Clusterin, a Novel DEC1 Target, Modulates DNA Damage ......modulates the sensitivity of the DNA...

Cell Death and Survival

Clusterin, a Novel DEC1 Target, Modulates DNADamage–Mediated Cell DeathXin Ming1, Chenyi Bao1, Tao Hong1, Ying Yang1, Xinbin Chen2, Yong-Sam Jung1,and Yingjuan Qian1

Abstract

Differentiated embryonic chondrocyte expressed gene 1(DEC1, also known as Sharp2/Stra13/BHLHE40) is a basichelix–loop–helix transcription factor that plays an impor-tant role in circadian rhythms, cell proliferation, apoptosis,cellular senescence, hypoxia response, and epithelial-to-mesenchymal transition of tumor cells. Secretory clusterin(sCLU) is a cytoprotective protein that guards against gen-otoxic stresses. Here, clusterin (CLU) was identified as anovel target gene of DEC1 and suppresses DNA damage–induced cell death in tumor cells. Mechanistically, based onchromatin immunoprecipitation and luciferase assays,DEC1 binds to and activates the promoter of the CLU gene.DEC1 and DNA-damaging agents induce sCLU expression,whereas DEC1 knockdown decreases the expression of sCLU

upon DNA damage. Moreover, the data demonstrate thatDEC1 inhibits, whereas sCLU knockdown enhances, DNAdamage–induced cell death in MCF7 breast cancer cells.Given that DEC1 and sCLU are frequently overexpressedin breast cancers, these data provide mechanistic insight intoDEC1 as a prosurvival factor by upregulating sCLU to reducethe DNA damage–induced apoptotic response. Together,this study reveals sCLU as a novel target of DEC1 whichmodulates the sensitivity of the DNA damage response.

Implications: DEC1 and sCLU are frequently overexpressedin breast cancer, and targeting the sCLU-mediated cytoprotec-tive signaling pathway may be a novel therapeutic approach.Mol Cancer Res; 16(11); 1641–51. �2018 AACR.

IntroductionConventional cancer therapies are based on surgery, radio-

therapy, and chemotherapy, but resistance to anticancer agentshas become one of the primary impediments to effectivecancer therapy (1). Understanding intracellular processesinduced by anticancer drugs that lead to resistance especiallysignal transduction pathways that precede gene expressioncould improve cancer therapeutics. Although there are manyfactors regarding multiple resistance that are still unknown,studies show that it may be mediated by different mechanisms,one of which is closely related to the expression of the clusterin(CLU) protein (2, 3).

The CLU gene is a highly conserved gene during evolutionand a single nine-exon gene, located on human chromosome 8(8p21-p12). However, CLU expression is complex, appearingas different cell compartments and widely distributed amongdifferent species (3–5). CLU has been implicated in multiple

biological functions for tumorigenesis, including apoptosis,DNA repair, cell adhesion, membrane recycling, and immuneregulation (4, 6, 7); however, its mechanism of action remainselusive. Two distinct promoters have been identified surround-ing the transcription start sites (TSS), P1 and P2. The canonicalCLU promoter P1 is located upstream of the exon I, drives theexpression of CLU isoform 1, which encodes the functionalCLU protein, and has a conventional TATA box element plussome potential cis-regulatory elements including activatingprotein-1 (AP1), activating protein-2 (AP2), specificity protein1 (SP1) motifs, and a sequence with high homology to the heat-shock response element (HSE; ref. 8). Researchers havereported several transcription factors contribute to the regula-tion of CLU gene. For example, CLU gene contains a Myb-binding site, which interacts with Myb-like protein 2 (B-MYB)and mediates B-MYB–dependent transactivation of the CLUpromoter P1 (9). Similarly, Y-box binding protein-1 (YB-1) canalso directly bind P1 and promote CLU expression uponendoplasmic reticulum (ER) stress (10). Twist-related protein1 (TWIST1) binds P1 and activates CLU expression followingtransforming growth factor-b or insulin growth factor-1 stim-ulation (11, 12). P2 is a TATA-less, GC-rich promoter, locateddownstream of P1 and surrounding TSS2, which functions as aweaker promoter than P1 (13, 14). Sterol regulatory elementbinding protein-1c (SREBP-1c) was recruited to CLU P2 prox-imal region by glucose and stimulated CLU expression (15).

There are two major isoforms of CLU in human cells, thenonglycosylated prodeath nuclear form (nCLU) and the highlyglycosylated secretory protein (sCLU), also known as apolipo-protein J (ApoJ), complement lysis inhibitor (CLI), sulfatedglycoprotein 2 (SGP-2; ref. 16), testosterone-repressed prostate

1MOE Joint International Research Laboratory of Animal Health and Food Safety,College of VeterinaryMedicine, Nanjing Agricultural University, Nanjing, JiangsuProvince, China. 2The Comparative Oncology Laboratory, Schools of VeterinaryMedicine and Medicine, University of California at Davis, Davis, California.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Yingjuan Qian, Nanjing Agricultural University, 1Weigang, Nanjing, Jiangsu Province 210095, China. Phone: 86-25-8439-9102;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0070

�2018 American Association for Cancer Research.

