Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo

Article abstract of DOI:10.1039/C6SC05368H

UNC-51-like kinase 1 (ULK1) is well-known to initiate autophagy, and the downregulation of ULK1 has been found in most breast cancer tissues. Thus, the activation of ULK1-modulated autophagy could be a promising strategy for breast cancer therapy. In this study, we found that ULK1 was remarkably downregulated in breast cancer tissue samples by The Cancer Genome Atlas (TCGA) analysis and tissue microarray (TMA) analysis, especially in triple negative breast cancer (TNBC). To design a ULK1 agonist, we integrated in silico screening and chemical synthesis to acquire a series of small molecule candidates. After rounds of kinase and anti-proliferative activity screening, we discovered the small molecule, LYN-1604, to be the best candidate for a ULK1 agonist. Additionally, we identified that three amino acid residues (LYS50, LEU53, and TYR89) were key to the activation site of LYN-1604 and ULK1 by site-directed mutagenesis and biochemical assays. Subsequently, we demonstrated that LYN-1604 could induce cell death, associated with autophagy by the ULK complex (ULK1-mATG13-FIP200-ATG101) in MDA-MB-231 cells. To further explore LYN-1604-induced autophagic mechanisms, we found some potential ULK1 interactors, such as ATF3, RAD21, and caspase3, by performing comparative microarray analysis. Intriguingly, we found that LYN-1604 induced cell death involved in ATF3, RAD21, and caspase3, accompanied by autophagy and apoptosis. Moreover, we demonstrated that LYN-1604 has potential for good therapeutic effects on TNBC by targeting ULK1-modulated cell death in vivo; thus making this ULK1 agonist a novel potential small-molecule drug candidate for future TNBC therapy.

Full text of DOI:10.1039/C6SC05368H

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Discovery of a small molecule targeting ULK1-modulating cell
death of triple negative breast cancer in vitro and in vivo
Lan Zhang#, Leilei Fu#, Shouyue Zhang, Jin Zhang, Yuqian Zhao, Yaxin Zheng, Gu He,
Shengyong Yang, Liang Ouyang*, Bo Liu*
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University, and Collaborative Innovation Center of Biotherapy, Chengdu 610041, China
_______________________________________________________________________
*
#These authors contributed equally to this work. Corresponding author. Tel./Fax:
86ꢀ28ꢀ85164063.
Eꢀmail
addresses:
liubo2400@163.com
(Bo
Liu),
or
ouyangliang@scu.edu.cn (Liang Ouyang).
1

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Abstract
UNCꢀ51ꢀlike kinase 1 (ULK1) is wellꢀknown to initiate autophagy, and downregulation of
ULK1 has been found in most breast cancer tissues. Thus, activation of ULK1ꢀmodulated
autophagy may be a promising strategy for breast cancer therapy. In this study, we found
that ULK1 was remarkably downregulated in breast cancer tissue samples by The Cancer
Genome Atlas (TCGA) analysis and tissue microarray (TMA) analysis, especially in triple
negative breast cancer (TNBC). To design an ULK1 agonist, we integrated in silico
screening and chemical synthesis to acquire series of candidate small molecules. After
rounds of kinase and antiꢀproliferative activity screening, we discovered a small molecule
named LYNꢀ1604 as the best candidate ULK1 agonist. Additionally, we identified that
three amino acid residues (LYS50, LEU53 and TYR89) were key to the activation site of
LYNꢀ1604 and ULK1 by siteꢀdirected mutagenesis and biochemical assays. Subsequently,
we demonstrated that LYNꢀ1604 could induce cell death, associated with autophagy by
the ULK complex (ULK1ꢀmATG13ꢀFIP200ꢀATG101) in MDAꢀMBꢀ231 cells. To further
explore LYNꢀ1604ꢀinduced autophagic mechanisms, we found some potential ULK1
interactors, such as ATF3, RAD21 and caspase3 by performing comparative microarray
analysis. Intriguingly, we found that LYNꢀ1604 induced cell death involved in ATF3,
RAD21 and caspase3, accompanied with autophagy and apoptosis. Moreover, we
demonstrated that LYNꢀ1604 had a good therapeutic potential on TNBC by targeting
ULK1ꢀmodulated cell death in vivo; thus making this ULK1 agonist to be utilized as a novel
candidate smallꢀmolecule drug for the future TNBC therapy.
Key words: UNCꢀ51ꢀlike kinase 1 (ULK1); Cell death; Autophagy; ULK1 agonist; Triple
negative breast cancer (TNBC)
Introduction
Macroautophagy (hereafter referred as autophagy) is a highly conserved cellular
selfꢀdigestion process by which cellular components are targeted to lysosomes for their
degradation. 1 Autophagy normally acts as a cellular stress response and a quality control
2

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mechanism to maintain cell survival under stress conditions, since it may provide energy
and metabolic precursors, as well as get rid of some damaged proteins or organelles.2 On
the other hand, increasing evidence for cell death by autophagy has been emerging
now.3,4 Although the accurate mechanisms to distinguish the role of autophagy for
cytoprotective or cell death still need to be addressed, there is evidence showing that both
excessive activation of autophagy and the selective removal of autophagy substrates will
lead to cell death.5ꢀ7
Of note, defects of the autophagic machinery, such as those imposed by the
heterozygous deletion of Beclinꢀ1 or the homozygous loss of autophagyꢀrelated 5 (ATG5)
predispose mice to malignant transformation, especially in breast cancer.8 UNCꢀ51 like
kinase 1 (ULK1), the mammalian homolog of ATG1, has been well known as the
autophagic initiator that may decide the subsequent cell fate.9 In autophagic process,
ULK1 is a component of the ULK complex (ULK1ꢀmATG13ꢀFIP200ꢀATG101) that is
essential for autophagy induction in different types of cancer, such as breast cancer.10,11
Additionally, characterization of ULK1 expression in breast cancer tissues has been
identified that low expression of ULK1 is associated with operable breast cancer
progression and thus being regarded as an adverse prognostic marker of survival for
patients.12 Besides, inhibiting mTOR signaling, the negative regulators of ULK1, may
result in the inhibition of breast cancer growth, suggesting the pivotal role of ULK1 in
breast cancer.13 As mentioned above, activating ULK1ꢀmodulated autophagy will be a
therapeutic strategy in breast cancer therapy.
