NF-�B Signaling Activation Induced by Chloroquine RequiresAutophagosome, p62 Protein, and c-Jun N-terminal Kinase(JNK) Signaling and Promotes Tumor Cell Resistance*

Received for publication, August 31, 2016, and in revised form, January 10, 2017 Published, JBC Papers in Press, January 12, 2017, DOI 10.1074/jbc.M116.756536

Seungwon Yang‡, Lei Qiang‡, Ashley Sample‡§, Palak Shah‡, and Yu-Ying He‡§1

From the ‡Department of Medicine, Section of Dermatology, and §Committee on Cancer Biology, University of Chicago,Chicago, Illinois 60637

Edited by George N. DeMartino

Macroautophagy (hereafter autophagy) is a catabolic cellularself-eating process by which unwanted organelles or proteinsare delivered to lysosomes for degradation through autophago-somes. Although the role of autophagy in cancer has been shownto be context-dependent, the role of autophagy in tumor cellsurvival has attracted great interest in targeting autophagy forcancer therapy. One family of potential autophagy blockers isthe quinoline-derived antimalarial family, including chloro-quine (CQ). However, the molecular basis for tumor cellresponse to CQ remains poorly understood. We show here thatin both squamous cell carcinoma cells and melanoma tumorcells, CQ induced NF-�B activation and the expression of itstarget genes HIF-1�, IL-8, BCL-2, and BCL-XL through theaccumulation of autophagosomes, p62, and JNK signaling. Theactivation of NF-�B further increased p62 gene expression.Either genetic knockdown of p62 or inhibition of NF-�B sensi-tized tumor cells to CQ, resulting in increased apoptotic celldeath following treatment. Our findings provide new molecularinsights into the CQ response in tumor cells and CQ resistancein cancer therapy. These findings may facilitate development ofimproved therapeutic strategies by targeting the p62/NF-�Bpathway.

Macroautophagy (hereafter autophagy) is an evolutionarilyconserved cellular self-eating process, in which proteins ororganelles are delivered to lysosomes for degradation (1,2). Autophagy can inhibit or promote tumor developmentdepending on the context (3– 6). Autophagy deficiency hasbeen reported to increase genome instability induced by oxida-tive stress or DNA damage, a well known factor for cancer ini-tiation and progression (2, 7, 8). However, autophagy has beenshown to promote cell survival and adaptation by protectingcells against various stress conditions such as anticancer treat-

ment and unfavorable tumor microenvironments such as anoi-kis, starvation, and hypoxic or oxidative conditions (9, 10).Increasing evidence has indicated that inhibition of autophagysuppresses tumor growth, invasion, and metastasis (11–13).These findings suggest autophagy inhibition as an attractivenew strategy to prevent and treat cancer.

One of the representative autophagy inhibitors is chloro-quine (CQ),2 a lysosomotropic drug approved by the UnitedStates Food and Drug Administration for the prophylactictreatment of malaria (14, 15) and the management of lupuserythematosus and rheumatoid arthritis (16, 17). Although ithas several side effects such as skin rash, muscle damage, andvision problems (18, 19), and an overdose can be lethal (20, 21),CQ has recently attracted considerable attention as an antitu-mor drug due to the potential biological effects on blockingautophagy in tumor cells (22–24). However, recent studies haveshown that CQ exhibits its antitumor activity independent ofautophagy inhibition (25), including normalizing the tumorvasculature (26). Several phase I and II clinical trials with CQsuggest that CQ can moderately improve the clinical activity ofradiation therapy and several chemotherapeutics (22–24). Incontrast, another antimalarial quinacrine is shown to inducecancer cell death through autophagy inhibition and p53-depen-dent inhibition of the oxidative pentose phosphate pathway(27).

It is possible that the limited anticancer efficacy of CQ iscaused by the induction of resistance pathways in tumor cells.However, how CQ induces resistance is unknown. In this study,we found that CQ induced NF-�B activation through autopha-gosome accumulation, p62, and JNK signaling, which mediatedCQ resistance in both squamous cell carcinoma (SCC) and mel-anoma cells.

Results

CQ Induces the Activation of NF-�B Activation and theExpression of Its Target Genes HIF-1� and IL-8 —To determinethe effect of CQ on skin tumor cells, we treated human Mel624melanoma cells with different concentrations of CQ. We foundthat CQ at 50, 75, and 100 �M induced apoptosis, whereas lowerconcentrations of CQ (10 and 25 �M) had no effect (Fig. 1, A and

* This work was supported by National Institutes of Health Grants ES024373and ES016936 from NIEHS (to Y.-Y. H.), American Cancer Society Grant RSG-13-078-01 (to Y.-Y. H.), University of Chicago Cancer Research Center GrantP30 CA014599, CTSA Grant UL1 TR000430, and the University of ChicagoFriends of Dermatology Endowment Fund. The authors declare that theyhave no conflicts of interest with the contents of this article. The content issolely the responsibility of the authors and does not necessarily representthe official views of the National Institutes of Health.

