NF-κB Signaling Activation Induced by Chloroquine Requires Autophagosome, p62 Protein, and c-Jun N-terminal Kinase (JNK) Signaling and Promotes Tumor Cell Resistance

Abstract

Macroautophagy (hereafter autophagy) is a catabolic cellular self-eating process by which unwanted organelles or proteins are delivered to lysosomes for degradation through autophagosomes. Although the role of autophagy in cancer has been shown to be context-dependent, the role of autophagy in tumor cell survival has attracted great interest in targeting autophagy for cancer therapy. One family of potential autophagy blockers is the quinoline-derived antimalarial family, including chloroquine (CQ). However, the molecular basis for tumor cell response to CQ remains poorly understood. We show here that in both squamous cell carcinoma cells and melanoma tumor cells, CQ induced NF-κB activation and the expression of its target genes HIF-1α, IL-8, BCL-2, and BCL-XL through the accumulation of autophagosomes, p62, and JNK signaling. The activation of NF-κB further increased p62 gene expression. Either genetic knockdown of p62 or inhibition of NF-κB sensitized tumor cells to CQ, resulting in increased apoptotic cell death following treatment. Our findings provide new molecular insights into the CQ response in tumor cells and CQ resistance in cancer therapy. These findings may facilitate development of improved therapeutic strategies by targeting the p62/NF-κB pathway.

Introduction

Macroautophagy (hereafter autophagy) is an evolutionarily conserved cellular self-eating process, in which proteins or organelles are delivered to lysosomes for degradation (1, 2). Autophagy can inhibit or promote tumor development depending on the context (3–6). Autophagy deficiency has been reported to increase genome instability induced by oxidative stress or DNA damage, a well known factor for cancer initiation and progression (2, 7, 8). However, autophagy has been shown to promote cell survival and adaptation by protecting cells against various stress conditions such as anticancer treatment and unfavorable tumor microenvironments such as anoikis, starvation, and hypoxic or oxidative conditions (9, 10). Increasing evidence has indicated that inhibition of autophagy suppresses tumor growth, invasion, and metastasis (11–13). These findings suggest autophagy inhibition as an attractive new strategy to prevent and treat cancer.

One of the representative autophagy inhibitors is chloroquine (CQ),² a lysosomotropic drug approved by the United States Food and Drug Administration for the prophylactic treatment of malaria (14, 15) and the management of lupus erythematosus and rheumatoid arthritis (16, 17). Although it has several side effects such as skin rash, muscle damage, and vision problems (18, 19), and an overdose can be lethal (20, 21), CQ has recently attracted considerable attention as an antitumor drug due to the potential biological effects on blocking autophagy in tumor cells (22–24). However, recent studies have shown that CQ exhibits its antitumor activity independent of autophagy inhibition (25), including normalizing the tumor vasculature (26). Several phase I and II clinical trials with CQ suggest that CQ can moderately improve the clinical activity of radiation therapy and several chemotherapeutics (22–24). In contrast, another antimalarial quinacrine is shown to induce cancer cell death through autophagy inhibition and p53-dependent inhibition of the oxidative pentose phosphate pathway (27).

It is possible that the limited anticancer efficacy of CQ is caused 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-κB activation through autophagosome accumulation, p62, and JNK signaling, which mediated CQ resistance in both squamous cell carcinoma (SCC) and melanoma cells.

Results

CQ Induces the Activation of NF-κB Activation and the Expression of Its Target Genes HIF-1α and IL-8

To determine the effect of CQ on skin tumor cells, we treated human Mel624 melanoma cells with different concentrations of CQ. We found that CQ at 50, 75, and 100 μm induced apoptosis, whereas lower concentrations of CQ (10 and 25 μm) had no effect (Fig. 1, A and B). However, the autophagic flux was blocked in the cells (Fig. 1, C and D). It is possible that CQ induces resistant pathways that suppress CQ-induced apoptosis in skin cancer cells.

FIGURE 1.