Herein, we found that ULK1 was remarkably downregulated in breast cancer tissues
(especially in TNBC) by TCGA analyses and TMA analysis, suggesting ULK1 may be a
potential new drug target in TNBC. Subsequently, we integrated in silico screening,
chemical synthesis, kinase and antiꢀproliferative activity screening, as well as siteꢀdirected
mutagenesis, to eventually acquire a potent ULK1 agonist (LYNꢀ1604). Moreover, we
demonstrated that LYNꢀ1604 induced cell death via the ULK complex, involved in ATF3,
RAD21 and caspase3 toward MDAꢀMBꢀ231 cells. In addition, we found that LYNꢀ1604
had a therapeutic potential by targeting ULK1ꢀmodulated cell death, associated with
3

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autophagy and apoptosis of TNBC in vivo, which together demonstrate that this ULK1
agonist would be utilized as a new candidate antiꢀTNBC drug.
Results and Discussion
TCGA-guided identification of ULK1 is remarkably downregulated in breast cancer,
especially TNBC
TNBC typically has a relatively poor outcome due to its inherently invasive and
metastatic clinical behavior; therefore, discovering a novel target and relevant
smallꢀmolecule drugs have been emerging as a focus on TNBC therapy. To explore the
function of ULK1 in TNBC, we investigated The Cancer Genome Atlas (TCGA) breast
cancer gene expression difference between TNBC (n = 55) and nonꢀTNBC tumors (n =
472). We found that ULK1 expression was downregulated in both nonꢀTNBC breast
cancer and TNBC. Interestingly, we demonstrated that ULK expression in TNBC tumors
was significantly lower (p=1.39Eꢀ06) than nonꢀTNBC ones (Fig. 1A). Consequently, we
mapped all remarkably changed autophagic regulators that could be annotated by Gene
Ontology (GO) into the core autophagic proteinꢀprotein interaction (PPI) network in TNBC.
This network was composed of 203 nodes and 579 interactions. Among them, we found
some low expressed autophagic positive regulators (e.g., ULK1, BECN1 and TBK1) that
could be identified as hub proteins (e.g., ULK1 has 22 interactors that are more than the
average number 5.695), suggesting that ULK1 may be a hub protein or potential target in
the autophagic network in TNBC (Fig. 1B). Moreover, we evaluated the expression of
ULK1 in tissue microarray (TMA) containing 40 samples of BRs with patientꢀmatched
adjacent tissues. Immunohistochemical analyses demonstrated decreased expression of
ULK1 in breast cancer samples compared with adjacent normal tissues (Fig. 1C and D).
Notably, the expression of ULK1 in TNBC tissues is much lower than nonꢀTNBC ones,
which displays a consistent result with TCGA analysis (p<0.0001) (Fig. 1D). These results
demonstrate that ULK1 is remarkably downregulated in TNBC.
More recently, the expression levels of ULK1 have been reported higher in
hepatocellular carcinoma (HCC), nasopharygeal carcinoma (NPC) and esophageal
4

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squamous cell carcinoma (ESCC) than those in adjacent tissues and significantly
associated with aggressive clinical feature.14ꢀ16 In contrast, both mRNA and protein levels
of ULK1 were found to be remarkably decreased in breast cancer tissues, and ULK1
expression is negatively correlated with tumor size, lymph node status and pathological
stage, suggesting that decreased expression of ULK1 is associated with breast cancer
progression.12 Thus, in our study, based upon TCGA analysis and TMA analysis, we
found that ULK1 was downregulated in breast cancer tissue samples, especially
remarkable in TNBC. In addition, we used a systematic biology network method to
construct an autophagic PPI network in TNBC, which revealed ULK1 as a hub
protein/potential target, not only at protein expression level, but at PPI network level.
Together, these results indicate that activating ULK1 may be a novel strategy for the
current TNBC therapy.
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Figure.1 Identification of ULK1 is remarkably downregulated in TNBC
(A) ULK1 expression was significantly lower in TNBC tumors than nonꢀTNBC (p=1.39Eꢀ06). (B) In the
core autophagic PPI network, ULK1, BECN1 and TBK1 were low expressed positive regulators of
autophagy. ULK1 was a hub protein with 22 interactors. (C) The representative immunoreactivity
intensity of ULK1 for negative (0), weakly positive (1), positive (2), strongly positive (3) staining. Scale bar
= 50 ꢁm. (D) ULK1 expression was downregulated in breast cancer tissues (TNBC: p<0.0001,
nonꢀTNBC: p=0.0478 ) compared with normal adjacent tissues (NAT), especially in TNBC (vs. nonꢀTNBC:
p=0.0314).
6

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Design, screening and synthesis of candidate ULK1 agonists
Based upon the hypothesis that mATG13 and/or FIP200 bind to hydrophobic pockets
of ULK1, there by switching the conformation of kinase domain to the onꢀstate, we
synthesized agonists to “lock in” their interaction (Fig. 2A). Firstly, we defined 9 candidate
binding pockets of kinase domain of ULK1 (PDB ID 4WNP) by Discovery Studio. As
shown in Fig.2B, we selected the nearest pocket (green) of the interaction site to perform
the virtual screening of agonists. Threeꢀstep molecular docking were performed on
FDAꢀapproved drugs from Drugbank, searching for leading compounds (Fig. 2C). Top500
hits were selected by LibDock protocol in the first step. Subsequently, Top20 (Uꢀ1~Uꢀ20)
hits determining by CDOCKER protocol were selected for in silico screening. As a result,
one
compound
(DB01127)
named
Uꢀ11
(1ꢀ(2ꢀ((4ꢀchlorophenyl)
methoxy)ꢀ2ꢀ(2,4ꢀdichlorophenyl) ethyl)ꢀ1hꢀimidazol) exhibited the best score (CDOCKER
energy 17.6299; CDOCKER interaction energy 25.5461), suggesting Uꢀ11 has a priority
as a candidate ULK1 agonist. Thus, we selected Uꢀ11 as a leading compound for
structure modifications and SAR analyses.
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Figure.2 In silico high-throughput screening of candidate small-molecule compounds targeting
ULK1
(A) Schematic drawing of the activation mechanism of ULK1 agonist. (B) Candidate binding pockets in
the structure of the kinase domain of ULK1. Pocket of inhibitors was shown in red and pocket of our
virtual screening was shown in green. (C) Virtual screening protocol for the identification of novel ULK1
agonist. (D) The top twenty candidate smallꢀmolecule compounds targeting ULK1 from Drugbank.