1 To whom corresponding should be addressed: Section of Dermatology,Dept. of Medicine, University of Chicago, Chicago, IL 60637. Tel.: 773-795-4696; Fax: 773-702-8398; E-mail: yyhe@medicine.bsd.uchicago.edu.

2 The abbreviations used are: CQ, chloroquine; MEF, mouse embryo fibro-blast; SCC, squamous cell carcinoma; BafA1, bafilomycin A1; CREB, cAMP-response element-binding protein; IKK, I�B kinase; BMS, BMS-345541.

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B). However, the autophagic flux was blocked in the cells (Fig. 1,C and D). It is possible that CQ induces resistant pathways thatsuppress CQ-induced apoptosis in skin cancer cells.

To determine whether a lower dose of CQ regulates levels ofmolecules associated with cancer-promoting or suppressingproperties, we carried out a screening analysis of known factorscontributing to cancer. We found that, in both Mel624 mela-noma cells and A431 squamous cell carcinoma (SCC) cells, CQincreased the protein levels (Fig. 1, C and D) and mRNA levelsof HIF-1� (Fig. 1, E and F), which are critical factors in skincancer development and progression and are associated withincreased tumor survival, growth, and angiogenesis (28, 29).Treatment with the protein synthesis inhibitor cycloheximideabolished CQ-induced HIF-1� up-regulation (Fig. 1G). Treat-ment with the proteasome inhibitor MG132 increased the pro-

tein levels of HIF-1� in cells treated with vehicle or CQ (Fig. 1,H and I), suggesting that HIF-1� was regulated mainly througha mechanism other than protein stability. In addition to HIF-1�, CQ also induced IL-8 expression (Fig. 1, J and K). Using acytokine array in conditioned medium from melanoma cells,we found that CQ increased the secreted level of IL-8 but hadmodest or no effect on other factors (Fig. 1, L and M). Similarly,bafilomycin A1 (BafA1), another lysosome inhibitor that blocksautophagic flux, also increased p62, HIF-1�, and IL-8 mRNAlevels (Fig. 1, N–P).

To determine how CQ up-regulates HIF-1� and IL-8 in skincancer cells, we assessed the potential role of transcription fac-tors, such as the candidates of upstream signal molecules ofHIF-1� and IL-8. These included cAMP-response element-binding protein (CREB), activator protein 1 (AP-1), and nuclear

FIGURE 1. CQ increases NF-�B activity and the expression of HIF-1� and IL-8 in melanoma and SCC cells. A and B, apoptotic cell death in Mel624 treatedwith the indicated concentration of CQ for 18 h. C and D, immunoblot analysis of HIF-1�, LC3-I/II, p62, and GAPDH (C), or �-actin (D) in Mel624 melanoma cells(C) and A431 SCC cells (D) treated with CQ (25 �M) for 24 h. E and F, real time PCR analysis of HIF-1� mRNA levels in Mel624 (E) and A431 cells (F) treated withCQ (25 �M) for 24 h. G, immunoblot analysis of HIF-1� and GAPDH in Mel624 melanoma cells treated with or without CQ (25 �M) and/or cycloheximide (100�g/ml) over a time course. H and I, immunoblot analysis of HIF-1� and GAPDH in Mel624 melanoma cells (H) or A431 cells (I) treated with or without CQ (25 �M)and/or MG132 (10 �M) over a time course. J and K, real time PCR analysis of IL-8 in Mel624 (H) and A431 cells (I) treated with CQ (25 �M) for 24 h. L, humanangiogenesis factor array analysis of conditioned medium derived from Mel624 cells incubated with or without CQ (25 �M) for 24 h. M, quantification of L. N–P,real time PCR analysis of p62 (N), HIF-1� (O), and IL-8 (P) in Mel624 treated with the indicated concentration of BafA1 for 24 h. Q, luciferase reporter analysis ofthe activities for CREB, AP-1, or NF-�B in Mel624 cells transfected with reporter vectors with specific response elements followed by treatment with or withoutCQ (25 �M) for 24 h. R, immunoblot analysis of p-IKK, IKK, and �-actin in Mel624 treated with or without CQ (25 �M) for the indicated time points. The results wereobtained from three independent experiments (mean � S.D. (error bars), n � 3; *, p � 0.05 between comparison groups (E, F, J, K, and Q) or with controls (N–P)(Student’s t test)).

