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

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.

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)(4)(5)(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 treat-ment 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)(12)(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)(23)(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)(23)(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-B activation through autophagosome accumulation, p62, and JNK signaling, which mediated CQ resistance in both squamous cell carcinoma (SCC) and melanoma cells.

CQ Induces the Activation of NF-B Activation and the
Expression of Its Target Genes HIF-1␣ 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 M induced apoptosis, whereas lower concentrations of CQ (10 and 25 M) had no effect (Fig. 1 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.
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-1␣ (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-1␣ up-regulation (Fig. 1G). Treatment 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 through a mechanism other than protein stability. In addition to HIF-1␣, 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-1␣, and IL-8 mRNA levels ( Fig. 1, N-P).
To determine how CQ up-regulates HIF-1␣ 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-1␣ and IL-8. These included cAMP-response elementbinding protein (CREB), activator protein 1 (AP-1), and nuclear

p62 and NF-B in Chloroquine Resistance
transcription factor-B (NF-B) (30 -32). In melanoma cells, CQ increased the transcriptional activity of NF-B and, to a much lesser extent, CREB and AP-1 (Fig. 1Q). It also increased the phosphorylation of IKK (Fig. 1R), which activates NF-B through phosphorylating and inducing degradation of the NF-B inhibitor (33,34). These results indicate that CQ increases the expression of HIF-1␣ and IL-8 and activates NF-B.
To determine the role of NF-B activity in the CQ-induced expression of HIF-1␣ 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-B p65 subunit, to inhibit NF-B activity. BMS or knockdown of RELA prevented the increases in the protein levels of HIF-1␣ and mRNA levels of HIF-1␣ 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-B activity. These results indicate that 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 Activation-To determine the mechanism by which CQ activates NF-B, 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-B 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-B 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-B activation.
p62 Up-regulation Is Required for CQ-induced NF-B Activation-Next, we assessed the role of p62 up-regulation, because induction of p62 by Ras activation has been shown to lead to NF-B 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 CQinduced 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-B activation (Fig. 4, E and F). In addition, p62 inhibition prevented CQ-induced HIF-1␣ upregulation 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-B activation.
NF-B 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.
To determine how CQ induces p62 expression, we analyzed the role of NF-B. Inhibiting the NF-B 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

p62 and NF-B in Chloroquine Resistance
FEBRUARY 24, 2017 • VOLUME 292 • NUMBER 8 further confirmed the role of NF-B with knockdown of RELA (Fig. 5, I and L). These data indicate that NF-B 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-B; CQ-induced p62 protein up-regulation triggers NF-B activation, and the activated NF-B induces p62 expression in turn.
JNK Signaling Is Critical for CQ-induced NF-B Activation and p62 Expression-Next, we determined the role of JNK signaling in CQ-induced NF-B activation, because previous studies have shown that JNK is required for high glucose-induced NF-B 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-B activation (Fig. 6E), HIF-1␣ 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-B activation. However, inhibiting NF-B by RELA knockdown had no effect on c-Jun phosphorylation (Fig. 6J), indicating that JNK is an upstream signal of NF-B.

Inhibition of Either p62 or the NF-B Pathway Sensitizes Cancer Cells to CQ-induced Cell
Killing-Next, we assessed the role of the p62/NF-B axis in CQ-induced cancer cell killing. In Mel624, A431, and MEF cells, CQ (25 M) 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.
Similar to p62 inhibition, pharmacological inhibition of NF-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 melanoma and A431 SCC cells. However, inhibition of NF-B 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-B pathway promotes cell survival and confers a resistance mechanism to CQ in skin cancer 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,  's t test)).

p62 and NF-B in Chloroquine Resistance
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-B through autophagosome accu-mulation, p62 up-regulation, and JNK signaling (Fig. 8N). NF-B activation in turn induced p62 expression and thus formed a positive feedback loop. Inhibiting either p62 or NF-B did sensitize tumor cells to CQ-induced apoptotic cell death.   's t test)).

p62 and NF-B in Chloroquine Resistance
FEBRUARY 24, 2017 • VOLUME 292 • NUMBER 8 Thus, inhibiting either p62 or NF-B 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-B pathway promotes cancer cell survival in response to CQ. CQ was found to induce 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 killing. The activation of p62 and NF-B 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-B pathway protected tumor cells against a CQ-induced intrinsic antitumor effect. Future investigation is needed to determine   's t test)).

p62 and NF-B in Chloroquine Resistance
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-B (35,39), NRF2 (41)(42)(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-B through p62 up-regulation. p62 up-regulation is required for NF-B 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-B in response to Toll-like receptor pathway activation (49). Intriguingly, we also show that the activation of NF-B 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-B activation in macrophages has been shown to induce p62 up-regulation, leading to mitophagy and thus limiting inflammasome activation (50). An NF-B response element has been identified in the p62 promoter (51). However, the functional significance of this element and how NF-B regulates p62 expression remain to be determined. Our findings underscore the critical role of the NF-B 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-B activation. CQ increased autophagosome abundance and inhibiting autophagosome formation abolished CQ-induced NF-B activation. In addition, CQ also activated JNK signaling, and inhibiting JNK signaling by JNK knockdown reduced CQ-induced NF-B activation. This is consistent with previous studies showing that high glucose-induced NF-B requires JNK signaling (36). It appears that CQ-induced NF-B signaling

p62 and NF-B in Chloroquine Resistance
FEBRUARY 24, 2017 • VOLUME 292 • NUMBER 8 requires p62, autophagosome, and JNK signaling. It is possible that, for CQ-induced NF-B activation, autophagosome serves as a signing hub and that p62 and JNK are also required.
In summary, we have demonstrated that CQ induces NF-B activation through autophagosome accumulation, p62 up-regulation, and JNK signaling. NF-B activation in turn increased p62 expression, thereby forming a positive feedback loop. Blocking either p62 or NF-B sensitizes tumor cells to CQinduced 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-B pathway.
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.