Next, a series of new UA1 derivatives UA1ꢀ01~12 containing different terminal
groups (R1 and R2 in Fig. 3, Scheme 1) were synthesized. On the basis of molecular
modeling, the nitrogen atom of the piperazine ring present a hydrogen bond acceptor in
an ideal orientation that interact with amino group of the LYS50 side chain and also
provide suitable substitution vectors for improving activity. Therefore, the chemical library
was enriched by different substituted phenyl or naphthylꢀbased hydrophobic ligands that
were identified as favorable functional groups for biological potency in the first round of
screening (Table S1), and compound UA1ꢀ03 exhibited the best activity (Enzymatic
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activity =153.64% at 100 nM and IC50 =20.94 ꢁM toward MDAꢀMBꢀ231 cells). Based on
the preliminary results, UA2 analogs UA2ꢀ01~13 containing specific 2ꢀnaphthyl and R3
(acylpiperazinꢀ1ꢀyl) were designed and prepared (Fig. 3, Scheme 2). Sequentially, we
found that the existence of methylene in UA2ꢀ10~13 played a key role for biological
potency which should be preserved in order to maintain the activity (Table S2), and the
mechanistic enzymatic potency of UA2ꢀ10~13 was outstanding (Enzymatic activity
=141.37~160.29% at 100 nM). To explore the impact of R2, we used a methylene to
expand the hinge. Additionally, the αꢀchlorinated ketone group of the scaffold provided a
straight forward entry for diversity generation from a list of available amines to afford
UA3ꢀ01~07 (Fig. 3, Scheme 3). Bulking up the Nꢀsubstitution had a contribution to
improve the activity (Table S3). Compound UA3ꢀ02 (hereafter referred to as LYNꢀ1604)
showed the best potency, which was 10ꢀfold improvement over UA1ꢀ03. Briefly, a
preferred compound, LYNꢀ1604, were finally found as potential ULK1 agonist (Enzymatic
activity =195.7% at 100nM and IC50=1.66 ꢁM against MDAꢀMBꢀ231 cells) through three
rounds of screening (Fig. 4A and B).
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Figure.3 Chemical synthesis of candidate ULK1 agonists
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(A) Scheme 1. Reagents and conditions: (a) 1ꢀbocꢀpiperazine or imidazole or benzimidazole or
1Hꢀ1,2,4ꢀtriazole, K2CO3, acetonitrile; (b) NaBH4, CH3OH, r.t.; (c) benzyl halogenated, KTB, TABA, KI,
CH3CN, reflux.
(B) Scheme 2. Reagents and conditions: (d) TFA, CH2Cl2, 0 °C; (e) Et3N, CH2Cl2, acyl chloride, r.t.
(C) Scheme 3. Reagents and conditions: (f) Et3N, CH2Cl2, r.t.; (g) Et3N, KI, CH3CN, secondary amines, r.t.
or 60 °C.
Figure.4 Biological evaluation of candidate ULK1 agonists toward human breast cancer cells
(A) Heat map of mean IC50 values of ULK1 agonists in breast cell lines. Cell viabilities were measured for
each compound with various concentrations by MTT assay, the IC50 values were calculated by Prism
6.0. (B) The luminescence (RLU) of ULK1 agonists at 100 nM were determined by ADPꢀGlo ULK1 kinase
assay. Blank: no ULK1 kinase (0% kinase activity), Control: containing ULK1 kinase (100% kinase
activity). Error bars represent the standard deviations of the results from three independent experiments.
Identification of LYN-1604 as a potent ULK1 agonist
To confirm LYNꢀ1604 is an ULK1 agonist (Fig.5A), the ligand structure with the most
favorable binding free energies and reasonable orientations were selected as the optimal
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docked conformation. We found that LYNꢀ1604 could formed hydrogen bonds with LYS50
and formed hydrophobic interaction with residues LEU53 and TYR89 (Fig. 5B and C). To
validate the capacity of LYNꢀ1604 that activates ULK1, we used the ADPꢀGlo™ Kinase
Assay to evaluate the EC50 value of LYNꢀ1604. Intriguingly, LYNꢀ1604 displayed a good
effect on ULK1 activation with an EC50 (the concentration required to reach 50% of the
maximal activation) of 18.94 nM, indicating that LYNꢀ1604 is a potent ULK1 agonist (Fig.
5D). To further determine the binding mode of activation site between ULK1 and
LYNꢀ1604, we generated three ULK1 mutants by siteꢀdirected mutagenesis. The lysine
residue, leucine residue and tyrosine residue in the binding pocket were mutated to
alanine (ULK1K50A, ULK1L53A, ULK1Y89A). ULK1 kinase activity tests indicated that both
ULK1K50A and ULK1L53A mutants had less reduced kinase activity in the presence of
LYNꢀ1604, compared to the wildꢀtype ULK1 protein (ULK1K50A: ꢀ11.93% at 10 nM, ꢀ8.73%
at 100 nM; ULK1L53A: ꢀ3.22% at 10 nM, ꢀ4.06% at 100 nM). But, ULK1Y89A was not
activated by LYNꢀ1604 totally (Fig. 5E). In vitro kinase assay revealed that the LYNꢀ1604
increased the phosphorylation of mATG13 at ser318 in wildꢀtype ULK1 transfected
HEKꢀ293T cells, indicating LYNꢀ1604 activates ULK1 in live cells. However, the ULK1K50A
and ULK1L53A mutants only resulted in negligible decrease of mATG13 phosphorylation in
the presence of LYNꢀ1604, whereas ULK1Y89A mutant significantly decreased the
phosphorylation of mATG13 (Fig. 5F). In addition, a known ULK1 inhibitor, SBIꢀ0206965
was used to determine the kinase activity induced by LYNꢀ1604. We found that
SBIꢀ0206965 (300 nM) could markedly reverse LYNꢀ1604ꢀinduced increase of ULK1
kinase activity (Fig. S1, ESI†). Subsequently, the binding properties of LYNꢀ1604 to
wildꢀtype and mutant ULK1 were determined by surface plasmon resonance (SPR)
analysis (Fig. 5G). We found that LYNꢀ1604 bound to wildꢀtype ULK1 with a binding
affinity in the nanomole range (KD = 291.4 nM), but the ULK1Y89A mutant protein caused a
sharp decrease in binding affinity with lower response and KD than wildꢀtype ULK1,
ULK1K50A and ULK1L53A mutants. Moreover, we compared the binding conformation of
LYNꢀ1604 with wildꢀtype and mutant ULK1 by molecular docking. Interestingly, LYNꢀ1604
had a remarkable change in binding conformation with ULK1Y89A mutant, compared to
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wildꢀtype ULK1 and the other two mutants (Fig.S2, ESI†). Then, we performed a 25ꢀns MD
simulation on LYNꢀ1604/ULK1 complex. The low rootꢀmeanꢀsquare deviation (RMSD)
fluctuations and the convergence of the energies, temperatures, and pressures of the
systems indicated that it is a wellꢀbehaved system. But, the LYNꢀ1604/ULK1Y89A complex
occurred conformational change after 10 ns simulation, suggesting TYR89 is a key amino
acid residue of ULK1 for binding with LYNꢀ1604 (Fig. 5H). Thus, these results indicate that
LYNꢀ1604 is a potent ULK1 agonist.
