d Regulates Death Receptor 5 Expression Induced by PS-341 ... · PS-341 through ATF4–ATF3/CHOP Axis in Human Lung Cancer Cells Linyan Xu, Ling Su, and Xiangguo Liu ... (p19/p17)

Therapeutic Discovery

PKCd Regulates Death Receptor 5 Expression Induced byPS-341 through ATF4–ATF3/CHOP Axis in Human LungCancer Cells

Linyan Xu, Ling Su, and Xiangguo Liu

AbstractPS-341 (bortezomib), a proteasome inhibitor, has been approved for the treatment ofmultiplemyeloma.Our

previous work has shown that PS-341 induces death receptor 5 (DR5)–dependent apoptosis and enhances

the TNF-related apoptosis-inducing ligand–induced apoptosis in human non–small cell lung cancer cells.

However, the definite mechanism remains undefined. In the present study, we reveal that PKCd and RSK2

mediate PS-341–induced DR5 upregulation, involving coactivation of endoplasmic reticulum (ER) stress. We

discovered that PS-341 activated ER stress through elevating the expression of BiP, p-eIF2a, IRE1a, ATF4,

ATF3, and CCAAT/enhancer-binding protein homologous protein (CHOP). Further study showed that DR5

upregulation was dependent on ATF4, ATF3, and CHOP expression. Silencing either one of the ATF4, ATF3,

and CHOP expression decreased DR5 upregulation and subsequent apoptosis. We determined that ATF4

regulated ATF3 and CHOP expression. Thereafter, ATF3 and CHOP formed a complex and regulated

DR5 expression. In addition, we discovered that the phosphorylation of extracellular signal-regulated

kinase (ERK) 1/2 and RSK2 were elevated after PS-341 treatment and inhibition of their phosphorylation

using MAP-ERK kinase 1/2 inhibitor decreased the DR5 level, indicating that ERK/RSK2 signaling is

involved in DR5 upregulation. Furthermore, we detected the cleavage of PKCd, and the blockage of PKCdexpression cut down DR5 upregulation and apoptosis. Importantly, knockdown of PKCd expression

decreased the induction of ER stress and the phosphorylation of ERK1/2 and RSK2, suggesting that PKCdregulates DR5 expression through ERK/RSK2 signaling and ATF4–CHOP/ATF3 axis. Collectively, we

show that PS-341 induces PKCd-dependent DR5 expression through activation of ERK/RSK2 and ER stress

signaling pathway. Mol Cancer Ther; 11(10); 2174–82. �2012 AACR.

IntroductionDeath receptor 5 (DR5, also named Apo2), one of the

TNF-related apoptosis-inducing ligand (TRAIL) recep-tors, belongs to the TNF receptor superfamily (1). It islocated on the cell surface, and becomes trimerized onbinding to its ligand TRAIL and recruits adapter pro-teins, such as Fas-associated death domain, then formsthe death inducing signaling complex, and eventuallyactivates caspase cascades (1). Nowadays, many studieshave shown that inducing the expression of DR5

contributes to certain cancer therapeutic agents-induced apoptosis and enhances TRAIL-induced apo-ptotic pathway (1).

CCAAT/enhancer-binding protein homologous pro-tein (CHOP), a downstream component of endoplasmicreticulum (ER) stress pathways, can be mediated bypancreatic ER kinase-like ER kinase-, activating transcrip-tion factor 6 (ATF6)-, and inositol-requiring enzyme 1a(IRE1a)–induced signal transduction pathways (2, 3).Many studies have documented that CHOP promotesapoptosis through upregulation of DR5 expressioncaused by many agents, such as lonafarnib (4), MG132(5), CDDO-Me (6) in a variety of cancer cells. Activatingtranscription factor 4 (ATF4) and ATF3 are stress respon-sive proteins, which can be induced by ER stress (7, 8).Some earlier studies have found that ATF3 can suppressCHOP gene transcription (9), whereas CHOP inhibitsATF3 protein function (10). However, some data showthat ATF3 can promote CHOP expression (11).

