19,20,21], thus inhibiting
tumor growth both in vitro and in vivo. MLN4924 has been
advanced to several Phase I clinical trials .
We have recently shown that inactivation of SCF E3 ubiquitin
ligases by siRNA knockdown of either RBX1 or RBX2 (also
known as SAG, Sensitive to Apoptosis Gene) sensitized human
cancer cells to radiation [23,24,25], and that Sag knockout also
sensitized mouse embryonic stem cells to radiation via inducing
apoptosis . Most recently, we found that MLN4924 could act
as a novel radiosensitizing agent by causing accumulation of
CDT1 and WEE1 to enhance radiation-induced DNA damage,
aneuploidy, G2/M arrest and apoptosis in pancreatic cancer cells
. Here we extended our MLN4924-radiosensitization study
PLoS ONE | www.plosone.org1 March 2012 | Volume 7 | Issue 3 | e34079
from human pancreatic cancer cells to breast cancer cells and
reported that MLN4924 is a effective radiosensitizing agent
against breast cancer cells via a p21-dependent mechanism.
Materials and Methods
MLN4924 was a gift from Millennium Pharmaceuticals, Inc.
(Cambridge, MA) . The compound was dissolved in DMSO to
make a 10 mM stock solution and kept in 220uC before use.
Breast cancer lines, including SK-BR-3, MCF-7, ZR-75-1 and
T-47D were obtained from ATCC and were grown in DMEM
(Gibco) with 10% fetal bovine serum.
Radiation exposure and clonogenic assay
Cells were seeded in 60-mm dishes at proper cell densities in
duplicate and exposed to different doses of radiation (Philips
RT250, Kimtron Medical) after 24 hrs pre-treatment with
MLN4924, followed by incubation at 37uC for 7 to 11 days.
Survival curves were fitted using the linear-quadratic equation,
and the mean inactivation dose was calculated . Irradiations
were performed in the Experimental Irradiation Core of the
University of Michigan Cancer Center.
siRNA knockdown of p21
The siRNA oligonucleotide target p21 expression is as follows:
si-p21 (59-GUGGACAGCGAGCAGCUGAUU-39), and scram-
bled control siRNA (siCONT: 59-AUUGUAUGCGAUCGCA-
GACUU-39). The oligoes were purchased from Dharmacon
(USA). Cells were transfected with siRNA using Lipfectamine
2000 and split 48 hrs later. One portion was used for clonogenic
assay and the other portion for immunoblotting (IB) .
Cells were exposed to various treatments and harvested for
Western blotting as described  using antibodies against CDT1,
WEE-1, CUL1 (Santa Cruz Biotechnology), ORC-1, p21, p27 (BD
Biosciences), NOXA (Calbiochem), c-H2AX (MILLPORE), PARP,
Caspase-3, BIM-EL, phospho-CHK1(S345), phospho-CHK2 (T68),
total CHK1 and CHK2 (Cell Signaling), and b-actin (Sigma).
FACS (Fluorescence-activated Cell Sorting) analysis
Cells were treated with MLN4924, or exposed to radiation
alone or in combination. Cells were harvested 24 or 48 hrs post
radiation and analyzed by flow cytometry .
DNA fragmentation assay
pretreated next day with DMSO (control) or MLN4924 (100 nM)
for 6 hrs before being exposed to radiation (6 Gy). The cells were
harvested for48 hrs post radiation by scraping, pelleted and lysed in
600 ml of lysis buffer (5 mM Tris-HCl, pH 8, 20 mM EDTA and
0.5% Triton X-100). The fragmented DNAs in the supernatant
after 14,000 rpm centrifugation were extracted with PCI (Phenol/
Chloroform/Isopropanol) (Fisher) and precipitated with ethanol,
followed by electrophoresis in a 1.8% agarose gel .
ANOVA were used with SPSS (Statistical Product and Service
Solution) software for statistical comparisons involving multiple
groups, followed by SNK post hoc test to determine significance of
each two group (p,0.05). Paired or unpaired two-tailed Student t
test was performed in comparison between two groups, using SAS
MLN4924 sensitized breast cancer cells to radiation
Our most recent study showed that MLN4924 is a potent
radiosensitizing agent against pancreatic cancer cells . Here
we extended the observation to breast cancer cells. We first
determined the efficacy of MLN4924 in inactivation of SCF E3
ligases as reflected by cullin deneddylation in multiple breast
cancer cells. As shown in Figure 1A, in all tested breast cancer
lines, a portion of cullin-1 was neddylated, and cullin-1
neddylation was completely inhibited after 6 hrs of exposure to
MLN4924 at 1 mM (Figure 1A). We next measured a time-
dependent inhibition of cullin-1 neddylation with a much lower
concentration of MLN4924 and found that MLN4924 at 30 nM
caused a remarkable inhibition of cullin-1 neddylation at 24 hrs,
and a complete inhibition at 72 hrs and thereafter up to 144 hrs
(Figure 1B). We therefore used 30 nM of MLN4924 in our
radiosensitization experiment and found that MLN4924 at such a
low concentration caused a remarkable radiosensitization in SK-
BR-3 cells with a sensitivity enhancement ratio (SER) of 1.75
(Figure 1C). Similar observation was made in MCF7 cells, but to a
lesser extent with a SER of 1.32 (Figure 1C). Thus, we conclude
that MLN4924 is a potent radiosensizer against breast cancer cells.
MLN4924 radiosensitization is associated with enhanced
G2/M arrest and apoptosis
To determine the nature of MLN4924 radiosensitization, we
performed cell cycle profile of two breast cancer cell lines treated
with MLN4924, radiation, alone or in combination using FACS
analysis. As shown in Figure 2A (left panel), treatment with
MLN4924 or radiation for 24 hrs remarkably arrested SK-BR-3
cells (harboring a mutant p53)  at the G2/M phase of cell
cycle (43% or 57% vs. control at 19%, respectively). The
combinational treatment further enhanced G2/M arrest with
86% of population arrested in the G2/M. The enhanced G2/M
arrest persisted up to 48 hrs (Figure 2A, right panel). Furthermore,
FACS analysis also showed that radiation, but not MLN4924,
induced apoptosis (as reflected by sub-G1 population) after 24-hrs
treatment, which was not enhanced by MLN4924 at 24-hrs, but
was enhanced at 48-hrs time point (Figure 2B). Consistenly,
significant induction of apoptosis was seen in SK-BR-3 cells
treated with radiation-MLN4924 combination, as demonstrated
by enhanced DNA fragmentation (Figure 2C) as well as PARP
cleavage and caspase-3 cleavage/activation (Figure 2D). However,
in wild type p53-containing MCF7 cells , MLN4924 induced
growth arrest at the G2/M phase of cell cycle at either 24-hrs
(45.7% vs. 22.4%) or 48-hrs (56.1% vs. 27.4%) time point, whereas
radiation induced G1 arrest in both time points (63.4% vs. 48.5%
and 57.3% vs. 45.2%, respectively) (Figure 3A). Compared to
MLN4924 treatment alone, MLN4924-radiation combination had
little effect on cell cycle profile (Figure 3A), but did cause a
significant induction of apoptosis (Figure 3B). These results
suggested that radiation-induced disruption of cell cycle progres-
sion in SK-BR-3 cells and apoptotic cell death in both SK-BR-3
and MCF7 cells, can be further enhanced by MLN4924.
Blockage of apoptosis failed to abrogate MLN4924
Since our recent work showed that activation of caspases,
followed by induction of apoptosis was causally related to
MLN4924 Radiosensitizes Breast Cancer Cells
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