Recently, some smallꢀmolecule agents such as compound 6, SBIꢀ0206965,
MRT67307 and MRT68921 have been reported as inhibitors of ULK1; however, these
inhibitors have not been widely used for cancer therapy.17ꢀ20 In addition, to our knowledge,
not any smallꢀmolecule agonist has been reported to activate ULK1 for potential cancer
therapy. Thus, LYNꢀ1604 is an ULK1 agonist with potent enzymatic activity for ULK1
activation and remarkable antiꢀproliferative effects against TNBC cell lines. Moreover, we
used ACTP, a webserver for predicting potential targets and relevant pathways of
autophagyꢀmodulating compounds21 to predict the potential autophagic target of
LYNꢀ1604. We found that ULK1 is the best predicted target of LYNꢀ1604 and prone to
activation pattern, which was in a good agreement with our aforementioned results. More
interestingly, some ULK1 inhibitor such as compound 6 is designed based on coꢀcrystal
structure of the inhibition pattern and its binding sites are determined as MET92, LYS46,
ASP165 and ASN143.17 Distinctive from them, LYNꢀ1604 is designed based upon the
xꢀray structure of ULK1, and we speculated that its activating binding sites are LYS50,
LEU53 and TYR89. By mutation of these residues, we found TYR89 is a key amino acid
residue of ULK1 for binding with LYNꢀ1604. Thus, LYNꢀ1604 as an ULK1 agonist, will be a
novel small molecule for TNBC therapy.
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Figure.5 Identification of LYN-1604 as a potent ULK1 agonist and its binding mode
(A) Chemical structure of ULK1 agonist LYNꢀ1604. (B, C) The binding model of LYNꢀ1604 with ULK1.
General (B) and detailed (C) views of the interactions between LYNꢀ1604 and ULK1. A detailed view
shows that LYNꢀ1604 formed hydrophobic interactions with binding pocket. (D) ULK1 kinase assay was
performed with LYNꢀ1604, to determine the level of ULK1 kinase activity. The luminescence changed
was normalized to the maximum response induced by LYNꢀ1604 and quantified as 0ꢀ100% ULK1 kinase
activity. The EC50 was calculated from the fitted curve. (E) Mutation at LYS50, LEU53 and TYR89 in the
binding pocket of ULK1 kinase were performed by siteꢀdirected mutagenesis. The wildꢀtype and mutant
ULK1 were expressed in insect cells and purified. ULK1 kinase activity was measured in the presence
and absence of LYNꢀ1604 using ADPꢀGlo assay. (F) In vitro kinase assay for the capability of wild type or
mutant ULK1 purified from 293T cells to phosphorylate purified mATG13. Phosphorylation of mATG13 at
ser318 was detected by a specific antibody and equal inputs of enzymes and substrate are shown on the
bottom. (G) Surface plasmon resonance measurements were carried out on a Biacore system to
evaluate the binding affinities of wild type or mutant ULK1 to LYNꢀ1604. (H) Molecular dynamics
simulation of LYNꢀ1604 binding to wild type or mutant ULK1. The binding conformation was stabilized
after 10 ns simulation excepted in LYNꢀ1604/ULK1Y89A complex.
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LYN-1604 induces cell death via the ULK complex in MDA-MB-231 cells
Next, we determined whether LYNꢀ1604ꢀinduced cell death is associated with
autophagy. Autophagic ratio was evaluated by MDC staining after treatment with
LYNꢀ1604. We found that LYNꢀ1604ꢀtreated cells displayed a remarkable green
fluorescence with MDC staining and the autophagy ratio was increased in a
doseꢀdependent manner (Fig. 6A and B). We also found that LYNꢀ1604 induced
remarkable upꢀregulation of Beclinꢀ1 and degradation of p62, as well as transformation of
LC3ꢀI to LC3ꢀII (Fig. 6C). To assess whether LYNꢀ1604ꢀinduced autophagy was a
continual process, Bafilomycin A1 (BafA1) was used for detection of p62 and LC3II/LC3I
levels. Obvious aggregation of LC3 puncta was observed in the presence of BafA1
compared to LYNꢀ1604ꢀtreated cells (Fig. 5D). We also found BafA1 could increase the
accumulation of p62 and LC3II, indicating LYNꢀ1604ꢀinduced autophagy is a continual
process (Fig. 6E). To determine whether autophagy plays a role in LYNꢀ1604ꢀinduced cell
death, the autophagy inhibitor 3ꢀmethyladenine (3ꢀMA), which inhibits autophagy by
blocking autophagosome formation via inhibiting type III phosphatidylinositol 3ꢀkinases
(PI3KIII), was employed to block the induction of autophagy. We found that the cell
viability was significantly increased following treatment with 3ꢀMA (p<0.001) (Fig. 5F).
Subsequently, flow cytometry analysis with AnnexinꢀV/PI demonstrated that 3ꢀMA could
suppress LYNꢀ1604ꢀinduced cell death (Fig. 5G). These results indicate that LYNꢀ1604
induces cell death in MDAꢀMBꢀ231 cells.
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Figure.6 LYN-1604 induces cell death in MDA-MB-231 cells
(A) MDAꢀMBꢀ231 cells were treated with 0.5, 1.0 and 2.0 ꢁM LYNꢀ1604, respectively, then stained with
MDC and observed by fluorescence microscope. Scale bar = 20 ꢁm. (B) MDAꢀMBꢀ231 cells were treated
with 0.5, 1.0 and 2.0 ꢁM LYNꢀ1604, respectively. Then cells were collected and analyzed by flow
cytometry, the MDC positive ratio was quantified. (C) Cells were treated with different concentrations of
LYNꢀ1604 for 24h, then the expressions of Beclinꢀ1, p62 and LC3 were detected by western blot analysis.
(D) MDAꢀMBꢀ231 cells were transfected with GFP/mRFPꢀLC3 plasmid, after coꢀincubation with 2.0 ꢁM
LYNꢀ1604 in the presence or absence of 10 nM Bafilomycin A1 (BafA1), the GFPꢀLC3 puncta were
observed by fluorescence microscope. Scale bar = 20 ꢁm. (E) MDAꢀMBꢀ231 cells were coꢀincubated with
2.0 ꢁM LYNꢀ1604 in the presence or absence of 10nM Bafilomycin A1 (BafA1), then the expression level
of p62 and LC3 were detected. βꢀactin was measured as loading control. (F) MDAꢀMBꢀ231 cells were
treated with 2.0 ꢁM LYNꢀ1604, 3ꢀMA (1 mM) was added 1 h before treatment of LYNꢀ1604. After above
treatment, cell viabilities were detected by MTT assay. ***, p<0.001 vs. LYNꢀ1604ꢀtreated group. (G)
MDAꢀMBꢀ231 cells were treated with 2 ꢁM LYNꢀ1604 for indicated times, apoptosis ratios were
determined by flow cytometry analysis of AnnexinꢀV/PI double staining. 3ꢀMA (1 mM) was added 1 h
before treatment of LYNꢀ1604.