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message 2 (TRPM-2), or secreted protein 40,40 (SP-40,40;refs. 5, 17), with a cytoprotective role after the occurrenceof various genotoxic stresses (3, 7, 18). The sCLU isoform is awell-documented secreted heterodimeric protein that representsthe predominant translation product of the human gene. Pre-cursor secretory CLU (psCLU) is a 449-amino acid polypeptideand is synthesized by translation from the initial AUG codonof full-length CLU mRNA at exon II, which produces a signalpeptide targeting the precursor to the ER. The psCLU (60-kDa) isproteolytically cleaved in ER to remove the signal peptide and togenerate two distinct peptides (a and b) held together by fivedisulfide bonds. The mature sCLU (80-kDa) protein is ultimatelysecreted from cells as a heavily glycosylated and heterodimericform with two 40-kDa a- and b-subunits. In contrast, the nCLUisoform is synthesized from an alternative splicing of exons I andIII (19), consequently resulting in lacking the N-terminal signalsequence of sCLU in exon II, does not undergo cleavage andglycosylation, and exclusively localizes in the nucleus whenexposure to cytotoxic stress (20, 21).

Due to the different subcellular localization and struc-ture of CLU isoforms, nCLU and sCLU exert distinct functionin determining the cell fate upon various stress signals.Exposure to different DNA-damaging agents such as paclitaxel,doxorubicin, or cisplatin usually results in an increased sCLUexpression, which has been related to an improved tumor cellsurvival and an enhanced treatment resistance (22–24). Incontrast, the inhibition of sCLU expression by antisense oli-gonucleotides (ASO) or siRNA has shown to enhance thechemosensitivities of various cell lines (25, 26), suggestingthat sCLU expression was a prominent resistance factor incancer cells. On the contrary, overexpression of nCLU has beenreported to induce G2–M phase arrest and caspase 3–indepen-dent apoptosis and lethality in MCF7 cells (27–29), whereassCLU overexpression does not affect the survival of MCF7 cells.This is due to the interaction between sCLU and activated Bax,thereby inhibiting cytochrome c release and apoptosis (30),whereas nCLU functions as a prosurvival factor through seques-tering Bcl-XL via a putative BH3 domain, releasing Bax, andpromoting apoptosis (31). These observations suggest thatsCLU has prosurvival activity, whereas nCLU has proapoptoticactivity. Therefore, the relative balance between sCLU andnCLU might control cell fate and play a key role in tumori-genesis. However, the molecular mechanism regulating theirexpression remains obscure, and the upstream regulators arestill need to be discovered.

DEC1/BHLHE40 (differentiated embryo-chondrocyte ex-pressed gene 1), also called Stra13 (stimulated with retinoicacid 13) in mouse and Sharp2 (enhancer of split and hairyrelated protein 2) in rat, belongs to the basic helix–loop–helixprotein family (32). DEC1 functions as a transcription repres-sor by directly binding to class B E-boxes of its target genes, suchas DEC2 (33) or as a transcription activator by binding to theSP1 site of its target genes, such as SURVIVIN or tumor protein73 (TAp73, p73 isoform containing transactivation domain;refs. 34, 35). Functionally, DEC1 is a critical regulator ofcircadian rhythm and plays an important role in a variety ofcellular processes such as senescence, apoptosis, differentiation,and epithelial-to-mesenchymal transition in response to vari-ous stimuli (36–39). Interestingly, we found that DEC1 mod-ulates p53-dependent cell survival through MIC-1 in responseto genotoxic stresses (40). In this study, we identified sCLU as a

novel downstream target of DEC1 that is also implicated inpromoting cell survival upon treatment with genotoxic agents.

Materials and MethodsPlasmids

Wild-type HA-tagged DEC1, DEC1-M (lacks residue 53–65in the DNA-binding domain), DEC1-DHLH (lacks HLHdomain), or DEC1-R58P (has a point mutation from arginineto proline at codon 58) expression vectors in pcDNA3 weredescribed previously (40). For tetracycline-inducible cell linegeneration, HA-tagged DEC1, wild-type DEC1 and DEC1-R58Pin pcDNA4/TO were constructed previously (36). Besides,pBabe-H1-siDEC1 was also constructed before for DEC1-inducible knockdown cell line generation (36). To generate aluciferase reporter under the control of the CLU promoter(nt –2000 to þ55, NM_001831), genomic DNA fragment wasamplified from MCF7 cells with the forward primer, CLU-KpnI-2000 (50-GAAGGTACCCATGGCAGGTAGTGAGCTCCCTG-30),and reverse primer, CLU-HindIII-55 (50-CACAAGCTTTTTGG-GGCTGGCTGCAAACCTGCAT-30) and ligated into pGL3-basicvector. Luciferase reporter under control of the SURVIVINpromoter was described previously (34).