The protein kinase C (PKC) family is importantin regulation of cell proliferation and apoptosis. PKCdis generally considered to be a proapoptotic protein(12, 13). Studies have revealed that PKCd can becleaved between the regulatory domain and catalytic

Authors' Affiliation: Key Laboratory for Experimental Teratology of theMinistry of Education and School of Life Sciences, Shandong University,Jinan, Shandong, China

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

L. Su and L. Xu contributed equally to this work.

Corresponding Author: Xiangguo Liu, Shandong University School of LifeSciences, Room 103, South Building, 27 Shanda South Road, Jinan,Shandong 250100, China. Phone: 86-531-88364330; Fax: 86-531-88565610; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0602

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(10) October 20122174

on October 28, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 30, 2012; DOI: 10.1158/1535-7163.MCT-12-0602

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domain when cells are exposed to diverse stimuli.Consequently, a 41 kDa fragment is generated, whichhas constitutive bioactivity and is responsible for apo-ptosis (14–16).Many preclinical experiments have shown that the

proteasome inhibitor PS-341 (Fig. 1A; ref. 17) alone orcombination with other agents have antitumor effectin various cancers, including lung cancer (17, 18). How-ever, the molecular mechanism underlying PS-341mediated apoptosis is not very clear now. Our previousstudies have shown that DR5 expression contributes toPS-341–mediated apoptosis in human non–small celllung cancer (NSCLC) cells (18). To further characterizethe mechanism by which PS-341 upregulates DR5expression in NSCLC cells, we explored the molecularmechanism of PS-341 on the induction of ER stress andPKCd.

Materials and MethodsReagents

The powder of pure PS-341 was purchased from LCLaboratories. DR5 antibody was purchased from ProSci.Caspase-3 antibody was purchased from Imgenex.Caspase-8, caspase-9, PARP, p-ERK, p-RSK2, BiP, IRE1a,p-eIF2a, and eIF2a antibodies were purchased from CellSignaling Technology. CHOP, ATF3, ATF4, and PKCdantibodies were purchased from Santa Cruz.

Cell cultureThe humanNSCLC cell lines A549, H1792, H460, H157,

and Calu-1 were originally obtained from the AmericanType Culture Collection. A549 and H1792 cell lines wereauthenticated in Microread Gene Technology by shorttandem repeat analysis on March 19, 2012. H460, H157,and Calu-1 have not been re-authenticated. The cells were

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Figure 1. PS-341 (A) enhancesCHOPexpression in adose-dependent and a time-dependentmanner (B), and inhibitingCHOPexpressionby siRNAdecreasesPS-341–induced DR5 upregulation and apoptosis (C and D). A, the chemical structure of PS-341. B, the indicated lung cancer cell lines were treatedwith 0, 100, 200 nmol/L PS-341 for 24 hours or treated with 50 nmol/L PS-341 for the indicated time. H460, A549 (C), and Calu-1 (C and D) cells wereseeded in 6-well plates and on the second day transfected with control (Ctrl) or CHOP siRNA. Forty-eight hours after the transfection, cells were treated withthe indicated concentration of PS-341 for 16 hours (C) or 24 hours (D). Then the cells were harvested andprepared forWestern blot analysis (C) or for detectionof apoptotic cells using Annexin V/7-AAD staining (D). CF, cleaved form.

PKCd Regulates DR5 via ER Stress Pathway

www.aacrjournals.org Mol Cancer Ther; 11(10) October 2012 2175




grown inmonolayer culture in RPMI 1640 with 5% FBS at37�C in a humidified atmosphere consisting of 5% CO2

and 95% air.

Western blot analysisThe preparation of whole cell protein lysates and the

Western blot analysis processes were described previous-ly (19).