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transcription factor-�B (NF-�B) (30 –32). In melanoma cells,CQ increased the transcriptional activity of NF-�B and, to amuch lesser extent, CREB and AP-1 (Fig. 1Q). It also increasedthe phosphorylation of IKK (Fig. 1R), which activates NF-�Bthrough phosphorylating and inducing degradation of theNF-�B inhibitor (33, 34). These results indicate that CQincreases the expression of HIF-1� and IL-8 and activatesNF-�B.

To determine the role of NF-�B activity in the CQ-inducedexpression of HIF-1� and IL-8, we treated cells with BMS-345541 (BMS), a specific inhibitor for IKK, or with siRNAknockdown of RELA, a nuclear factor NF-�B p65 subunit, toinhibit NF-�B activity. BMS or knockdown of RELA preventedthe increases in the protein levels of HIF-1� and mRNA levelsof HIF-1� and the mRNA levels of IL-8 in both melanoma andSCC cells (Fig. 2, A–I). We additionally verified that knock-down of RELA prevented the increases in the mRNA levels ofBCL-2 (Fig. 2J) and BCL-XL (Fig. 2K), which are antiapoptoticmolecules controlled by NF-�B activity. These results indicatethat NF-�B activation is required for CQ-increased HIF-1A,IL-8, BCL-2, and BCL-XL expression.

Autophagosome Is Required for CQ-induced NF-�B Activa-tion—To determine the mechanism by which CQ activatesNF-�B, we first examined the role of autophagosome abun-dance, because CQ inhibits the lysosomal degradation ofautophagosome. In Mel624 melanoma cells, knockdown of theessential autophagy gene ATG5 or ATG7 increased the p62protein level, although it decreased LC3-II formation (Fig. 3A),confirming an inhibition of autophagosome formation. ATG5or ATG7 knockdown decreased NF-�B activity (Fig. 3B). InMEF cells, genetic ATG5 or ATG7 deficiency increased the p62protein level, although it decreased LC3-II formation (Fig. 3C),confirming an inhibition of autophagosome formation. ATG5or ATG7 deficiency decreased CQ-induced NF-�B activity (Fig.

3D). Immunofluorescence analysis showed that CQ increasedthe number of LC3 puncta (Fig. 3E), indicating an increase inautophagosome abundance. These LC3 puncta did not colocal-ize with LAMP1, a lysosome marker, indicating that inhibitinglysosome increased autophagosome abundance and that theautophagosome was not fused with lysosome. These data indi-cate that autophagosome is required for CQ-induced NF-�Bactivation.

p62 Up-regulation Is Required for CQ-induced NF-�BActivation—Next, we assessed the role of p62 up-regulation,because induction of p62 by Ras activation has been shown tolead to NF-�B activation and thus promote tumorigenesis (35).Indeed, CQ increased the p62 protein levels in both melanomaand SCC cells (Fig. 4, A and B). p62 knockdown prevented CQ-induced IKK phosphorylation (Fig. 4, C and D). siRNA knock-down of p62 in Mel624 cells or genetic p62 deletion in MEFcells inhibited CQ-induced NF-�B activation (Fig. 4, E and F).In addition, p62 inhibition prevented CQ-induced HIF-1� up-regulation in Mel624 (Fig. 4, G and H) and MEF cells (Fig. 4, Iand J). These results indicate that p62 is required for CQ-in-duced NF-�B activation.

NF-�B Positively Regulates p62 Expression as a Positive Feed-back Loop—CQ induced the accumulation of p62 protein dueto autolysosomal blockade. In addition, we found that CQincreased the mRNA level of p62 in both melanoma and SCCcells (Fig. 5, A and B). The results indicated that CQ not onlyincreased p62 protein stability but also increased p62 expres-sion, both of which can increase the p62 protein level.

To determine how CQ induces p62 expression, we analyzedthe role of NF-�B. Inhibiting the NF-�B activity by its inhibitorBMS decreased the basal protein and mRNA levels of p62 inboth melanoma and SCC cells (Fig. 5, C–F). BMS preventedCQ-induced p62 up-regulation at the mRNA and protein levelsin both melanoma and SCC cells (Fig. 5, G, H, J, and K). We