Cell linesMCF7, RKO, and H1299 were cultured in DMEM supple-

mentedwith 10%FBS at 37�Cwith 5%CO2.MCF7-DEC1 (clones6 and 16) and RKO-DEC1 (clone 7) were generated to expresswild-type DEC1 upon tetracycline induction, whereas MCF7-siDEC1 (clones 1 and 34) were generated to express DEC1 siRNAupon tetracycline induction. Due to different integration sites,MCF7-DEC1 (clone 6) expresses higher level of DEC1 and lesssensitive than clone 16 to camptothecin (CPT)-induced celldeath. Similarly, the knockdown efficiency in MCF7-siDEC1(Clone 1) cell line was better than that of MCF7-siDEC1 (clone34) cell line. MCF7-DEC1-R58P (clone 2) and MCF7-HA-DEC1(clone 2) were generated to express DEC1-R58P and HA-taggedDEC1 when treated with tetracycline. All cell lines were used aspreviously described (36, 40).

Western blot analysisWhole cell extracts were prepared with 2 � SDS sample buffer

and boiled for 10 minutes at 96�C. The antibody againstDEC1 was purchased from Bethyl laboratories (A300-649A).Anti–clusterin-a (B5, sc-5289) and anti-p53 (FL393, sc-6243)were purchased from Santa Cruz Biotechnology. Anti-actin waspurchased from Sigma. Goat anti-mouse IgG Fc HRP and goatanti-rabbit IgG Fc HRP were purchased from Millipore.

RNA isolation and RT-PCRTotal RNA was extracted using TRIzol reagent (Sigma) accord-

ing to themanufacturer's protocol. The cDNAwas prepared usingthe iScript Reverse Transcription Supermix Kit (Bio-Rad). The PCRwas carried out with 2� Taq Master Mix (Vazyme) with thefollowing conditions: denaturation at 94�C for 5 minutes, fol-lowed by 25 cycles of denaturation at 94�C for 30 seconds,annealing at 55�C for 30 seconds, and extension at 72�C for 30seconds. Specific primers were designed to target DEC1, CLU,sCLU, nCLU, and Actin (Supplementary Table S1) using thePrimer Premier and checked with BLAST before use to avoidamplification of genomic DNA or pseudogenes.

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siRNADEC1 siRNA-512, DEC1 siRNA-342, DEC1 siRNA-802, and

sCLU siRNA and scramble RNA were purchased from Biotend.The sequence of siRNA oligomers against the sCLU mRNAleader ER signal peptide (si-sCLU) was described previously(41). The nuclear CLU (nCLU) isoform is synthesized froman alternative splicing of exons I and III, therefore lacking theN-terminal signal sequence of sCLU in exon II. The sequenceof this siRNA is targeting the exon II which is sCLU exclusive.The sequences of siRNAs used in this study were described inSupplementary Table S2.

Luciferase reporter assayThe dual-luciferase assay was performed in triplicate according

to the manufacturer's instruction (Promega). To determinewhether DEC1 activates CLU promoter, 0.25 mg of a luciferasereporter, 0.25 mg of empty pcDNA3, or pcDNA3 that expressesDEC1orDEC1-M and3ng of an internal controlRenilla luciferaseassay vector pRL-CMV (Promega) were transfected into p53-nullH1299 cells byusing the lip2000 transfection reagent according tothe manufacturer's instruction (Invitrogen). Cells were seeded at2 � 104 per well in 24-well plates 24 hours before transfection.Luciferase activity was measured with the dual luciferase Kit andGloMax-96Microplate Luminometer (Promega). The fold changein relative luciferase activity is a product of the luciferase activityinduced by DEC1 protein divided by that induced by an emptypcDNA3 vector.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assay was per-

formed as previously described (42). The binding of DEC1 toCLU promoter was detected with primers, CLU-CHIP-287F: 50-GGGTCAAGAGAAGTGGCGCTTGTG-30, CLU-CHIP-90R: 50-AGCTGTGCTGGCTCCAGGAGAGA-30. Primers that were usedto amplify the DEC2 and GAPDH promoters were used aspreviously described (36, 43).

