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.

Keywords: NF-κB; autophagy; chloroquine; melanoma; p62 (sequestosome 1(SQSTM1)); tumor cell biology.

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)).

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)).

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)).

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)).

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)).

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)).

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)).

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.