CQ increases NF-κB activity and the expression of HIF-1α and IL-8 in melanoma and SCC cells. A and B, apoptotic cell death in Mel624 treated with the indicated concentration of CQ for 18 h. C and D, immunoblot analysis of HIF-1α, LC3-I/II, p62, and GAPDH (C), or β-actin (D) in Mel624 melanoma cells (C) and A431 SCC cells (D) treated with CQ (25 μm) for 24 h. E and F, real time PCR analysis of HIF-1α mRNA levels in Mel624 (E) and A431 cells (F) treated with CQ (25 μm) for 24 h. G, immunoblot analysis of HIF-1α and GAPDH in Mel624 melanoma cells treated with or without CQ (25 μm) and/or cycloheximide (100 μg/ml) over a time course. H and I, immunoblot analysis of HIF-1α and GAPDH in Mel624 melanoma cells (H) or A431 cells (I) treated with or without CQ (25 μm) and/or MG132 (10 μm) 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 μm) for 24 h. L, human angiogenesis factor array analysis of conditioned medium derived from Mel624 cells incubated with or without CQ (25 μm) for 24 h. M, quantification of L. N–P, real time PCR analysis of p62 (N), HIF-1α (O), and IL-8 (P) in Mel624 treated with the indicated concentration of BafA1 for 24 h. Q, luciferase reporter analysis of the activities for CREB, AP-1, or NF-κB in Mel624 cells transfected with reporter vectors with specific response elements followed by treatment with or without CQ (25 μm) for 24 h. R, immunoblot analysis of p-IKK, IKK, and β-actin in Mel624 treated with or without CQ (25 μm) for the indicated time points. The results were obtained 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)).

To determine whether a lower dose of CQ regulates levels of molecules associated with cancer-promoting or suppressing properties, we carried out a screening analysis of known factors contributing to cancer. We found that, in both Mel624 melanoma cells and A431 squamous cell carcinoma (SCC) cells, CQ increased the protein levels (Fig. 1, C and D) and mRNA levels of HIF-1α (Fig. 1, E and F), which are critical factors in skin cancer development and progression and are associated with increased tumor survival, growth, and angiogenesis (28, 29). Treatment with the protein synthesis inhibitor cycloheximide abolished CQ-induced HIF-1α up-regulation (Fig. 1G). Treatment with the proteasome inhibitor MG132 increased the protein levels of HIF-1α in cells treated with vehicle or CQ (Fig. 1, H and I), suggesting that HIF-1α was regulated mainly through a mechanism other than protein stability. In addition to HIF-1α, CQ also induced IL-8 expression (Fig. 1, J and K). Using a cytokine array in conditioned medium from melanoma cells, we found that CQ increased the secreted level of IL-8 but had modest or no effect on other factors (Fig. 1, L and M). Similarly, bafilomycin A1 (BafA1), another lysosome inhibitor that blocks autophagic flux, also increased p62, HIF-1α, and IL-8 mRNA levels (Fig. 1, N–P).

To determine how CQ up-regulates HIF-1α and IL-8 in skin cancer cells, we assessed the potential role of transcription factors, such as the candidates of upstream signal molecules of HIF-1α and IL-8. These included cAMP-response element-binding protein (CREB), activator protein 1 (AP-1), and nuclear transcription factor-κB (NF-κB) (30–32). In melanoma cells, CQ increased the transcriptional activity of NF-κB and, to a much lesser extent, CREB and AP-1 (Fig. 1Q). It also increased the phosphorylation of IKK (Fig. 1R), which activates NF-κB through phosphorylating and inducing degradation of the NF-κB inhibitor (33, 34). These results indicate that CQ increases the expression of HIF-1α and IL-8 and activates NF-κB.