Colony formation assayMCF7-DEC1-16 and MCF7-siDEC1-1 cells were seeded at 3 �

105 per well in 6-cm dishes 24 hours before transfection, then50 nmol/L scramble or siRNA-sCLU was transfected with Meta-fectene (Biontex) according to the manufacturer's instruction. At24or 48hours after transfection, transfected cellsMCF7-siDEC1-1or MCF7-DEC1-16 were induced with or without tetracycline toexpress DEC1 siRNA or DEC1 for 48 or 12 hours, and then cellswere split to 6-well plate at 4,000 per well uninduced or inducedto express DEC1 siRNA or DEC1 for 12 hours, and followed byuntreated or treated with CPT (250 nmol/L) for 9 hours. Forcontrols, cellswere seeded at 1,000perwell andmock treatedwithDMSO. The assay was performed as described (37).

FACS and DNA histogram analysisTo measure the effect of sCLU on DNA damage–induced cell

death, cells were seeded at 3 � 105 per well in 6-cm dishes24 hours before transfection, then 50 nmol/L scramble orsiRNA-sCLU was transfected with Metafectene (Biontex) accord-ing to the manufacturer's instruction. At 24 hours (MCF7-siDEC1-1) or 48 hours (MCF7-DEC1-16) after transfection,transfected cells were split into 12-well plate with or withouttetracycline to induce DEC1 or DEC1 siRNA expression for 12 or48 hours, and then treated or untreated with camptothecin

(CPT) at 1 mmol/L for 30 hours. Both floating and attached cellswere collected and fixed in precooled (–20�C) ethanol (70%)overnight followed by propidium iodide staining. Samples wereanalyzed by FACS Calibur flow cytometer (BD Biosciences).

ResultsClusterin expression can be upregulated by DEC1 induction

To identify novel genes regulated by DEC1, cDNAmicroarrayassay was performed using MCF7 cells uninduced or induced toexpress DEC1 by tetracycline. We found that CLU mRNA waselevated by DEC1. To confirm this, RT-PCR was performed withprimers (CLU-732F and CLU-1140R) designed to amplify totalCLU. We found that total CLU mRNA was increased whenDEC1 was induced for 24 and 48 hours in both MCF7-DEC1-6and 16 cells (Fig. 1A, CLU panel). Next, because nCLU is lackingexon II compared with sCLU, primers targeting from exon I toexon IV can be used to distinguish sCLU and nCLU (19).Therefore, the size of amplified nCLU would be 126-bp shorterthan sCLU when CLU-Ex1-F and CLU-Ex4-R were used for PCR.We found that sCLU was increased upon DEC1 induction(Fig. 1A, sCLU panel). However, nCLU cannot be detected dueto the endogenous level of nCLU was significantly lower thansCLU. Therefore, we focused on studying the regulation ofsCLU by DEC1. We found both psCLU and mature sCLU pro-teins in MCF7 cells were elevated upon induction of wild-typeDEC1 (Fig. 1B) but not mutant DEC1-R58P (Fig. 1C). To ruleout cell type–specific effects, DEC1 was induced by tetracyclinein RKO-DEC1-7 cells. Consistent with the above observation,the levels of psCLU and mature sCLU were increased (Fig. 1D).A previous report has shown that p53 can suppress the level ofsCLU upon ionization-induced DNA damage (44). In order torule out the effect of p53 on sCLU, p53-null H1299 cells weretransiently transfected with HA-tagged DEC1 (wild-type),DEC1-M, DEC1-DHLH, or DEC1-R58P (Fig. 1E). Similarly,psCLU was increased in H1299 upon DEC1-WT and DEC1-DHLH overexpression, but not DEC1-M or DEC1-R58P(Fig. 1F).

Conversely, DEC1 knockdown by tetracycline decreased totalCLU and sCLU mRNA and precursor sCLU protein levels inMCF7 cells (Fig. 2A and B). Consistent with the above observa-tions, psCLU can also be downregulated by transient transfec-tion with DEC1 siRNA for 72 hours in both MCF7 and H1299cells (Fig. 2C and D).

DEC1 binds to the CLU promoter region and regulates CLUtranscription

DEC1 has been reported to act as an activator by binding tothe SP1 sites in its target genes, such as SURVIVIN, DNp63, andTAp73 (34, 35, 37). Thus, if CLU is a direct target of DEC1, oneor more SP1 sites should exist in the CLU gene. To test this, wesearched for putative SP1 sites within the proximal promoterregion for CLU and found seven possible SP1 sites (Fig. 3A). Todetermine if these SP1 sites are responsive to DEC1, a DNAfragment from the CLU promoter (–2,000 to þ55) was clonedinto pGL3-basic luciferase reporter and designated pGL3-CLU-2000. Next, the luciferase reporter assay was performed, andit showed that the luciferase activity was enhanced by DEC1but not DEC-M (Fig. 3B). As a control, the SURVIVIN promoterwas also activated by DEC1 but not by DEC1-M (Fig. 3C),consistent with a previous report (34).