Considering that the ULK complex (ULK1ꢀmATG13ꢀFIP200ꢀATG101) is closely
related to autophagosome formation, we detected the expressions of ULK1, mATG13,
FIP200 and ATG101 in LYNꢀ1604ꢀtreated cells. Firstly, we found LYNꢀ1604 significantly
upregulated the activity of ULK1 in the cytoplasm as determined with an increasing
fluorescence of antiꢀpꢀULK1 (Fig. 7A). We also revealed that LYNꢀ1604 treatment induced
upregulation of pꢀULK1 (ser317) and downregulation of pꢀULK1 (ser757), as well as the
increase in phosphorylation of its downstream substrate mATG13 at ser318 (Fig. 7B). In
addition, we detected whether LYNꢀ1604 activates AMPK. LYNꢀ1604 decreased the
phosphorylation of ACC and AMPK (Fig. S3, ESI†), indicating that LYNꢀ1604 decreases
AMPK activity, which may be due to the negative regulation of ULK1 on AMPK activity.22
To examine whether LYNꢀ1604 induced autophagy by targeting ULK1, the specific
smallꢀinterfering RNA was applied for silencing ULK1 expression. We found that the LC3B
puncta were almost disappeared in ULK1ꢀsiRNA treated cells comparing with the
controlꢀsiRNA treated cells (Fig. 7C). Subsequently, we detected the expression of the
ULK complex, LC3 and p62. Silencing of ULK1 resulted in a prominent inactivation of
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autophagy (Fig. 7D and E). Moreover, we knocked down another autophagyꢀrelated gene
ATG5 by siRNA. Interestingly, we found that silencing of ATG5 did not disrupt the
activation of ULK1 but could block the degradation of p62 and lipidation of LC3 (Fig. 7F).
We determined the cell viability by knocking down of ULK1 or ATG5, respectively. The cell
viabilities were increased after silencing of ULK1 or ATG5 compared to the negative
control group (Fig. 7G). Simultaneously, the ULK1 inhibitor SBIꢀ0206965 also could
decrease LYNꢀ1604ꢀinduced cell death (Fig. S4, ESI†). These results suggest that
LYNꢀ1604 can induce ATG5ꢀdependent autophagy via the ULK complex.
Of note, autophagy is regarded as a doubleꢀedged sword in the survival of cancer.
And, it is well known that ULK1ꢀactivated autophagy is mainly dependent on the ULK
complex in different types of cancer cells.23,24 Although there are a number of
smallꢀmolecule agents have been reported to induce cell death, associated with
autophagy in breast cancer cells. For instance, YM155 is a potent survivin inhibitor that
can modulate autophagy and induce autophagyꢀdependent cell death in breast cancer
cells.25 Ivermectin, a broadꢀspectrum antiꢀparasitic drug, has been found that induces
cytostatic autophagy by blocking PAK1/Akt axis in breast cancer.26 However, these small
molecules either have no specific targets or possess some other complex mechanisms
which are not directly regulated by autophagyꢀrelated (ATG) proteins. Distinctive from
aboveꢀmentioned studies, LYNꢀ1604 could induce ATG5ꢀdependent autophagy in TNBC
cells by targeting the ULK complex, which may provide an autophagyꢀmodulating agent
for potential TNBC therapeutics.
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Figure.7 LYN-1604 induces ATG5-dependent autophagy via the ULK complex
(A) LYNꢀ1604 could obvious upꢀregulate the expression of pꢀULK1. The expression of pꢀULK1 (Ser317)
was detected by immunocytochemistry. Red: TRITCꢀantiꢀpꢀULK1; Blue: DAPI. (B) The expressions of
ULK complex were detected by western blot analysis. βꢀactin was measured as loading control. (C)
MDAꢀMBꢀ231 cells were transfected with and control or ULK1 siRNA, followed by treatment with 2.0 ꢁM
LYNꢀ1604 for 24h. Then, the expression of LC3B puncta were detected by immunocytochemistry. Green:
antiꢀLC3B; Blue: DAPI. (D and E) The cells were transfected with control or ULK1 siRNA, followed by
treatment with 2.0 ꢁM LYNꢀ1604 for 24h. Then the expression levels of ULK complex (D), as well as p62
and LC3 (E) were determined by western blot analysis. βꢀactin was measured as loading control. (F) The
cells were transfected with control or ATG5 siRNA, followed by treatment with 2.0 ꢁM LYNꢀ1604 for 24h.
Then the expression levels of ATG5, ULK1, pꢀULK1 (ser317, ser757), p62 and LC3 were determined by
western blot analysis. βꢀactin was measured as loading control. (G) The cells were transfected with
control, ATG5 or ULK1 siRNA, respectively. After treatment with 2.0 ꢁM LYNꢀ1604 for 24h, cell viabilities
were measured by MTT assay.
Microarray-based analyses of potential ULK1 interactors in LYN-1604-induced
MDA-MB231 cell autophagy
Subsequently, the different effect of ULK1 knockdown or amplifying on gene
expression by transcriptional analysis was investigated. We performed microarray
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analysis on MDAꢀMBꢀ231 cells treated with ULK1 siRNA or transfected with pcDNA
3.1ꢀULK1. The analysis of significant differentially expressed genes (log fold change > 0.5)
identified 491 genes downꢀregulated and 1,001 genes upꢀregulated after ULK1
knockdown, while 712 genes downꢀregulated and 1,268 genes upꢀregulated after ULK1
amplified (Fig. 8A). 35 genes with opposite expression changes were predicted to be
closely involved in ULK1ꢀregulated network in breast cancer (Fig. 8B). Interestingly, 5
genes (ATF3, TTC23L, PLK4, CASP3 and RAD21) showed the opposite expression
changes simultaneously after ULK1 knockdown or overexpression, meanwhile other
genes showed expression changes in different extent, indicating that these genes may be
directly interacted with ULK1. Moreover, the PPI network of ULK1 was modified to the
microarrayꢀbased ULK1 network into the context of TNBC, which consisted of 23 possible
ULK1ꢀinteracting genes. According to microarrayꢀbased analyses of core ULK1ꢀregulated
autophagic subnetwork, we found that activating transcription factor 3 (ATF3),
doubleꢀstrandꢀbreak repair protein rad21 homolog (RAD21) and caspase3 had significant
changes in the expressions and were closely correlated with ULK1 (Fig. 8C). Thus, it
suggests the key role of ULK1 with its potential interactors in autophagy of TNBC cells. To
validate the potential interactors and intricate mechanisms of ULK1, we used the ULK1
agonist LYNꢀ1604, which is similar with ULK1 overexpression plasmid, to activate
ULK1ꢀmodulating cell death, associated with autophagy.