To determine the role of NF-κB activity in the CQ-induced expression of HIF-1α and IL-8, we treated cells with BMS-345541 (BMS), a specific inhibitor for IKK, or with siRNA knockdown of RELA, a nuclear factor NF-κB p65 subunit, to inhibit NF-κB activity. BMS or knockdown of RELA prevented the increases in the protein levels of HIF-1α and mRNA levels of HIF-1α and the mRNA levels of IL-8 in both melanoma and SCC cells (Fig. 2, A–I). We additionally verified that knockdown of RELA prevented the increases in the mRNA levels of BCL-2 (Fig. 2J) and BCL-XL (Fig. 2K), which are antiapoptotic molecules controlled by NF-κB activity. These results indicate that NF-κB activation is required for CQ-increased HIF-1A, IL-8, BCL-2, and BCL-XL expression.

FIGURE 2.

CQ regulates HIF-1α, IL-8, BCL-2, and BCL-XL expression through NF-κB activation. A and B, immunoblot analysis of HIF-1α and GAPDH in Mel624 (A) and A431 cells (B) treated with CQ (10 μm) for 24 h in the presence or absence of the NF-κB pathway inhibitor BMS (2 μm). C and D, real time PCR analysis of HIF-1α and IL-8 mRNA levels in Mel624 cells treated with or without CQ (25 μm) for 6 h in the presence or absence of BMS (5 μm). E and F, real time PCR analysis of HIF-1α and IL-8 mRNA levels in A431 cells treated with or without CQ (25 μm) for 6 h in the presence or absence of BMS (2 μm). G, immunoblot analysis of HIF-1α, RELA, and GAPDH in Mel624 transfected with si-control or si-RELA followed by treatment with CQ (10 μm) for 24 h. H–K, real time PCR analysis of HIF-1α, 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 or without CQ (25 μm) for 24 h. 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)).

Autophagosome Is Required for CQ-induced NF-κB Activation

To determine the mechanism by which CQ activates NF-κB, we first examined the role of autophagosome abundance, because CQ inhibits the lysosomal degradation of autophagosome. In Mel624 melanoma cells, knockdown of the essential autophagy gene ATG5 or ATG7 increased the p62 protein level, although it decreased LC3-II formation (Fig. 3A), confirming an inhibition of autophagosome formation. ATG5 or ATG7 knockdown decreased NF-κB activity (Fig. 3B). In MEF cells, genetic ATG5 or ATG7 deficiency increased the p62 protein level, although it decreased LC3-II formation (Fig. 3C), confirming an inhibition of autophagosome formation. ATG5 or ATG7 deficiency decreased CQ-induced NF-κB activity (Fig. 3D). Immunofluorescence analysis showed that CQ increased the number of LC3 puncta (Fig. 3E), indicating an increase in autophagosome abundance. These LC3 puncta did not colocalize with LAMP1, a lysosome marker, indicating that inhibiting lysosome increased autophagosome abundance and that the autophagosome was not fused with lysosome. These data indicate that autophagosome is required for CQ-induced NF-κB activation.

FIGURE 3.

Role of autophagosome in CQ-induced NF-κB activation. A, immunoblot analysis of p62, LC3-I/II, and GAPDH in Mel624 cells stably infected with a lentiviral vector expressing negative control shRNA (sh-NC) or shRNA targeting ATG5 (sh-ATG5) or ATG7 (sh-ATG7). B, luciferase reporter assay of NF-κB activity in 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-κB activity in WT, ATG5-KO, or ATG7-KO MEF cells treated with or without CQ (25 μm) for 24 h. E, immunofluorescence analysis of LC3 and LAMP1 in WT, ATG5-KO, or ATG7-KO MEF cells treated with or without CQ (25 μm) 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)).

p62 Up-regulation Is Required for CQ-induced NF-κB Activation

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

FIGURE 4.

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

NF-κB Positively Regulates p62 Expression as a Positive Feedback Loop

CQ induced the accumulation of p62 protein due to autolysosomal blockade. In addition, we found that CQ increased the mRNA level of p62 in both melanoma and SCC cells (Fig. 5, A and B). The results indicated that CQ not only increased p62 protein stability but also increased p62 expression, both of which can increase the p62 protein level.

FIGURE 5.