DEC1 Regulates sCLU-Mediated Cytoprotective Activity

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Figure 1.

DEC1 overexpression upregulates Clusterin. A, The mRNA levels of total CLU and sCLU are induced by DEC1 overexpression. RT-PCR was preparedwith RNAs purified from MCF7 cells that were untreated (–) or treated (þ) with tetracycline (2 mg/mL) to express DEC1 for 24 and 48 hours. B, sCLUexpression is upregulated by DEC1 overexpression in MCF7 cells. Western blot samples were prepared from MCF7 cells that were uninduced or inducedto express DEC1 for 24 hours. C, sCLU expression cannot be upregulated by DEC1-R58P overexpression. Western blot samples were prepared fromMCF7 cells that were uninduced or induced to express DEC1-R58P for 24 hours. D, sCLU expression is increased by DEC1 in RKO cells. Western blotsamples were prepared from RKO cells that were uninduced (–) or induced (þ) to express DEC1 for 12 and 24 hours. E, Schematic presentation of the DEC1and its mutants. B, The basic domain (DNA-binding domain); HLH, helix–loop–helix domain; orange, orange domain; P-rich, proline-rich domain. F, sCLUexpression is increased by DEC1 in p53-null H1299 cells. Western blot samples were prepared from H1299 cells that were transiently transfected withpcDNA3 or pcDNA3-DEC1 (wild-type and DEC1 mutants) for 48 hours.

Figure 2.

DEC1 knockdown decreases the expression of precursor secretory clusterin. A, The mRNA levels of total CLU and sCLU were decreased by DEC1knockdown. RT-PCR was prepared with RNAs purified from MCF7 cells that were untreated (–) or treated (þ) with tetracycline (2 mg/mL) to expressDEC1 siRNA for 72 hours. B, sCLU expression is downregulated by DEC1 knockdown. Western blot samples were prepared from MCF7 cells that wereuninduced (–) or induced (þ) to express DEC1 siRNA for 48 hours. C and D, Western blot samples were prepared from MCF7 and H1299 cells that weretransiently transfected with 50 nmol/L scramble or DEC1 siRNA for 72 hours.

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To further examine whether the DEC1 protein can bind tothe CLU gene in vivo, a ChIP assay was performed with theprimers shown in Fig. 3D. The binding of DEC1 proteins to theDEC2 promoter was chosen as a positive control (Fig. 3E,middle plot). In addition, a region within the GAPDH pro-moter was used as a control for nonspecific binding. We foundthat DEC1 was able to associate with the CLU promoter, andthe precipitated CLU gene fragments were significantlyincreased upon DEC1 overexpression. As expected, the DEC2promoter was recognized by DEC1, but not GAPDH (Fig. 3E).Taken together, these data suggest that CLU is a direct targetgene of DEC1.

DEC1 is partially required for DNA damage–induced sCLUsCLU expression is induced by various extracellular stresses

in MCF7 cells, including ionizing radiation, ultraviolet radia-

tion, topoisomerase inhibitors, microtubule stabilizers, as wellas other reagents that do not cause direct damage to DNA (44).We tested whether sCLU is regulated by DNA damage in cellstreated with chemotherapy drugs, CPT, or etoposide (ETP).Both CPT (an inhibitor of topoisomerase I) and ETP (aninhibitor of topoisomerase II) can induce DNA double-strandbreaks. We found that the expression level of sCLU wasincreased along with the upregulation of DEC1 upon CPT andETP treatment in both MCF7 and H1299 cells (Fig. 4A and B).As expected, DEC1 and p53 were increased upon CPT and ETPtreatment in a time-dependent manner. Next, we wanted toexamine whether DEC1 is required for inducing sCLU uponDNA damage. To address this, DEC1 was inducibly knockeddown by tetracycline in MCF7-siDEC1 cells. We found thatDNA damage–induced upregulation of sCLU was significantlydiminished by DEC1 knockdown (Fig. 4C). Similar results were

Figure 3.