FIGURE 2. CQ regulates HIF-1�, IL-8, BCL-2, and BCL-XL expression through NF-�B activation. A and B, immunoblot analysis of HIF-1� and GAPDH inMel624 (A) and A431 cells (B) treated with CQ (10 �M) for 24 h in the presence or absence of the NF-�B pathway inhibitor BMS (2 �M). C and D, real time PCRanalysis of HIF-1� and IL-8 mRNA levels in Mel624 cells treated with or without CQ (25 �M) for 6 h in the presence or absence of BMS (5 �M). E and F, real timePCR analysis of HIF-1� and IL-8 mRNA levels in A431 cells treated with or without CQ (25 �M) for 6 h in the presence or absence of BMS (2 �M). G, immunoblotanalysis of HIF-1�, RELA, and GAPDH in Mel624 transfected with si-control or si-RELA followed by treatment with CQ (10 �M) for 24 h. H–K, real time PCR analysisof HIF-1�, IL-8, BCL-2, and BCL-XL mRNA levels in Mel624 cells transfected with control siRNA or siRNA targeting RELA (si-RELA), followed by treatment with orwithout CQ (25 �M) for 24 h. The results were obtained from three independent experiments (mean � S.D. (error bars), n � 3; *, p � 0.05 between comparisongroups (Student’s t test)).

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further confirmed the role of NF-�B with knockdown of RELA(Fig. 5, I and L). These data indicate that NF-�B is required forCQ-induced p62 expression and p62 up-regulation. Theseresults demonstrate that CQ induces a positive feedback loopbetween p62 and NF-�B; CQ-induced p62 protein up-regu-lation triggers NF-�B activation, and the activated NF-�Binduces p62 expression in turn.

JNK Signaling Is Critical for CQ-induced NF-�B Activationand p62 Expression—Next, we determined the role of JNK sig-naling in CQ-induced NF-�B activation, because previous stud-ies have shown that JNK is required for high glucose-inducedNF-�B activation in kidney epithelial cells (36). In both Mel624and A431 cells, CQ induced phosphorylation of c-Jun (Fig. 6, Aand B), a substrate of JNK kinase. In comparison, ATG5 orATG7 deficiency had little effect on c-Jun phosphorylation (Fig.6C), indicating that inhibiting autophagosome formation is dis-pensable for JNK activation. Inhibiting JNK signaling by JNKknockdown reduced CQ-induced p62 expression (Fig. 6D),NF-�B activation (Fig. 6E), HIF-1� protein level (Fig. 6, F andG), and BCL-2 and BCL-XL mRNA level (Fig. 6, H and I). Theseresults indicate that JNK signaling is critical for CQ-inducedNF-�B activation. However, inhibiting NF-�B by RELA knock-down had no effect on c-Jun phosphorylation (Fig. 6J), indicat-ing that JNK is an upstream signal of NF-�B.

Inhibition of Either p62 or the NF-�B Pathway Sensitizes Can-cer Cells to CQ-induced Cell Killing—Next, we assessed the roleof the p62/NF-�B axis in CQ-induced cancer cell killing. InMel624, A431, and MEF cells, CQ (25 �M) alone or p62 inhibi-tion did not induce the activation of caspase-3 (Fig. 7, A–C), anindicator of apoptosis, or affect cell viability (Fig. 7, D–F). How-ever, inhibition of p62 by shRNA knockdown or genetic dele-tion sensitized the cells to CQ-induced caspase-3 activation(Fig. 7, A–C) and decreased cell viability (Fig. 7, D–H). Theseresults indicate that p62 up-regulation mediates CQ resistance.

Similar to p62 inhibition, pharmacological inhibition ofNF-�B alone or CQ did not affect caspase-3 activation (Fig. 8,A–C) or cell viability (Fig. 8, D–I) in Mel624 and A375 mela-noma and A431 SCC cells. However, inhibition of NF-�B byBMS or RELA knockdown sensitized the cells to CQ-inducedcaspase-3 activation (Fig. 8, A–C) and decreased cell viability(Fig. 8, D–M). These results indicate that CQ-induced activa-tion of the NF-�B pathway promotes cell survival and confers aresistance mechanism to CQ in skin cancer cells.

Discussion

Autophagy can facilitate adaptation and survival of tumorcells in various stressful environments such as anticancer treat-ment and anoikis, hypoxia, and oxidation or starvation. Hence,

FIGURE 3. Role of autophagosome in CQ-induced NF-�B activation. A, immunoblot analysis of p62, LC3-I/II, and GAPDH in Mel624 cells stably infected witha lentiviral vector expressing negative control shRNA (sh-NC) or shRNA targeting ATG5 (sh-ATG5) or ATG7 (sh-ATG7). B, luciferase reporter assay of NF-�B activityin Mel624 cells stably infected with a lentiviral vector expressing sh-NC, sh-ATG5, or sh-ATG7. C, immunoblot analysis of p62, LC3-I/II, and GAPDH in wild-type(WT), ATG5-deficient (ATG5-KO), or ATG7-deficient (ATG7-KO) mouse embryonic fibroblast (MEF) cells. D, luciferase reporter assay of NF-�B activity in WT,ATG5-KO, or ATG7-KO MEF cells treated with or without CQ (25 �M) for 24 h. E, immunofluorescence analysis of LC3 and LAMP1 in WT, ATG5-KO, or ATG7-KOMEF cells treated with or without CQ (25 �M) for 18 h. Blue indicates DAPI nuclear counterstain. The results were obtained from three independent experiments(mean � S.D. (error bars), n � 3; *, p � 0.05 between comparison groups (Student’s t test)).