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Figure.8 Cell-specific microarray-based analyses of core ULK1-regulated network in MDA-MB-231
cells
(A) Expression data of microarray analyses from two individual experiments. MDAꢀMBꢀ231 cells were
treated with ULK1 siRNA or transfected with pcDNA 3.1ꢀULK1. Significant differentially expressed genes
with opposite expression change were circled by red dash line. (B) 35 opposite expression changed
genes. (C) Microarrayꢀbased ULK1 subnetwork in breast cancer, 23 opposite expression changed genes
(blue) were involved in this subnetwork. ATF3, RAD21 and caspase3 were verified that could be
regulated by ULK1.
As caspase3 is known as the apoptotic executor, the morphologic changes of
MDAꢀMBꢀ231 cells were observed with fluorescent dye. Interestingly, the apoptotic
phenomenon was not obvious, which was determined by Hoechst 33258 staining (Fig.
9A). Susequently, we checked expressions of caspase3 and its cleavage substrate PARP,
then found they were slightly changed only with high concentration of LYNꢀ1604 treatment,
which suggests that LYNꢀ1604 induces cell death, associated with autophagy and
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apoptosis (Fig. 9B). We also found that LYNꢀ1604 obviously upꢀregulated of ATF3 and
downꢀregulated RAD21 (Fig. 9C). However, LYNꢀ1604 had no effects on the expressions
of cleavedꢀcaspase3, ATF3 and RAD21, when ULK1 was knocked down by siRNA (Fig.
9D). These results indicate that caspase3, ATF3 and RAD21 are all involved in
LYNꢀ1604ꢀinduced cell death, which may be simultaneously regulated by ULK1 (Fig. 9E).
ULK1 can be directly regulated by AMPK and mTORC1 via phosphorylation.27,28
Subsequently, ULK1 and its complex are activated and modulate necessary downstream
signals, including phosphorylation of Beclinꢀ1 and activation of VPS34 lipid kinase.29,30 To
explore the intricate mechanisms, we constructed the ULK1ꢀregulated network by
performing microarray analyses in TNBC cells. Intriguingly, we found several autophagic
proteins such as ATF3, RAD21 and even the apoptotic protein caspase3 that could be
regulated by ULK1, indicating that ULK1 plays its dual roles in autophagyꢀinitiating and
promoting apoptosis with some potential new interactors in TNBC. Of note, ATF3 is an
autophagyꢀrelated protein; therefore, activation of ATF3 can promote the process of
autophagy.31 RAD21 is important and closely related with breast cancer cell growth and
apoptosis, thus inhibiting RAD21 can inhibit cancer cell proliferation.32,33 In our study, we
showed that LYNꢀ1604 could modulate ATF3 and RAD21 through ULK1 in MDAꢀMBꢀ231
cells, which is in good agreement with our aboveꢀmentioned microarrayꢀbased analyses
of ULK1 in TNBC. In addition, LYNꢀ1604 can also increase cleavage of caspase3 and
induce apoptosis. We speculate that ULK1 probably regulate caspase3 activity to induce
apoptosis, or ULK1 may be a substrate, which could be cleaved by caspase3. However,
the internal relations between ULK1 and caspase3 still need to be explored. Together,
these findings would provide some potential mechanisms for ULK1 agonists to induce cell
death in TNBC therapy.
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Figure.9 LYN-1604 induces autophagy involved in ATF3, RAD21 and caspase3
(A) After treatment with LYNꢀ1604, cells were stained with Hoechst 33258. Morphologic changes were
observed under fluorescence microscope. Scale bar = 20ꢁm. (B) After LYNꢀ1604 treatment, the
expression levels of caspase3 and PARP were determined by western blot analysis. βꢀactin was
measured as loading control. (C) After LYNꢀ1604 treatment, the expression levels of ATF3 and RAD21
were determined by western blot analysis. βꢀactin was measured as loading control. (D) The cells were
transfected with control or ULK1 siRNA, followed by treatment with 2.0 ꢁM LYNꢀ1604 for 24h. Then the
expression levels of cleavedꢀcaspase3, ATF3 and RAD21 were determined by western blot analysis.
βꢀactin was measured as loading control. (E) The schematic model of LYNꢀ1604ꢀinduced cell death
pathways, associated with autophagy and apoptosis.
LYN-1604 inhibits the growth of xenograft TNBC by targeting ULK1-modulated cell
death
To evaluate the efficacy of LYNꢀ1604 in vivo, we established a MDAꢀMBꢀ231
xenograft model. Based on the results of tumor volume and weight, we found that
LYNꢀ1604 could significantly inhibited the growth of xenograft MDAꢀMBꢀ231 cells (Fig.
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10A and B). The body weights of mice were stable, with no obvious distinctions between
LYNꢀ1604ꢀtreated and control mice (Fig. 10C). By the end of the experiment, the liver and
spleen weight indexes of mice were slightly increased in part of the groups, while the
kidney weight index was not affected in all dose groups (Fig. 10D). To confirm the
mechanisms for the therapeutic efficacy of LYNꢀ1604 in vivo, we examined the
immunoreactivity of ULK1 and pꢀULK1 in tumor tissues, and found both of them showed
increasing trend in LYNꢀ1604ꢀtreated tumor tissues (Fig. 10E and F). Next, we carried out
western blot analysis to clarify the mechanism of LYNꢀ1604 in vivo. It revealed that
LYNꢀ1604 induced remarkable autophagy in vivo, which was consistent with the
immunohistochemical analysis and the aforementioned in vitro results (Fig. 10G and H).
Thus, these results suggest that LYNꢀ1604 has a good therapeutic potential by targeting
ULK1ꢀmodulated cell death of TNBC in vivo, thus making an ULK1 agonist to be utilized
as a new drug for TNBC therapy.
More recently, some agents have been broadly
used for treatment of TNBC by modulating autophagy. Everolimus, an oral mTOR inhibitor,
can effectively inhibit cell growth in different subtypes of TNBC cells through
PI3K/Akt/mTOR cascade, which also demonstrates antitumor activity in a mouse
xenograft model of basalꢀlike breast cancer, rather than nonꢀbasal breast cancer.34
Chloroquine, an autophagy inhibitor could prevent lysosomeꢀfusion in autophagy process,
has been reported that can inhibit xenograft tumor growth of TNBC, especially in
combination with other anticancer drugs such as panobinostat, epirubicin and
carboplatin.35ꢀ37 Beyond these small molecules, LYNꢀ1604 can directly activate ULK1, the
initiator of autophagy, thereby inducing cell death to inhibit growth of TNBC in vitro and in
vivo. More importantly, ULK1 is a promising therapeutic target of TNBC, which is a
specific and potent target for TNBC rather than other breast cancer, thus making an
agonist of ULK1, LYNꢀ1604 to be utilized as a candidate smallꢀmolecule drug for the
future TNBC therapy.