DEC1 activates the clusterinpromoter activity. A, Schematicpresentation of the luciferasereporter driven by the CLU (nt–2,000 to þ55). The locations ofvarious transcription factor–binding sites were marked bydifferent symbols. B and C,Potential SP1 sites in theCLU/Survivin gene are responsiveto DEC1 but not to mutant DEC1.The luciferase assay wasperformed as described in"Materials and Methods."D, Schematic presentation of theCLU, DEC2, and GAPDH promoterswith location of the potentialDEC1-responsive elements andPCR primers used for ChIP assay.E, DEC1 binds to the CLU promoterin vivo. MCF7 cells that wereuninduced (–) or induced (þ) toexpress HA-tagged DEC1 werecross-linked with formaldehydeand then sonicated. Chromatinwas immunoprecipitated (IP) withanti-HA or a control IgG. Thebinding of DEC1 to the CLU, DEC2,and GAPDH promoters wasmeasured by PCR.






observed when DEC1 was transiently knocked down byDEC1 siRNA in H1299 cells along with scramble siRNA as acontrol (Fig. 4D). Therefore, inhibition of DEC1 can attenuatethe level of sCLU upon DNA damage. Furthermore, we foundthat the activation of sCLU upon CPT treatment was enhancedby DEC1 overexpression in MCF7 cells (Fig. 4E). Consistentwith this, sCLU was also induced by DEC1 not DEC1-Mtransient transfection in H1299 cells (Fig. 4F). These dataindicate that DEC1 is contributing to sCLU upregulation uponDNA damage.

sCLU is partially required for DEC1 to inhibit DNAdamage–induced cell death

sCLU overexpression has been detected in many chemore-sistant cancers, and DEC1 has also been shown to inhibitDNA damage–induced cell death. Here, we examined whetherDEC1 modulates DNA damage–induced cell death through

sCLU. sCLU was transiently knocked down in MCF7-DEC1-16cells that were induced to express DEC1 along with orwithout CPT treatment. As expected, DEC1 was increased bytetracycline, and sCLU was decreased upon siRNA transfection(Fig. 5A). To determine whether sCLU and DEC1 play a rolein growth suppression by CPT treatment, the colony forma-tion assay was performed in MCF7 cells in which DEC1 canbe inducibly expressed with or without sCLU knockdown.We showed that although MCF7 cell proliferation wasdecreased by CPT (Fig. 5B), cell growth can be recovered byectopic expression of DEC1. However, the ability of DEC1to promote colony growth upon CPT treatment can be attenu-ated by sCLU knockdown (Fig. 5B). In addition, DNA histo-gram analysis was performed to measure the percentage ofthe sub-G1 population. We found that the sub-G1 popula-tion of DEC1 ectopic expressed cells was decreased 29.95%compared with treatment of CPT alone (44.4% in CPT-treated

Figure 4.

DEC1 partially contributes to DNAdamage–induced sCLU. A and B, sCLUexpression is upregulated upon CPTand ETP treatment. Western blotsamples were prepared from MCF7and H1299 cells that were treated withCPT (250 nmol/L) or ETP (100 mmol/L)for 0, 6, 12, and 24 hours, respectively.sCLU, DEC1, p53, and actin weredetected by indicated antibodies. C,Western blot samples were preparedfrom MCF7 cells that were uninducedor induced to express DEC1 siRNA for72 hours along with or without CPT(250 nmol/L) treated for 12 or 24hours. D, Western blot samples wereprepared from H1299 cells that weretransiently transfected with scramblesiRNA or DEC1 siRNA for 72 hoursalong with or without CPT(250 nmol/L) treatment for 12 hours. E,Western blot samples were preparedfrom MCF7 cells that were uninducedor induced to express DEC1 for 24 hourand then followed by CPT treatmentfor 24 hours. F, Western blot sampleswere prepared from H1299 cells thatwere transiently transfected withempty vector pcDNA3-KHA, KHA-DEC1, or KHA-DEC1-M to express DEC1or DEC1-M for 24 hours and thenfollowed by CPT (250 nmol/L)treatment for 24 hours.

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vs. 31.1% in CPT with DEC1-treated; Fig. 5C). Transientknockdown of sCLU had no effect, whereas upon treatmentwith CPT, the sub-G1 population was increased comparedwith scramble transfection (44.4% in CPT vs. 51.57% insi-sCLU with CPT-treated). However, the sub-G1 populationof DEC1 overexpression upon CPT treatment was 48.03%,which was only decreased 6.86%, compared with CPTalone, due to the downregulation of sCLU (Fig. 5C). AlthoughMCF7-DEC1 clone 6 is less sensitive than clone 16 to CPT-induced cell death, consistent results can be observed (Sup-plementary Fig. S1A and S1B). Therefore, sCLU contributesto the inhibition of DEC1-mediated cell death upon DNA-damaging agent.

To further confirm this, we also examined the cell viabilityupon CPT treatment when DEC1 was knockdown. Western blotanalysis showed that comparable levels of DEC1 and sCLUprotein were decreased in MCF-siDEC1-1 (Fig. 6A). We foundthat the ability of CPT to suppress colony formation wasadvanced by DEC1 knockdown. However, this sensitizationcannot be further increased by knockdown of both proteins(Fig. 6B). In addition, FACS analysis also showed the sub-G1population was increased from 11.05% to 16.73% by DEC1knockdown upon CPT treatment and kept almost the samelevel when together with sCLU knockdown (Fig. 6C). Takentogether, these data indicated that sCLU was partially requiredfor DEC1 to inhibit DNA damage–induced cell death.