Silencing of gene expression using small interferingRNA

The small interfering RNA (siRNA) duplexes forcontrol, ATF4, ATF3, and CHOP genes were describedpreviously (11). PKCd siRNA duplexes target thesequence of 50-AAGGCTACAAATGCAGGCAAT-30.The siRNAs were synthesized by GenePharma. High-Perfect Transfection Reagent (Qiagen) was used totransfect siRNAs into cells following the manufacture’sprotocol. Gene silencing effects were evaluated byWestern blot analysis.

Plasmid constructionCHOPandATF3 coding regionswere amplifiedbyPCR

fromHCT116 genomic DNAusing the following primers:

CHOP sense, 50-CACTAGTGCCGCCACCATGGCAGC-TGAGTCATTGCCTTTC-30,

antisense, 50-CGGGCCCTCATGCTTGGTGCAGATTCA-CC-30;

ATF3 sense, 50-CACTAGTGCCGCCACCATGATGCTT-CAACACCCAG-30,

antisense, 50-CGGGCCCTTAGCTCTGCAATGTTCCTT-C-30.

The procedures of plasmid constructions weredescribed previously (20), and the resultant constructswere named pLenti-CHOP and pLenti-ATF3, respec-tively.

Plasmid transient transfection andimmunoprecipitation

H157 cells were seeded in 10 cm dishes and transfectedwith pLenti-CHOP and pLenti-ATF3 plasmids using X-tremeGENE HP DNA Transfection Reagent (Roche) fol-lowing the manufacture’s protocol. Then cells were har-vested and subjected to immunoprecipitationwith CHOPantibody (Santa Cruz Biotechnology Inc., sc-7351). Theprocedures of immunoprecipitation were described pre-viously (21).

Apoptosis assaysApoptosis was evaluated by Annexin V-PE/7-AAD

Apoptosis Detection Kit purchased from BD Biosciencesfollowing the manufacture’s protocol. Caspase activationwas detected by Western blot analysis.

ResultsCHOP contributes to PS-341–induced DR5upregulation and apoptosis

Studies have revealed that some antitumor agentscan upregulate CHOP expression to induce cell apoptosis(4–6). Consistently, we found PS-341 strongly promoted

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Figure 2. Silencing of ATF3 expression attenuates the ability of PS-341 to activate caspases (A) and increase apoptosis (B). A549, H157 (A), and Calu-1 (A andB) cells were seeded in 6-well plates and on the second day transfected with control (Ctrl) or ATF3 siRNA. Forty-eight hours after the transfection, cells weretreated with the indicated concentration of PS-341 for 16 hours (A) or 24 hours (B). Then the cells were harvested and prepared forWestern blot analysis (A) orfor detection of apoptotic cells using Annexin V/7-AAD staining (B). CF, cleaved form.

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CHOP expression in NSCLC cells in a dose-dependentfashion (Fig. 1B). In the time course assay, we detectedCHOP expression occurred at 4 hours, reached a peak at12 hours (H460 and H157 cells) or 24 hours (Calu-1cells; Fig. 1B). Besides, we noticed that CHOP expressionwas always earlier than DR5 upregulation (Fig. 1B), so wequestioned whether CHOP would play an importantrole in PS-341–induced DR5 upregulation. We usedsiRNA to silence CHOP expression and found that PS-341–induced DR5 levels were dramatically decreased(Fig. 1C). Moreover, we examined weaker cleavage ofcaspase-8, caspase-9, caspase-3, and PARP in CHOPknockdown cells after exposure to PS-341 (Fig. 1C). Con-sistently, the early apoptosis was detected in CHOPsiRNA-transfected cells byAnnexin V staining–flow cyto-metry (Fig. 1D). Taken together, we conclude that CHOPexpression contributes to PS-341–induced DR5 upregula-tion and apoptosis.