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Figure.10 LYN-1604 has a therapeutic potential by targeting ULK-modulated cell death in vivo
Antitumor activities of LYNꢀ1604, tumor volume (A) and tumor weight (B) were expressed as the mean ±
SEM, ***p<0.001, **p<0.05 vs. control group (n=6). Representative tumors from mice after vehicle and
LYNꢀ1604 treatment. The toxicity of LYNꢀ1604. Relative body weight (C), liver index, spleen index and
kidney index (D) were evaluated at the end of treatment. (E) The expression of pꢀULK1 (Ser317) in
representative tumor section of mice after vehicle and median dose LYNꢀ1604 treatment were
determined by immunohistochemistry (×100 magnification), ***p<0.001 vs. control group. (F) Quantitative
analysis of ULK1 and pꢀULK1 positive ratios. (G) Western blot analysis of tumor tissues from vehicle or
LYNꢀ1604 treated mice for expression of ULK1, pꢀULK1, LC3, Beclinꢀ1 and PARP, βꢀactin was
measured as loading control. (H) Quantification of immunoblot shown in (G).
Conclusion
Based upon TCGA and TMA analyses, we found that ULK1 was more remarkably
downregulated in TNBC tissues than other breast cancer ones, indicating that ULK1 may
be a new target in TNBC. Combining with in silico highꢀthroughput screening, series
synthesis, kinase and antiꢀproliferative activity screening, as well as siteꢀdirected
mutagenesis, we discovered a potent ULK1 agonist (LYNꢀ1604). Moreover, we
demonstrated that LYNꢀ1604 could activate ULK1 and ULK complex
(ULK1ꢀmATG13ꢀFIP200ꢀATG101), and also trigger cell death via ATG5 involvement, as
well as ATF3, RAD21 and caspase3. Additionally, we found that LYNꢀ1604 had a good
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therapeutic potential on TNBC by targeting ULK1ꢀmodulated cell death in vivo.
In summary, our integrative TCGAꢀ, microarrayꢀbased and experimental findings
would together provide us a better understanding of how ULK1, as a potential antiꢀTNBC
target can regulate cell death by the ULK complex (ULK1ꢀmATG13ꢀFIP200ꢀATG101) and
other potential new interactors (ATF3, RAD21 and caspase3). More importantly, such
results may help us discovering this ULK1 agonist (LYNꢀ1604) with its remarkable
antiꢀtumor activity and cell death mechanisms in vitro and in vivo that would be further
exploited as a novel smallꢀmolecule candidate drug for the future TNBC therapeutics.
Experimental
TCGA analysis and network construction
TCGAꢀbased gene expression profile was measured using the Agilent G4502A_07_3
platform. Log2 lowess normalized (cy5/cy3) collapsed by gene symbol count was used as
gene level expression estimates in this study. A total of 527 breast cancer patients (55
TNBC and 472 nonꢀTNBC) with gene expression datasets were studied. The core
autophagic PPI network was download from ARN database.38 Only PPIs annotated as
Interaction between autophagy proteins were used. The PPI network was constructed
using Cytoscape.39
Tissue microarray (TMA) analysis
The breast cancer tissue microarray (BR804a) was obtained from Cybrdi, Inc.
Detection of ULK1 was performed by antiꢀULK1 antibody (1:200) as previously described.
26,40
The microarray contained breast cancer tumor tissues from 40 individuals plus
matched adjacent normal tissues. The immunostaining intensity was indicated by four
grades (0, negative; 1, weakly positive; 2, positive; 3, strongly positive) and the
percentage of cell staining at each intensity level was graded as proportion of
stainingꢀpositive cells was divided into five grades 0 (<5%), 1 (5ꢀ25%), 2 (26ꢀ50%), 3
(51ꢀ75%), 4 (>75%). The final score was calculated as intensity score × the proportion of
stainingꢀpositive cells.
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Cell culture, antibodies and reagents
HEKꢀ293T, MDAꢀMBꢀ231, MDAꢀMBꢀ468 and MCFꢀ7 cells were purchased from
American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in
DMEM with 10% fetal bovine serum and incubated with 5% CO2.
Antibodies used in this study were as follow: ULK1 (8054, CST, MA, USA), ULK1
(ab128859, Abcam, UK), pꢀULK1ser317 (12753, CST), pꢀULK1ser757 (14202, CST), mAtg13
(13273, CST), pꢀmATG13Ser318 (PAB19948, Abnova, Taiwan), ATG101 (13492, CST),
FIP200 (12436, CST), AMPKα (5831, CST), pꢀAMPKαthr172 (2535, CST), ACC (3676,
CST), pꢀACCser79 (11818, CST), Beclinꢀ1 (3495, CST), LC3B (3868, CST), p62 (5114,
CST), ATG5 (12994, CST), PARP (9532, CST), caspaseꢀ3 (9665, CST), ATF3 (ab58668,
Abcam), RAD21 (12673, CST), βꢀactin (66009ꢀ1ꢀIg, Proteintech, IL, USA).
MTT (M2128), Hoechst 33258 (14530), MDC (30432) and GSTꢀtagged mATG13
(SRP5341) were purchased from SigmaꢀAldrich (St. Louis, MO, USA). Bafilomycin A1
(ab120497) was purchased from Abcam. The plasmid encoding mycꢀhULK1 (#31961)
was purchased from Addgene and subcloned to pRK5ꢀFlag. Flagꢀtagged ULK1 mutants
(K50A, L53A and Y89A) were generated by siteꢀdirected mutagenesis with the
Quikchange II siteꢀdirected mutagenesis kit (Stratagene).
Molecular docking and molecular dynamics (MD) simulations
The initial three dimensional geometric coordinates of the Xꢀray crystal structure of
ULK1 (PDB code: 4WNP) was downloaded from the Protein Databank (PDB). And, we
constructed the screening library containing 6,583 small molecule compounds from the
latest version of Drugbank (http://www.drugbank.ca/). The activators were constructed
using the Accelrys Discovery Studio (version 3.5; Accelrys, SanDiego, CA, USA)
molecular modeling software and were energy minimized with the CHARMm force field.
Docking and molecular dynamics (MD) simulations were performed according to our
previous reports.41,42 The LibDock and CDOCKER protocol were employed as docking
approach to conduct semiꢀflexible docking. MD simulations were carried out for
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LYNꢀ1604ꢀULK1WT
and
LYNꢀ1604ꢀULK1K50A
,
LYNꢀ1604ꢀULK1L53A
and
LYNꢀ1604ꢀULK1Y89A complexes using GROMACS (version 4.5.5) software package.43 All
MD simulations videos were supplied in the supplementary files.