Figure 5.

sCLU contributes to DEC1 inhibition onDNA damage–induced cell death. A,Western blot samples were preparedfrom MCF7-DEC1-16 cells transfectedwith scramble siRNA or sCLU siRNA(50 nmol/L) for 48 hours that werethen uninduced or induced to expressDEC1 for 12 hours. B, (Left) Colonyformation assay was performed withMCF7-DEC1-16 cells transfected withscramble siRNA or sCLU siRNA (50nmol/L) for 48 hours that were thenuntreated (–) or treated (þ) withtetracycline for 12 hours along withmock treatment or CPT (250 nmol/L)treatment for 9 hours, and thenmaintained for 15 days. Colonies werestainedwith 0.02% crystal violet (left).Quantification of colonies (right)presented as the ratio of coloniesformed in cells treated withtetracycline vs. that in control cells(mean� SD; n¼ 3). C,DNA histogramanalysis was performed with MCF7-DEC1-16 cells as in B. The assay isdescribed in "Materials and Methods"(mean � SD; n ¼ 3). Data arerepresentative of three independentexperiments performed in triplicate.






CLU and DEC1 are coexpressed in human breast cancerspecimens

To determine the clinical relevance of CLU and DEC1 expres-sion, we first analyzed the CLU and DEC1 protein expressionin clinical specimens from the Human Protein Atlas (www.proteinatlas.org). DEC1 and CLU were found to be markedlyincreased in breast cancer, with weak expression in normalbreast tissue (Fig. 7A). After searching Garnett cell line database(Compendia Biosciences, www.oncomine.org), we also foundthat the CLU mRNA level in breast cancer is higher than thatin other types of cancers (P ¼ 1.52E–7, fold ¼ 3.040, n ¼ 39;Fig. 7B). Furthermore, we found that both the CLU and DEC1

mRNA levels in a Hodgkin's lymphoma sample were signifi-cantly higher than that in normal lymphocytes (Fig. 7C and D).Taken together, these data suggest that CLU acts as an effector ofDEC1 in the regulation of survival and oncogenesis.

DiscussionIn this study, we found that sCLU is a novel target of DEC1

and contributes to the cytoprotective function of DEC1 uponCPT treatment. Specifically, both CLUmRNA and sCLU proteincan be upregulated by overexpressed DEC1. Conversely, lackof DEC1 negatively affects sCLU levels. We also found that

Figure 6.

Lack of sCLUpromotes cell death uponDNA-damaging agent.A,Western blotsamples were prepared from MCF7-siDEC1-1 cells transfected withscramble siRNA or sCLU siRNA (50nmol/L) for 24 hours that were thenuninduced or induced to express DEC1siRNA for 48 hours. B, (Left) Colonyformation assay was performed withMCF7-siDEC1-1 cells transfected withscramble siRNA or sCLU siRNA (50nmol/L) for 24 hours that were thenuntreated (�) or treated (þ) withtetracycline for 48 hours along withmock treatment or treatment with CPT(250 nmol/L) for 9 hours, and thenmaintained for 15 days. Colonies werestained with 0.02% crystal violet (left).Quantification of colonies (right)presented as the ratio of coloniesformed in cells treated withtetracycline vs. that in control cells(mean� SD; n¼ 3). C, DNA histogramanalysis was performed with MCF7-siDEC1-1 cells as in B. The assay isdescribed in "Materials and Methods"(mean � SD; n ¼ 3). Data arerepresentative of three independentexperiments performed in triplicate.

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sCLU is induced by DEC1 in a p53-independent manner.Moreover, we identified potential DEC1 response elements inthe promoter of CLU, and CLU promoter can be activated andbound by DEC1. All of these features suggest that CLU is a bonafide DEC1 target. Next, we examined the possibility that DEC1requires the cooperation of sCLU to fulfill its functions, and wefound that sCLU contributes to DEC1-mediated antiapoptoticactivity, because depletion of sCLU partially disrupts the pro-tective effect of DEC1 overexpression. Consistently, MCF7 cellsare sensitized to chemotherapy when DEC1 or sCLU is knockeddown. In addition, knockdown does not cause further sensiti-zation, indicating that DEC1 promotes survival via regulatingsCLU (Fig. 6B and C). Therefore, we hypothesize that sCLU islikely to be a functional mediator of DEC1 cytoprotectivefunction.