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inhibitors of the autophagy pathway such as CQ have thepotential for cancer therapy. However, how tumor cellsrespond to CQ is still poorly understood. In this study, we haveshown that CQ activates NF-�B through autophagosome accu-

mulation, p62 up-regulation, and JNK signaling (Fig. 8N).NF-�B activation in turn induced p62 expression and thusformed a positive feedback loop. Inhibiting either p62 or NF-�Bdid sensitize tumor cells to CQ-induced apoptotic cell death.

FIGURE 4. p62 is required for CQ-induced NF-�B activation. A and B, immunoblot analysis of p62 and GAPDH in Mel624 (A) and A431 (B) cells treated withor without CQ (25 �M) for 24 h. C and D, immunoblot analysis of p62 (C), p-IKK (D), IKK (D), and GAPDH in Mel624 cells transfected with control siRNA or siRNAtargeting p62 (si-p62), followed by treatment with or without CQ (25 �M) for 8 h. E, luciferase reporter assay of NF-�B activity in WT and p62 knock-out (KO) MEFcells transfected with the reporter vector with specific NF-�B response elements (NF-�B-RE) followed by treatment with or without CQ (10 �M) for 18 h. F,luciferase reporter assay of NF-�B activity Mel624 cells transfected with si-control or si-p62 followed by transfection with the NF-�B-RE reporter vector and thentreatment with or without CQ (25 �M) for 12 h. G, immunoblot analysis of p62 and GAPDH in A375 cells stably infected with sh-negative control (sh-NC) orsh-p62. H, immunoblot analysis of HIF-1� and GAPDH in sh-NC and sh-p62 A375 cells treated with or without CQ (25 �M) for 24 h. I, immunoblot analysis of p62and �-actin in WT and p62 KO MEF cells treated with or without CQ (25 �M) for 24 h. J, immunoblot analysis of HIF-1� and �-actin in WT and p62 KO cells treatedwith the indicated concentrations of CQ for 2 h. The results were obtained from three independent experiments (mean � S.D. (error bars), n � 3; *, p � 0.05between comparison groups (Student’s t test)).

FIGURE 5. NF-�B regulates p62 expression. A and B, real time PCR analysis of p62 in Mel624 (A) and A431 (B) cells treated with or without CQ (25 �M) for 24 h.C, immunoblot analysis of p62 and GAPDH in Mel624 cells treated with BMS (5 �M) for 24 h. D, real time PCR analysis of p62 in Mel624 cells treated with BMS (5�M) for 24 h. E, immunoblot analysis of p62 and GAPDH in A431 cells treated with BMS (2 �M) for 24 h. F, real time PCR analysis of p62 in A431 cells treated withBMS (2 �M) for 24 h. G and H, immunoblot analysis of p62 and GAPDH in Mel624 cells treated with BMS (5 �M) (G) and A431 cells treated with BMS (2 �M) (H) for24 h. I, immunoblot analysis of p62, RELA, and GAPDH in Mel624 cells transfected with si-control or si-RELA followed by treatment with CQ (25 �M) for 24 h. J andK, real time PCR analysis of p62 in Mel624 cells (J) and A431 cells (K) treated with CQ, BMS, the combination of both for 24 h. L, real time PCR analysis of p62 inMel624 cells transfected with si-control or si-RELA followed by treatment with CQ (25 �M) for 24 h. The results were obtained from three independentexperiments (mean � S.D. (error bars), n � 3; *, p � 0.05 between comparison groups (Student’s t test)).

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Thus, inhibiting either p62 or NF-�B may provide improvedanticancer efficacy. Our findings demonstrate a novel mecha-nism by which tumor cells evade CQ-induced killing and thussuggest a resistance mechanism (Fig. 8N).