NF-κB 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 μm) for 24 h. C, immunoblot analysis of p62 and GAPDH in Mel624 cells treated with BMS (5 μm) for 24 h. D, real time PCR analysis of p62 in Mel624 cells treated with BMS (5 μm) for 24 h. E, immunoblot analysis of p62 and GAPDH in A431 cells treated with BMS (2 μm) for 24 h. F, real time PCR analysis of p62 in A431 cells treated with BMS (2 μm) for 24 h. G and H, immunoblot analysis of p62 and GAPDH in Mel624 cells treated with BMS (5 μm) (G) and A431 cells treated with BMS (2 μm) (H) for 24 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 μm) for 24 h. J and K, 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 in Mel624 cells transfected with si-control or si-RELA followed by treatment with CQ (25 μm) for 24 h. 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)).

To determine how CQ induces p62 expression, we analyzed the role of NF-κB. Inhibiting the NF-κB activity by its inhibitor BMS decreased the basal protein and mRNA levels of p62 in both melanoma and SCC cells (Fig. 5, C–F). BMS prevented CQ-induced p62 up-regulation at the mRNA and protein levels in both melanoma and SCC cells (Fig. 5, G, H, J, and K). We further confirmed the role of NF-κB with knockdown of RELA (Fig. 5, I and L). These data indicate that NF-κB is required for CQ-induced p62 expression and p62 up-regulation. These results demonstrate that CQ induces a positive feedback loop between p62 and NF-κB; CQ-induced p62 protein up-regulation triggers NF-κB activation, and the activated NF-κB induces p62 expression in turn.

JNK Signaling Is Critical for CQ-induced NF-κB Activation and p62 Expression

Next, we determined the role of JNK signaling in CQ-induced NF-κB activation, because previous studies have shown that JNK is required for high glucose-induced NF-κB activation in kidney epithelial cells (36). In both Mel624 and A431 cells, CQ induced phosphorylation of c-Jun (Fig. 6, A and B), a substrate of JNK kinase. In comparison, ATG5 or ATG7 deficiency had little effect on c-Jun phosphorylation (Fig. 6C), indicating that inhibiting autophagosome formation is dispensable for JNK activation. Inhibiting JNK signaling by JNK knockdown reduced CQ-induced p62 expression (Fig. 6D), NF-κB activation (Fig. 6E), HIF-1α protein level (Fig. 6, F and G), and BCL-2 and BCL-XL mRNA level (Fig. 6, H and I). These results indicate that JNK signaling is critical for CQ-induced NF-κB activation. However, inhibiting NF-κB by RELA knockdown had no effect on c-Jun phosphorylation (Fig. 6J), indicating that JNK is an upstream signal of NF-κB.

FIGURE 6.

Role of JNK signaling in CQ-induced NF-κB activation and p62 expression. A and B, immunoblot analysis of p-c-Jun, c-Jun, and GAPDH in Mel624 cells (A) and A431 cells (B) treated with or without CQ (25 μm) for 24 h. C, immunoblot analysis of p-c-Jun, c-Jun, and GAPDH in sh-NC, sh-ATG5, and sh-ATG7 Mel624 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 with or without CQ (25 μm) for 18 h. E, luciferase reporter assay of NF-κB activity in Mel624 cells transfected with control siRNA or siRNA targeting JNK (si-JNK), followed by treatment with or without CQ (25 μm) for 18 h. F and G, immunoblot analysis of p62, HIF-1α, and GAPDH in Mel624 cells transfected with control siRNA or siRNA targeting JNK (si-JNK), followed by treatment with or without CQ (25 μm) for 18 h. H and I, real time PCR analysis of BCL-2 and BCL-XL mRNA in Mel624 cells transfected with control siRNA or siRNA targeting JNK (si-JNK), followed by treatment with or without CQ (25 μm) for 18 h. J, immunoblot analysis of 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 μm) 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's t test)).