PS-341 activates ER stressBecause CHOP is the key member of ER stress (2, 3),

we wondered whether PS-341 could activate ER stressin human NSCLC cells. We examined the expression ofATF4, ATF3, BiP, p-eIF2a, and IRE1a, which wereregarded as key protein markers of ER stress and foundthat their expression were all elevated in a dose-depen-dent (Supplementary Fig. S1A) and a time-dependent

manner (Supplementary Fig. S1B), indicating PS-341activates ER stress. Studies have shown that p53 reg-ulates ATF3 expression (22), but we found that thelevels of ATF3 were increased in all tested cells nomatter the p53 status (Supplementary Fig. S1), indicat-ing that PS-341–induced ATF3 expression is p53-independent.

ATF3 expression is required for PS-341–inducedapoptosis

Because PS-341 upregulated ATF3 expression (Supple-mentary Fig. S1), we then questioned whether ATF3 hadan effect on DR5 induction and apoptosis. Western blotanalysis showed that the levels of DR5 and the cleavedforms of caspase-8, caspase-9, caspase-3, and PARP wereall decreased in ATF3 siRNA-transfected cells (Fig. 2A).By Annexin V staining–flow cytometry, the percentage ofapoptotic cells in control siRNA-transfected cells was41%, whereas only 26% in ATF3 siRNA-transfected cells(Fig. 2B). So, we come to a conclusion that ATF3 expres-sion is important for PS-341–induced DR5 upregulationand apoptosis.

Inhibition of ATF4 expression decreases DR5expression and PS-341–induced apoptosis

It is known that ATF4 could enhance CHOP expression(2, 3), so we explored whether PS-341–induced ATF4

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Figure 3. Silencing ATF4 expression decreases PS-341–induced caspases activation (A) and apoptosis (B). H460 and A549 (A) and H157 (A and B) cells wereseeded in 6-well plates andon the secondday transfectedwith control (Ctrl) or ATF4 siRNA. Forty-eight hours after the transfection, cells were treatedwith theindicated concentration of PS-341 for 16 hours (A) or 24 hours (B). Then the cells were harvested and prepared forWestern blot analysis (A) or for detection ofapoptotic cells using Annexin V/7-AAD staining (B). CF, cleaved form.






upregulation would contribute to DR5 expression. To dothis, we successfully blocked PS-341–induced ATF4expression using ATF4 siRNA in H460, A549, and H157cells (Fig. 3A). We found that the inhibition of ATF4upregulation decreased the expression of DR5 andCHOPand the cleavage of caspase-8, caspase-9, caspase-3, andPARP after exposure to PS-341 (Fig. 3A). Besides, wenoticed that after abrogating ATF4 expression, PS-341–induced ATF3 expression was also reduced (Fig. 3A).Accordingly, the percentage of apoptotic cells decreasedfrom 29% to 19% (Fig. 3B). Collectively, these resultsindicate that ATF4 expression contributes to DR5 upre-gulation and is involved in PS-341–mediated apoptosis.

The relationship among ATF4, ATF3, and CHOPWe have shown that ATF4, ATF3, and CHOP contrib-

uted to DR5 expression, but the relationship among themis not clarified. In our experiments, we showed that ATF4expression stimulated CHOP and ATF3 expression, astheir expressionwas reduced inATF4-inhibited cells (Fig.3A). Then,we considered the relationship betweenCHOPand ATF3. In H157 and Calu-1 cells, when ATF3 expres-sion was suppressed, CHOP expression was weaklyinhibited, whereas ATF4 expression was unaltered (Fig.4A); when CHOP was knocked down, we detected thelightly depressed level inATF3 expression,whereasATF4was unaffected (Fig. 4B). To further investigate the phys-ical relationship betweenATF3 andCHOP,we conductedimmunoprecipitation experiment (Fig. 4C). The lentiviralplasmids containing CHOP andATF3 were cotransfectedinH157 cells and subjected to immunoprecipitation usingCHOP antibody and detected ATF3 by Western blotanalysis. We found that ATF3 was associated with CHOP(Fig. 4C). Taken together, we speculate that PS-341 treat-ment induces ATF4 upregulation, and subsequently as atranscriptional factor, promotes ATF3 and CHOP expres-sion.WhileATF3andCHOPformacomplex, andperhapsthey together regulate DR5 expression, as DR5 promotercontains a functional CHOP binding site (4, 23).