We found that activation of the p62/NF-�B pathway pro-motes cancer cell survival in response to CQ. CQ was found toinduce p62 protein stabilization and thus activate NF-�B,which leads to the induction of p62 expression. Inhibiting

either p62 or NF-�B sensitized tumor cells to CQ-induced kill-ing. The activation of p62 and NF-�B promoted CQ resistance.Our findings are consistent with recent findings that CQ didnot affect tumor cells directly in vivo but showed an antitu-mor effect normalizing the tumor vasculature (26). It is pos-sible that in vivo the induction of the p62/NF-�B pathwayprotected tumor cells against a CQ-induced intrinsic antitu-mor effect. Future investigation is needed to determine

FIGURE 6. Role of JNK signaling in CQ-induced NF-�B activation and p62 expression. A and B, immunoblot analysis of p-c-Jun, c-Jun, and GAPDH in Mel624cells (A) and A431 cells (B) treated with or without CQ (25 �M) for 24 h. C, immunoblot analysis of p-c-Jun, c-Jun, and GAPDH in sh-NC, sh-ATG5, and sh-ATG7Mel624 cells. D, real time PCR analysis of p62 mRNA in Mel624 cells transfected with control siRNA or siRNA targeting JNK (si-JNK), followed by treatment withor without CQ (25 �M) for 18 h. E, luciferase reporter assay of NF-�B activity in Mel624 cells transfected with control siRNA or siRNA targeting JNK (si-JNK),followed by treatment with or without CQ (25 �M) for 18 h. F and G, immunoblot analysis of p62, HIF-1�, and GAPDH in Mel624 cells transfected with controlsiRNA or siRNA targeting JNK (si-JNK), followed by treatment with or without CQ (25 �M) for 18 h. H and I, real time PCR analysis of BCL-2 and BCL-XL mRNA inMel624 cells transfected with control siRNA or siRNA targeting JNK (si-JNK), followed by treatment with or without CQ (25 �M) for 18 h. J, immunoblot analysisof p-c-Jun, c-Jun, RELA, and GAPDH in Mel624 cells transfected with control siRNA or siRNA targeting JNK (si-JNK), followed by treatment with or without CQ (25�M) for 18 h. The results were obtained from three independent experiments (mean � S.D. (error bars), n � 3; *, p � 0.05 between comparison groups (Student’st test)).

FIGURE 7. Loss of p62 sensitizes cancer cells to CQ-induced apoptotic cell death. A–C, immunoblot analysis of active caspase-3 and GAPDH in sh-NC orsh-p62 A375 cells treated with or without CQ (25 �M) for 24 h (A), sh-NC or sh-p62 A431 cells treated with or without CQ (25 �M) for 38 h (B), and WT or p62-KOMEF cells treated with or without CQ (25 �M) for 24 h (C). D–F, cell viability analysis of sh-NC or sh-p62 A375 cells (D), sh-NC or sh-p62 A431 cells (E), and WT orp62 KO MEF cells (F) cultured with 1% FBS followed by treatment with the indicated concentration of CQ for 24 h. G and H, analysis of apoptotic cell death insh-NC or sh-p62 A375 cells treated with or without CQ (25 �M) for 18 h. The results were obtained from three independent experiments (mean � S.D. (errorbars), n � 3; *, p � 0.05 between comparison groups (Student’s t test)).

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whether other autophagy inhibitors induced similar resis-tance mechanisms.

As a selective autophagy substrate and signaling adaptor, p62plays multifunctional roles in autophagy and signal transduc-tion in carcinogenesis and cancer progression (37, 38) throughregulating multiple signaling pathways, including NF-�B (35,39), NRF2 (41– 43), and Twist1 (44). Up-regulation of p62 hasbeen shown in a number of human cancers (37, 38). Inhibitingp62 reduced cell proliferation, invasion, and migration (35, 37,44). In addition, recent studies have demonstrated that p62 pro-tects cells from apoptosis (46 – 48). We found that in both SCCand melanoma cells, CQ induced p62 protein up-regulationand p62 expression. Knockdown of p62 was shown to sensitizecells to CQ-induced cell death. These data indicate that p62induction is critical for cell survival following CQ treatment.

In addition, we found that CQ induces NF-�B through p62up-regulation. p62 up-regulation is required for NF-�B activa-tion. It is possible that CQ-induced p62 promoted polyubiquiti-nation of tumor necrosis factor (TNF) receptor-associated fac-tor 6 (TRAF6) as demonstrated in Ras activation during lungtumorigenesis (35). Recent studies have also shown that p62 isrequired for the activation of NF-�B in response to Toll-likereceptor pathway activation (49). Intriguingly, we also show

that the activation of NF-�B in turn further induced p62 geneexpression in response to CQ treatment. It has been shownthat TLR2/6 activation induces p62 expression through theNADPH oxidase pathway (49). In addition, inflammatoryNF-�B activation in macrophages has been shown to inducep62 up-regulation, leading to mitophagy and thus limitinginflammasome activation (50). An NF-�B response element hasbeen identified in the p62 promoter (51). However, the func-tional significance of this element and how NF-�B regulatesp62 expression remain to be determined. Our findings under-score the critical role of the NF-�B signaling in tumor cell sur-vival following CQ treatment. The role of other CQ-inducedtranscription factors such as CREB and AP-1 requires furtherinvestigation.