Inhibition of Either p62 or the NF-κB Pathway Sensitizes Cancer Cells to CQ-induced Cell Killing

Next, we assessed the role of the p62/NF-κB axis in CQ-induced cancer cell killing. In Mel624, A431, and MEF cells, CQ (25 μm) alone or p62 inhibition did not induce the activation of caspase-3 (Fig. 7, A–C), an indicator of apoptosis, or affect cell viability (Fig. 7, D–F). However, inhibition of p62 by shRNA knockdown or genetic deletion sensitized the cells to CQ-induced caspase-3 activation (Fig. 7, A–C) and decreased cell viability (Fig. 7, D–H). These results indicate that p62 up-regulation mediates CQ resistance.

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 or sh-p62 A375 cells treated with or without CQ (25 μm) for 24 h (A), sh-NC or sh-p62 A431 cells treated with or without CQ (25 μm) for 38 h (B), and WT or p62-KO MEF cells treated with or without CQ (25 μm) 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 or p62 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 in sh-NC or sh-p62 A375 cells treated with or without CQ (25 μm) 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's t test)).

Similar to p62 inhibition, pharmacological inhibition of NF-κB alone or CQ did not affect caspase-3 activation (Fig. 8, A–C) or cell viability (Fig. 8, D–I) in Mel624 and A375 melanoma and A431 SCC cells. However, inhibition of NF-κB by BMS or RELA knockdown sensitized the cells to CQ-induced caspase-3 activation (Fig. 8, A–C) and decreased cell viability (Fig. 8, D–M). These results indicate that CQ-induced activation of the NF-κB pathway promotes cell survival and confers a resistance mechanism to CQ in skin cancer cells.

FIGURE 8.

Blocking the NF-κB pathway sensitizes cancer cells to CQ-induced apoptotic cell death. A–C, immunoblot analysis of active caspase-3 and GAPDH in Mel624 (A), A375 (B), and A431 (C) cells treated with CQ (25 μm) for 24 h in the presence or absence of BMS (5 μm for A and B and 2 μm 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 (J and 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 μm) 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's t test)). N, schematic for the role of the p62/NF-κB feedback loop in CQ resistance in tumor cells.

Discussion

Autophagy can facilitate adaptation and survival of tumor cells in various stressful environments such as anticancer treatment and anoikis, hypoxia, and oxidation or starvation. Hence, inhibitors of the autophagy pathway such as CQ have the potential for cancer therapy. However, how tumor cells respond to CQ is still poorly understood. In this study, we have shown that CQ activates NF-κB through autophagosome accumulation, p62 up-regulation, and JNK signaling (Fig. 8N). NF-κB activation in turn induced p62 expression and thus formed a positive feedback loop. Inhibiting either p62 or NF-κB did sensitize tumor cells to CQ-induced apoptotic cell death. Thus, inhibiting either p62 or NF-κB may provide improved anticancer efficacy. Our findings demonstrate a novel mechanism by which tumor cells evade CQ-induced killing and thus suggest a resistance mechanism (Fig. 8N).

We found that activation of the p62/NF-κB pathway promotes cancer cell survival in response to CQ. CQ was found to induce p62 protein stabilization and thus activate NF-κB, which leads to the induction of p62 expression. Inhibiting either p62 or NF-κB sensitized tumor cells to CQ-induced killing. The activation of p62 and NF-κB promoted CQ resistance. Our findings are consistent with recent findings that CQ did not affect tumor cells directly in vivo but showed an antitumor effect normalizing the tumor vasculature (26). It is possible that in vivo the induction of the p62/NF-κB pathway protected tumor cells against a CQ-induced intrinsic antitumor effect. Future investigation is needed to determine whether other autophagy inhibitors induced similar resistance mechanisms.

As a selective autophagy substrate and signaling adaptor, p62 plays multifunctional roles in autophagy and signal transduction in carcinogenesis and cancer progression (37, 38) through regulating multiple signaling pathways, including NF-κB (35, 39), NRF2 (41–43), and Twist1 (44). Up-regulation of p62 has been shown in a number of human cancers (37, 38). Inhibiting p62 reduced cell proliferation, invasion, and migration (35, 37, 44). In addition, recent studies have demonstrated that p62 protects cells from apoptosis (46–48). We found that in both SCC and melanoma cells, CQ induced p62 protein up-regulation and p62 expression. Knockdown of p62 was shown to sensitize cells to CQ-induced cell death. These data indicate that p62 induction is critical for cell survival following CQ treatment.