ERK/RSK2 regulates DR5 expression in PS-341–induced apoptosis

Given that ERK/RSK2 signaling promotes apoptoticinduction in some experiments (23, 24), we furtheraddressed their roles in cells exposed to PS-341. Alongwith the increased dose and time, the phosphorylation ofERK1/2 and RSK2 was all augmented (Fig. 5A and B).When using MAP–ERK kinase inhibitor, U0126, to sup-press p-ERK and p-RSK2 phosphorylation, we foundthat DR5 induction was diminished (Fig. 5C), indicatingthat ERK/RSK2 contributes to PS-341–mediated DR5expression.

PKCd contributes to PS-341–induced DR5expression and apoptosis

Because PKCd is conducive to apoptosis, we investi-gated the expression of PKCd in PS-341–treated cells.Using Western blot analysis, the 41 kDa fragment of

PKCd was detected in all tested cells, and the levelswere increased along with the enhanced concentrationand time (Fig. 6A), suggesting PS-341 activates PKCd.Then, we explored the role of PKCd on DR5 expressionwhen cells were exposed to PS-341. After inhibition ofPKCd expression by siRNA, we found that DR5 expres-sion was decreased (Fig. 6B), suggesting PKCd contri-butes to DR5 induction. Furthermore, we found thecleaved forms of caspase-8, caspase-9, caspase-3, andPARP were all reduced (Fig. 6C), and the percentage ofapoptotic cells decreased from 41% to 23% (Fig. 6D).Therefore, we concluded that PKCd conducted amarkedly function in PS-341–mediated apoptosis. Inthe meantime, we noted that the phosphorylation ofERK1/2 and RSK2 in PKCd siRNA-transfected cellswas also suppressed (Fig. 6B), suggesting that PKCd


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Figure 4. ATF3 and CHOP minimally modulate each other (A and B) whilephysically interactwith eachother (C).H157andCalu-1cellswere seededin 6-well plates and on the second day transfected with control (Ctrl) orATF3 (A) or CHOP (B) siRNA. Forty-eight hours after the transfection, cellswere treatedwith 100nmol/L PS-341 for 16 hours, and then the cellswereharvested and prepared for Western blot analysis (A and B). H157 cellswere seeded in 10 cm dishes and on the second day cotransfected withpLenti-CHOP and pLenti-ATF3 plasmids. Forty-eight hours later, cellswere harvested and subjected to immunoprecipitation using CHOPantibody (Santa Cruz Biotechnology Inc., cat#: sc-7351). Bound proteinwere eluted andprepared forWestern blot analysiswithATF3 (SantaCruzBiotechnology Inc., cat#: sc-188) and CHOP antibody (C).

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regulates DR5 expression maybe through ERK/RSK2signaling. In addition, we studied the effect of PKCdon ER stress. As compared with cells expressing PKCd,the expression of ATF4, ATF3, and CHOP were allattenuated in PKCd-abolished cells (Fig. 6B), manifest-ing that the regulation of PKCd on DR5 expression isthrough ER stress pathway. Taken together, we specu-late that PKCd regulates DR5 expression and apoptosisvia coactivation of ERK/RSK2 signaling and subse-quent ER stress.

DiscussionPS-341 has shown to induce apoptosis in various

kinds of cancer cells, but the molecular mechanism isnot very clear (17, 18). Our previous studies have shownthat DR5 contributes to PS-341–mediated apoptosis inhuman NSCLC cells (18). In this study, we further showthat PKCd regulates DR5 expression through ERK/RSK2 signaling and ATF4–CHOP/ATF3 axis. First,PS-341 induced CHOP upregulation, and CHOP wasrequired for DR5 expression. Second, PS-341 activatedER stress, and both ATF3 and ATF4 contributed to DR5induction and apoptotic sensitivity to PS-341. Third, p-ERK and p-RSK2 expressions were enhanced by PS-341,and ERK/RSK2 signaling was conducive to DR5 induc-tion. Finally, PS-341 cleaved PKCd, and PKCd regulatedDR5 expression through ERK/RSK2 signaling and ERstress.