In addition to p62, we found that autophagosome abundanceand JNK signaling are also critical for CQ-induced NF-�B acti-vation. CQ increased autophagosome abundance and inhibit-ing autophagosome formation abolished CQ-induced NF-�Bactivation. In addition, CQ also activated JNK signaling, andinhibiting JNK signaling by JNK knockdown reduced CQ-in-duced NF-�B activation. This is consistent with previous stud-ies showing that high glucose-induced NF-�B requires JNKsignaling (36). It appears that CQ-induced NF-�B signaling

FIGURE 8. Blocking the NF-�B pathway sensitizes cancer cells to CQ-induced apoptotic cell death. A–C, immunoblot analysis of active caspase-3 andGAPDH in Mel624 (A), A375 (B), and A431 (C) cells treated with CQ (25 �M) for 24 h in the presence or absence of BMS (5 �M for A and B and 2 �M for C). D–F,microscopic cellular morphology of cells as treated in A–C. G–I, cell viability assays for cells treated as in A–C. J–M, analysis of apoptotic cell death in Mel624 (Jand K) and A375 (L and M) cells transfected with control siRNA or siRNA targeting RELA (si-RELA), followed by treatment with or without CQ (25 �M) for 18 h. Theresults were obtained from three independent experiments (mean � S.D. (error bars), n � 3; *, p � 0.05 between comparison groups (Student’s t test)). N,schematic for the role of the p62/NF-�B feedback loop in CQ resistance in tumor cells.

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requires p62, autophagosome, and JNK signaling. It is possiblethat, for CQ-induced NF-�B activation, autophagosome servesas a signing hub and that p62 and JNK are also required.

In summary, we have demonstrated that CQ induces NF-�Bactivation through autophagosome accumulation, p62 up-reg-ulation, and JNK signaling. NF-�B activation in turn increasedp62 expression, thereby forming a positive feedback loop.Blocking either p62 or NF-�B sensitizes tumor cells to CQ-induced cell killing. Our findings provide new molecularinsights into the CQ response in tumor cells and CQ resistancein cancer therapy. These findings may facilitate development ofimproved therapeutic strategies by targeting the p62/NF-�Bpathway.

Materials and Methods

Cell Culture—Mel624 melanoma cells, A375 (human amela-notic melanoma cells), A431 (human squamous carcinomacells), WT, and p62 KO of mouse embryo fibroblast (MEF) cellswere obtained from Dr. Seungmin Hwang, University of Chi-cago, IL. They were maintained in a monolayer culture in 95%air and 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s medium(Invitrogen) supplemented with 10% fetal bovine serum(HyClone, Logan, UT), 100 units/ml penicillin, and 100 �g/mlstreptomycin (Invitrogen).

Lentiviral Production and Infection—pLKO.1 sh-p62 (human)was obtained from Sigma. Lentivirus was produced by co-trans-fection into HEK-293T cells (human embryonic kidney cells)with lentiviral constructs together with the pCMVdelta8.2packaging plasmid and pVSV-G envelope plasmid usingGenJetTM Plus DNA In Vitro Transfection Reagent (Signagen,Ijamsville, MD, as described previously (44, 52). Virus-contain-ing supernatants were collected 24 – 48 h after transfection andused to infect recipients. Target cells were infected in the pres-ence of Polybrene (8 �g/ml) (Sigma) and selected with puromy-cin (Santa Cruz Biotechnology, Santa Cruz, CA) at 1 �g/ml for6 days.

Immunoblotting—Immunoblotting was performed as describedpreviously (7, 44). Antibodies used were as follows: p62 (ProgenBiotechnik GmbH, Heidelberg, Germany); HIF-1A (R&D Sys-tems, Minneapolis, MN); p-IKK�/�, IKK, p-c-Jun, c-Jun, andRELA (Cell Signaling Technology, Beverly, MA); and GAPDHand �-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Cytokine Array Analysis—Conditioned medium derivedfrom Mel624 incubated with or without CQ (25 �M) for 24 hwas prepared in Dulbecco’s modified Eagle’s medium (Invitro-gen) supplemented with 1% fetal bovine serum (HyClone,Logan, UT). Equal volumes (1 ml) of each conditioned mediumwere incubated with the pre-coated Proteome Profiler arraymembrane for human angiogenesis antibody (R&D Systems,Minneapolis, MN) and processed according to the manufactu-rer’s instructions.