DEC1 and sCLU are both stress-activated survival factors func-tionally associated with anticancer treatment resistance. In breastcancer, both are correlated with tumor grade and progressionfrom primary carcinoma to invasive breast cancer (45, 46). Inaddition, both DEC1 and sCLU are linked to cytotoxic resistanceinduced by CPT and ETP (29, 40, 47, 48). However, the mech-

anism of sCLU induction upon genotoxic stress is still unknown.Based on previous studies, it was determined that sCLU can beregulated by YB-1 through binding to E-box region followingER stress, and the promoter region of CLU is highly conserved(10, 49). Although YB-1 is a transcription factor for CLU induc-tion, YB-1 overexpression cannot reverse decreases in paclitaxel-induced apoptosis following CLU depletion (10). This led usto investigate the relationship between DEC1 and sCLU. In thisstudy, we identified CLU as a novel downstream target ofDEC1 in regulating DNA damage–induced cell death. DEC1 caninduce sCLU protein expression under both normal and stressedconditions.However, knockdownofDEC1 canpartially attenuatesCLU expression at early time points upon DNA damage treat-ment. This indicates that many other factors might be involvedinCLU induction in response toDNAdamage agents, such as YB-1(10). Therefore, we can postulate that DEC1 is required but notessential for sCLU induction.

In order to develop novel therapeutic strategies for cancer,many approaches are used to unravel the activation mechanismof sCLU. Inhibiting the expression of specific prosurvivalgenes can be performed by ASOs, and CLU is being targeted

Figure 7.

CLU and DEC1 show similar expression pattern in human cancers. A, The expression patterns of DEC1 and CLU protein in breast cancer specimenscompared with normal breast. B, The mRNA level of CLU is upregulated in breast cancer. 1, breast cancer (n ¼ 39); 2, colorectal cancer (n ¼ 40);3, head and neck cancer (n ¼ 33); 4, leukemia (n ¼ 66); 5, melanoma (n ¼ 41); 6, myeloma (n ¼ 9). C and D, The mRNA levels of DEC1 and CLU areupregulated in Hodgkin's lymphoma specimens compared with normal. 1, centroblast (n ¼ 5); 2, memory B lymphocyte (n ¼ 5); 3, na€�ve pregerminalcenter B-lymphocyte (n ¼ 5); 4, plasma cell (n ¼ 5); 5, small cleaved follicle center cell (n ¼ 5); 6, Hodgkin's lymphoma (n ¼ 12). These data aretaken from Oncomine database (https://www.oncomine.org).





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by using Custirsen (OGX-011). Previous reports showed thatCustirsen enhances the toxicity of the antitumor agent taxane(50–52). Custirsen-mediated CLU downregulation can sensi-tize breast and prostate cancer cells to paclitaxel and docetaxel,respectively (53, 54). Therefore, CLU could be a therapeutictarget in cancer.

In conclusion, we have shown that ectopic overexpression ofDEC1 can inhibit cell death, whereas knockdown of DEC1 pro-motes cell death in MCF7 cells. sCLU as a downstream target ofDEC1 contributes to this process. However, sCLU knockdown canonly partially attenuate DEC1 inhibition on CPT-induced celldeath, implying other targets are involved in this process. Morestudies are also required to clarify the dual pro- and antiapoptoticroles of CLU and its signaling pathway, which may provide aninsight into possible therapeutic as well as diagnostic uses.Because CLU is a multifunctional and extracellular chaperone,we hypothesize that, in DEC1-overexpressed tumor cells, thebalance of CLU isoforms in response to DNA damage likelycontributes to conferring therapeutic resistance. Thus, futurestudies to analyze CLU isoforms in human tumors may provideadditional data regarding the role of CLU in other cancers inaddition to its role in breast cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: X. Ming, Y.-S. Jung, Y. QianDevelopment of methodology: X. Ming, Y.-S. Jung, Y. QianAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. MingAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Ming, C. Bao, X. Chen, Y.-S. Jung, Y. QianWriting, review, and/or revision of themanuscript:X.Ming, Y.-S. Jung, Y.QianAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Hong, Y. Yang, Y.-S. Jung, Y. QianStudy supervision: Y.-S. Jung, Y. Qian

AcknowledgmentsThe authors thank Professor Ruqian Zhao for the help with luciferase assay

and FACS assay, and all of our lab members for the help with the experiments.This work was supported by the National Natural Science Foundation of

China (Grant No. 31472218), the Fundamental Research Funds for theCentral Universities (KYHW201702), and the Priority Academic ProgramDevelopment of Jiangsu Higher Education Institutions (PAPD).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 23, 2018; revised April 28, 2018; accepted June 20, 2018;published first July 12, 2018.

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2018;16:1641-1651. Published OnlineFirst July 12, 2018.Mol Cancer Res Xin Ming, Chenyi Bao, Tao Hong, et al. Cell Death

Mediated−Clusterin, a Novel DEC1 Target, Modulates DNA Damage

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