ADP-Glo™ Kinase Assay
ULK1 kinase activity assay was performed using ADPꢀGlo™ Kinase Assay + ULK1
Kinase Enzyme System (Promega, Madison, WI, USA) as previously described.44
ADPꢀGlo™ Kinase Assay is a luminescent kinase assay that measures ADP formed from
a kinase reaction; ADP is converted into ATP, which is converted into light by UltraꢀGlo™
Luciferase. The luminescent signal positively correlates with kinase activity. The ULK1
kinase enzyme, substrate, ATP and compound were diluted in Kinase Buffer (40 mM Tris
pH 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 50 µM DTT). Then 1 µl of compound or (5%
DMSO), 2 µl of ULK1 kinase enzyme or purified wildꢀtype and mutant ULK1 (K50A, L53A,
Y89A) (10 ng), 2 µl of MBP (0.1 µg/µl)/ATP (10 µM) mix were added to the wells of 384
low volume plate. After incubation at room temperature for 60 minutes, 5 µl of ADPꢀGlo™
Reagent was added to per well. The plates were incubated at room temperature for 40
minutes, then 10 µl of Kinase Detection Reagent were added. After incubation at room
temperature for 30 minutes, the luminescence were recorded. The EC50 values were
calculated using nonlinear regression with normalized doseꢀresponse fit using Prism
software (GraphPad Software, San Diego, CA, USA). To exclude the increased kinase
activity might be due to aggregation. 0.02% TritonꢀX was used as a detergent for
eliminating the interference from nonꢀspecific compounds. The result was shown in Fig.
S5.
In vitro ULK1 kinase assay
The kinase assay of ULK1 was performed according to previous report.45,46 To assay
ULK1 kinase activity, Flagꢀtagged ULK1WT, ULK1K50A, ULK1L53A or ULK1Y89A mutants of
expressed in 293T cells was immunoprecipitated by antiꢀFlag antibody, and then
incubated with 200 ng purified GSTꢀtagged mATG13 at 37°C for 20 minutes in a kinase
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reaction containing 20 mM HEPES (pH 7.5), 20 mM MgCl 2 , 20 mM NaF, 0.05 mM DTT,
and 40 ꢁM ATP at a final volume of 20 ꢁl. The reaction was stopped by the addition of
sample buffer, boiled and analyzed by Western blot with pꢀmATG13 (ser318) antibody.
Surface Plasmon Resonance (SPR) analysis
The binding capacity of LYNꢀ1604 with ULK1 and ULK1 mutations (K50A, L53A,
Y89A) were assessed using a SPR analysis as the reference’s description.47 The
measurements were performed on a Biacore S51 device (GE Healthcare, Uppsala). For
immobilization, PBS with 0.05% Tween 20, pH 7.4, was used as a background bu er. The
standard amine coupling procedure was applied. A series S Sensor Chip CM5 was
activated with a mixture of 0.1 M NHS (Nꢀhydroxysuccinimide) and 0.4 M EDC
(1ꢀethylꢀ3ꢀ(3ꢀ(dimethylamino)propyl) carbodiimideꢀhydrochloride) at a flow rate of 10 ꢁL
minꢀ1 for 10 min. Then, a 50 ꢁg/mL solution of hCAII in 10 mM sodium acetate (pH 5.2)
with 30 ꢁM ethoxzolamide was injected for 2 min followed by an injection of 1 M
ethanolamine hydrochloride (pH 8.5) for 7 min. For measuring the compound, PBS with
0.05% Tween 20 and 2% DMSO, pH 7.4, was used as the running bu er. The compound
was injected at a flow rate of 30 ꢁL minꢀ1 for 120 s. Then, the dissociation was monitored
for 360 s and the dissociation constant at equilibrium (KD) was evaluated with the Biacore
S51 evaluation software (version: 1.2.1).
Cell viability assay
Cells were dispensed in 96ꢀwell plates at a density of 5 × 104 cells/ml. After 24 h
incubation, cells were treated with different concentrations of compounds for the indicated
time periods. Cell viability was measured by the MTT assay.
Autophagy and apoptosis assays
For autophagy assay, MDAꢀMBꢀ231 cells were transfected with GFP/mRFPꢀLC3
(Kindly provided by Prof. Canhua Huang, Sichuan University) or stained with MDC (0.05
mM) then observed under a fluorescence microscope. MDC positive ratio (here referring
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to autophagic ratio) was measured by FACScan flow cytometry (Becton Dickinson,
Franklin Lakes, NJ). For apoptosis assay, MDAꢀMBꢀ231 cells were stained with Hoechst
33258 in the dark at 37°C for 30min, then observed under a fluorescence microscope.
Immunofluorescence analysis
For immunofluorescence staining, nonspecific antibody binding was blocked by
incubating with PBS containing 1.5% goat serum. The MDAꢀMBꢀ231 cells were
sequentially incubated, starting with pꢀULK1 antibody (1:200) and LC3B antibody (1:200)
diluted in PBS containing 1% BSA incubated overnight at 4°C, followed by addition of
fluorescentꢀlabeled secondary antibodies (TRITC, ab6718; FITC, ab6717) for 1 h at room
temperature.
Western blot
Cells or tumor tissues were treated with LYNꢀ1604 for indicated times. Both adherent
and floating cells were collected. Western blot analysis was carried out. Briefly, the cell
pellets were resuspended with lysis buffer consisting of Hepes 50 mM pH 7.4,
TritonꢀXꢀ100 1%, sodium orthovanada 2 mM, sodium fluoride 100 mM, edetic acid 1 mM,
PMSF 1 mM, aprotinin (Sigma, MO, USA) 10 mg/L and leupeptin (Sigma) 10 mg/L and
lysed at 4°C for 1 h. After 12,000 rpm centrifugation for 15 min, the protein content of
supernatant was determined by the Bioꢀ Rad DC protein assay (BioꢀRad Laboratories,
Hercules, CA, USA). Equal amounts of the total protein were separated by 10ꢀ15%
SDSꢀPAGE and transferred to PVDF membranes, the membranes were soaked in
blocking buffer (5% skimmed milk or BSA). Proteins were detected using primary
antibodies, followed by HRPꢀconjugated secondary antibody and visualized by using ECL
as the HRP substrate. Quantification of immunoblot was performed by Quantity One 4.4.
siRNA transfection
Cells were transfected with ULK1, ATG5 and control siRNAs at 100nM final
concentration using Lipofectamine 2000 reagent (Invitrogen) according to the
manufacturer’s instructions. The transfected cells were used for subsequent experiments
29