In addition, we found that CQ induces NF-κB through p62 up-regulation. p62 up-regulation is required for NF-κB activation. It is possible that CQ-induced p62 promoted polyubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) as demonstrated in Ras activation during lung tumorigenesis (35). Recent studies have also shown that p62 is required for the activation of NF-κB in response to Toll-like receptor pathway activation (49). Intriguingly, we also show that the activation of NF-κB in turn further induced p62 gene expression in response to CQ treatment. It has been shown that TLR2/6 activation induces p62 expression through the NADPH oxidase pathway (49). In addition, inflammatory NF-κB activation in macrophages has been shown to induce p62 up-regulation, leading to mitophagy and thus limiting inflammasome activation (50). An NF-κB response element has been identified in the p62 promoter (51). However, the functional significance of this element and how NF-κB regulates p62 expression remain to be determined. Our findings underscore the critical role of the NF-κB signaling in tumor cell survival following CQ treatment. The role of other CQ-induced transcription factors such as CREB and AP-1 requires further investigation.

In addition to p62, we found that autophagosome abundance and JNK signaling are also critical for CQ-induced NF-κB activation. CQ increased autophagosome abundance and inhibiting autophagosome formation abolished CQ-induced NF-κB activation. In addition, CQ also activated JNK signaling, and inhibiting JNK signaling by JNK knockdown reduced CQ-induced NF-κB activation. This is consistent with previous studies showing that high glucose-induced NF-κB requires JNK signaling (36). It appears that CQ-induced NF-κB signaling requires p62, autophagosome, and JNK signaling. It is possible that, for CQ-induced NF-κB activation, autophagosome serves as a signing hub and that p62 and JNK are also required.

In summary, we have demonstrated that CQ induces NF-κB activation through autophagosome accumulation, p62 up-regulation, and JNK signaling. NF-κB activation in turn increased p62 expression, thereby forming a positive feedback loop. Blocking either p62 or NF-κB sensitizes tumor cells to CQ-induced cell killing. Our findings provide new molecular insights into the CQ response in tumor cells and CQ resistance in cancer therapy. These findings may facilitate development of improved therapeutic strategies by targeting the p62/NF-κB pathway.

Materials and Methods

Cell Culture

Mel624 melanoma cells, A375 (human amelanotic melanoma cells), A431 (human squamous carcinoma cells), WT, and p62 KO of mouse embryo fibroblast (MEF) cells were obtained from Dr. Seungmin Hwang, University of Chicago, 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 μg/ml streptomycin (Invitrogen).

Lentiviral Production and Infection

pLKO.1 sh-p62 (human) was obtained from Sigma. Lentivirus was produced by co-transfection into HEK-293T cells (human embryonic kidney cells) with lentiviral constructs together with the pCMVdelta8.2 packaging plasmid and pVSV-G envelope plasmid using GenJet^TM Plus DNA In Vitro Transfection Reagent (Signagen, Ijamsville, MD, as described previously (44, 52). Virus-containing supernatants were collected 24–48 h after transfection and used to infect recipients. Target cells were infected in the presence of Polybrene (8 μg/ml) (Sigma) and selected with puromycin (Santa Cruz Biotechnology, Santa Cruz, CA) at 1 μg/ml for 6 days.

Immunoblotting

Immunoblotting was performed as described previously (7, 44). Antibodies used were as follows: p62 (Progen Biotechnik GmbH, Heidelberg, Germany); HIF-1A (R&D Systems, Minneapolis, MN); p-IKKα/β, IKK, p-c-Jun, c-Jun, and RELA (Cell Signaling Technology, Beverly, MA); and GAPDH and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Cytokine Array Analysis

Conditioned medium derived from Mel624 incubated with or without CQ (25 μm) for 24 h was prepared in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 1% fetal bovine serum (HyClone, Logan, UT). Equal volumes (1 ml) of each conditioned medium were incubated with the pre-coated Proteome Profiler array membrane for human angiogenesis antibody (R&D Systems, Minneapolis, MN) and processed according to the manufacturer's instructions.