Studies have shown that ATF4 facilitates DR5 expres-sion in sorafenib and salermide-mediated cell death(11, 25). Consistently, we discovered that abolishedATF4 expression effectively cut down DR5 upregula-tion and the cleavage of caspase-8, caspase-9, caspase-3,and PARP and the apoptosis detected was inhibited(Fig. 3). Obviously, ATF4 is a key component of DR5induction and apoptosis. Data have shown that ATF3plays a dichotomous role in regulating gene transcrip-tion, apoptosis, and cell cycle (26). In our experiments,silenced ATF3 expression decreased the apoptosisinduced by PS-341, and the levels of DR5 were lowerin ATF3-inhibited cells (Fig. 2). This was also in agree-ment with our previous finding that ATF3 exerts pos-itive function in salermide-induced DR5 expression andapoptosis (11).

Su and Kilberg (27) have shown that ATF4 interactswith CHOP in vivo by yeast 2-hybrid screen and immu-noprecipitation.Wanghas shown thatATF3 can also formcomplex with ATF4 in vivo (28). Because our resultsshowed that ATF4, ATF3, and CHOP all contributed toDR5 expression, we examined the relationship amongATF4, ATF3, and CHOP in PS-341–induced DR5 upregu-lation in NSCLC cells. In the present study, we found thatATF3 andCHOPwere the downstream of ATF4 (Fig. 3A).However, ATF3 and CHOP minimally modulated eachother (Fig. 4A and B). By immunoprecipitation, we foundthat ATF3 and CHOP had physically interacted with each

Figure 5. PS-341 induces thephosphorylation of ERK1/2 andRSK2 in a dose-dependent (A) and atime-dependent (B) manner, andinhibiting the phosphorylation ofERK1/2 and RSK2 decreases DR5induction (C). The indicated lungcancer cell lines were treated with 0,100, 200 nmol/L PS-341 for 24 hours(A) or treated with 50 nmol/L PS-341for the indicated time (B). A549,H157, and Calu-1 cells were treatedwith 10 mmol/L U0126 for 30minutesbefore treating with 0, 100,200 nmol/L PS-341 for 8 hours. Thencells were harvested and preparedfor Western blot analysis.

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other in vivo (Fig. 4C),which also agreedwith study ofHaiand colleagues that ATF3 and CHOP can form a hetero-dimer (9). Our and others’ studies have shown that DR5promoter contains theCHOPbinding site (4, 23, 29), sowepresume that ATF3 regulates DR5 expression throughinteraction with CHOP directly. Because ATF4 can inter-act with ATF3 and CHOP, respectively, whether ATF4,ATF3, and CHOP form a complex to guide DR5 expres-sion is not clear.

PKCd can be cleaved by caspase-3 and generates acatalytic fragment, which is necessary for apoptosis(14–16). In the present study, we determined that PS-341 induced the cleavage of PKCd in a dose- and time-dependent pattern (Fig. 6A), suggesting that PKCd wasactivated by PS-341. Work of others has determinedthat PKCd causes the activation of death receptorligands and activates the extrinsic apoptotic pathway(30). Studies also show that PKCd contributes to urso-deoxycholic acid–induced DR5 expression in humangastric cancer cells (31). In the present study, weshowed inhibition of PKCd resulted in the decrement

of DR5 expression (Fig. 6B), proving DR5 inductionis dependent on PKCd in lung cancer cells. Further-more, PKCd knockdown decreased the apoptosisinduced by PS-341 (Fig. 6C and D), indicating thatPKCd functions as a proapoptotic protein in PS-341–induced apoptosis.