Luciferase Reporter Assays—Cells were transfected with 1 �gof pGL3 AP-1-Luc, pGL3 CREB-Luc, or pGL3 NF-�B-Luc and0.025 �g of pRL-TK, which is used as a transfection efficiencycontrol (Promega, Madison, WI), using GenJetTM Plus DNA invitro transfection reagent (Signagen, Ijamsville, MD) accordingto the manufacturer’s instructions. Luciferase reporter assayswere performed as described previously (7, 44).

siRNA Transfection—Cells were transfected with si-control,si-RNA (Cell Signaling Technology, Beverly, MA), targetingp62 (Cell Signaling Technology), targeting RELA (Dharmacon,Lafayette, CO), and targeting JNK (Santa Cruz Biotechnology,Santa Cruz, CA) using GenMuteTM siRNA transfection reagent(Signagen, Ijamsville, MD) according to the manufacturer’sinstructions.

Quantitative Real Time PCR—Quantitative real time PCRassays were performed using a CFX Connect real time system(Bio-Rad) with iQ SYBR Green Supermix (Bio-Rad). Thethreshold cycle number (CQ) for each sample was determinedin triplicate. The CQ for values for HIF-1A, IL-8, BCL-2, BCL-XL, and p62 were normalized against GAPDH or �-actin asdescribed previously (7, 44, 52). Amplification primers were asfollows: 5�-GTT TAC TAA AGG ACA AGT CAC C-3� (for-ward) and 5�-TTC TGT TTG TTG AAG GGA G-3� (reverse)for the HIF-1A gene; 5�-ATG ACT TCC AAG CTG GCC GTGGCT-3� (forward) and 5�-TCT CAG CCC TCT TCA AAAACT TCT-3� (reverse) for the human IL-8 gene; 5�-CAG AGAAGC CCA TGG ACA G-3� (forward) and 5�-AGC TGC CTTGTA CCC ACA TC-3� (reverse) for the human p62 gene;5�-GTG GAT GAC TGA GTA CCT GAA C-3� (forward) and5�-GCC AGG AGA AAT CAA ACA GAG G-3� (reverse) forBCL-2 gene; 5�-GAC ATC CCA GCT CCA CAT C-3� (forward)and 5�-GTT CCC ATA GAG TTC CAC AAA AG-3� (reverse)for BCL-XL gene; 5�-ATC GGA ACG GTG AAG GTG ACA-3�(forward) and 5�-ATG GCA AGG GAC TTC CTG TAA C-3�(reverse) for the human �-actin gene; and 5�-ACC ACA GTCCAT GCC ATC AC-3� (forward) and 5�-TCC ACC ACC CTGTTG CTG TA-3� (reverse) for the human GAPDH gene.

Cell Viability Assay—Cell viability was assessed with CellCounting Kit-8 (CCK-8) (Sigma). The CCK-8 analysis was per-formed following the manufacturer’s protocol as described pre-viously (40, 45, 53).

Immunofluorescence—The cells were fixed with 4% parafor-maldehyde/PBS for 30 min and permeabilized in 0.5% TritonX-100/PBS for 20 min. The cells were then washed with PBS.PBS supplemented with 5% normal goat serum (Invitrogen)was used as a blocking solution for 30 min. After removal of theblocking solution, the cells were incubated with LC3-AlexaFluor� 488-conjugated antibody (Cell Signaling Technology,Beverly, MA) and LAMP1-Cy3-conjugated antibody (Sigma)for 1 h at 37 °C. The cells were washed three times with 0.1%Triton X-100/TBS for 10 min. The cells were then fixed in Pro-long Gold Antifade with DAPI (Invitrogen) to visualize the cellnuclei and observed under a fluorescence microscope (Olym-pus IX71).

Flow Cytometric Analysis of Apoptosis—Apoptotic cell deathwas determined using the annexin V-FITC apoptosis detectionkit (eBioscience, San Diego), according to the manufacturer’sinstructions. Cell samples were then analyzed by BD FACSCali-bur flow cytometer (BD Biosciences).

Statistical Analyses—Statistical analyses were performedusing Prism 5 (GraphPad). Data were expressed as the mean ofat least three independent experiments and analyzed byStudent’s t test. Error bars indicate the S.D. of the means. p �0.05 was considered statistically significant.

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Author Contributions—S. Y. and Y.-Y. H. conceived and coordi-nated the study and wrote the paper. S. Y. designed, performed, andanalyzed the experiments shown in all figures. L. Q., A. S., and P. S.provided technical assistance and contributed to the preparation ofthe figures. All authors reviewed the results and approved the finalversion of the manuscript.

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Seungwon Yang, Lei Qiang, Ashley Sample, Palak Shah and Yu-Ying HeCell Resistance

p62 Protein, and c-Jun N-terminal Kinase (JNK) Signaling and Promotes Tumor B Signaling Activation Induced by Chloroquine Requires Autophagosome,κNF-

doi: 10.1074/jbc.M116.756536 originally published online January 12, 20172017, 292:3379-3388.J. Biol. Chem. 

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