Luciferase Reporter Assays

Cells were transfected with 1 μg of pGL3 AP-1-Luc, pGL3 CREB-Luc, or pGL3 NF-κB-Luc and 0.025 μg of pRL-TK, which is used as a transfection efficiency control (Promega, Madison, WI), using GenJet^TM Plus DNA in vitro transfection reagent (Signagen, Ijamsville, MD) according to the manufacturer's instructions. Luciferase reporter assays were performed as described previously (7, 44).

siRNA Transfection

Cells were transfected with si-control, si-RNA (Cell Signaling Technology, Beverly, MA), targeting p62 (Cell Signaling Technology), targeting RELA (Dharmacon, Lafayette, CO), and targeting JNK (Santa Cruz Biotechnology, Santa Cruz, CA) using GenMute^TM siRNA transfection reagent (Signagen, Ijamsville, MD) according to the manufacturer's instructions.

Quantitative Real Time PCR

Quantitative real time PCR assays were performed using a CFX Connect real time system (Bio-Rad) with iQ SYBR Green Supermix (Bio-Rad). The threshold cycle number (CQ) for each sample was determined in triplicate. The CQ for values for HIF-1A, IL-8, BCL-2, BCL-XL, and p62 were normalized against GAPDH or β-actin as described previously (7, 44, 52). Amplification primers were as follows: 5′-GTT TAC TAA AGG ACA AGT CAC C-3′ (forward) and 5′-TTC TGT TTG TTG AAG GGA G-3′ (reverse) for the HIF-1A gene; 5′-ATG ACT TCC AAG CTG GCC GTG GCT-3′ (forward) and 5′-TCT CAG CCC TCT TCA AAA ACT TCT-3′ (reverse) for the human IL-8 gene; 5′-CAG AGA AGC CCA TGG ACA G-3′ (forward) and 5′-AGC TGC CTT GTA CCC ACA TC-3′ (reverse) for the human p62 gene; 5′-GTG GAT GAC TGA GTA CCT GAA C-3′ (forward) and 5′-GCC AGG AGA AAT CAA ACA GAG G-3′ (reverse) for BCL-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 β-actin gene; and 5′-ACC ACA GTC CAT GCC ATC AC-3′ (forward) and 5′-TCC ACC ACC CTG TTG CTG TA-3′ (reverse) for the human GAPDH gene.

Cell Viability Assay

Cell viability was assessed with Cell Counting Kit-8 (CCK-8) (Sigma). The CCK-8 analysis was performed following the manufacturer's protocol as described previously (40, 45, 53).

Immunofluorescence

The cells were fixed with 4% paraformaldehyde/PBS for 30 min and permeabilized in 0.5% Triton X-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 the blocking solution, the cells were incubated with LC3-Alexa Fluor® 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 Prolong Gold Antifade with DAPI (Invitrogen) to visualize the cell nuclei and observed under a fluorescence microscope (Olympus IX71).

Flow Cytometric Analysis of Apoptosis

Apoptotic cell death was determined using the annexin V-FITC apoptosis detection kit (eBioscience, San Diego), according to the manufacturer's instructions. Cell samples were then analyzed by BD FACSCalibur flow cytometer (BD Biosciences).

Statistical Analyses

Statistical analyses were performed using Prism 5 (GraphPad). Data were expressed as the mean of at least three independent experiments and analyzed by Student's t test. Error bars indicate the S.D. of the means. p < 0.05 was considered statistically significant.

Author Contributions

S. Y. and Y.-Y. H. conceived and coordinated the study and wrote the paper. S. Y. designed, performed, and analyzed the experiments shown in all figures. L. Q., A. S., and P. S. provided technical assistance and contributed to the preparation of the figures. All authors reviewed the results and approved the final version of the manuscript.