Some data show that PKCd contributes to ER stressinduction, and inhibited PKCd expression decreasesthapsigargin-induced CHOP expression (32). Consis-tently, we detected that the upregulation of ATF4,ATF3, and CHOP were markedly suppressed in PKCdsiRNA-transfected cells (Fig. 6B), suggesting that PKCdis the upstream of ER stress. Then, we inferred thatPKCd regulated DR5 expression relies on ER stressactivation. In addition, we found that the phosphory-lation of ERK1/2 and RSK2 were decreased when PKCdexpression was inhibited (Fig. 6B), suggesting thatERK/RSK2 signaling is associated with PKCd-depen-dent DR5 upregulation. This result is consistent with thefact that PKCd is involved in the activation of ERK (33).Several studies have suggested that PKCd activates ERK

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Figure 6. PS-341 stimulates the cleavage of PKCd in a dose-dependent and time-dependent manner (A), and silencing of PKCd expression by siRNAattenuatesDR5 induction (B) and apoptosis induced by PS-341 (C andD). The indicated lung cancer cell lineswere treatedwith 0, 100, 200 nmol/L PS-341 for24 hours or treated with 50 nmol/L PS-341 for the indicated time (A). H157 (B and C) and Calu-1 (B-D) cells were seeded in 6-well plates and on thesecond day transfected with control (Ctrl) or PKCd siRNA. Forty-eight hours after the transfection, cells were treated with 100 nmol/L PS-341 for 16 hours(B and C) or 24 hours (D). Then the cells were harvested and prepared for Western blot analysis (B and C) or for detection of apoptotic cells using AnnexinV/7-AAD staining (D). CF, cleaved form.

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dependently of Raf (34). Recent studies have shown thatRas and B-Raf induce DR5 expression in an ERK/RSK2–dependent manner (35). So, it is necessary to investigatewhether PKCd regulates ERK/RSK2 expression throughactivation of Ras and Raf.In summary, we showed that PS-341 activates PKCd,

and thereafter regulates ERK/RSK2 pathway and ERstress, which contributes to DR5 induction via ATF4–ATF3/CHOP axis in human NSCLC cells.

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

Authors' ContributionsConception and design: L. Su, X. LiuDevelopment of methodology: L. Xu, L. Su, X. LiuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Xu, L. SuAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): L. Xu, L. Su, X. LiuWriting, review, and/or revision of the manuscript: L. Xu, L. Su, X. Liu

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. SuStudy supervision: X. Liu

AcknowledgmentsThe authors thank Dr. D. Zhong for helpful technical support.

Grant SupportThis work was supported by grants from the National Natural Science

Foundation of China (30971479, 31071215 to X. Liu and 81000947 to L. Su),the Doctoral Fund of Ministry of Education of China (20090131110002, X.Liu), theProgram forNewCenturyExcellent Talents inUniversity (NCET-10-0521, X. Liu), the Promotive Research Fund for Excellent Young andMiddle-aged Scientists of Shandong Province (BS2009YY004, X. Liu), andthe Shandong Provincial Natural Science Foundation (2010GSF10218 andJQ201007, X. Liu).

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 June 15, 2012; revised July 25, 2012; accepted July 25, 2012;published OnlineFirst July 30, 2012.

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Xu et al.





2012;11:2174-2182. Published OnlineFirst July 30, 2012.Mol Cancer Ther Linyan Xu, Ling Su and Xiangguo Liu

ATF3/CHOP Axis in Human Lung Cancer Cells−through ATF4 Regulates Death Receptor 5 Expression Induced by PS-341 δPKC

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d Regulates Death Receptor 5 Expression Induced by PS-341 ... · PS-341 through ATF4–ATF3/CHOP Axis in Human Lung Cancer Cells Linyan Xu, Ling Su, and Xiangguo Liu ... (p19/p17)