AK4 promotes nasopharyngeal carcinoma metastasis and chemoresistance by activating NLRP3 inflammatory complex

Abstract

Metastasis is the main cause of treatment failure in nasopharyngeal carcinoma (NPC). Our previous study developed a transcriptomics-based gene signature (AK4, CPAMD8, DDAH1, and CRTR1) to predict metastasis in NPC and identify candidates that could benefit from induction chemotherapy (IC). Of these, adenylate kinase 4 (AK4) is a potent oncogene involved in the malignant progression of a variety of tumors. This study investigated the expression and mechanism of action of AK4, a member of the AK family of enzymes, in NPC. Quantitative real-time PCR, western blotting, and immunohistochemistry revealed that AK4 was upregulated in NPC and correlated with metastasis and chemoresistance. Stable ectopic overexpression of AK4 in NPC cell lines conferred resistance to taxol-induced apoptosis, promoted the migration, invasion, and EMT phenotype, and induced IL-1β secretion by activating the NLRP3 signaling pathway; knockdown of AK4 had the opposite effects. Mechanistically, AK4 co-localized with NNT, upregulated NLRP3 and IL-1β, and consequently altered NPC cell metastasis and chemoresistance. AK4 may play a role in the development of NPC and represent a potential therapeutic target.

Fig. 1. AK4 is upregulated in nasopharyngeal carcinoma (NPC) and correlated with metastasis and chemoresistance.

AK4 expression in NPC tumor and normal tissue samples in array express microarray data (GEO accession number: GSE12452 and GSE53819; http://www.ncbi.nlm.nih.gov/geo/) (A, B), The Cancer Genome Atlas (head and neck) tumor, and normal tissue microarray data (https://cancergenome.nih.gov/) (C). GSEA plot showing that AK4 expression is positively correlated with NPC-activated gene signatures (SENGUPTA_NASOPHARYNGEAL_CARCINOMA_UP) and inversely correlated with NPC-suppressed gene signatures (SENGUPTA_NASOPHARYNGEAL_CARCINOMA_DN) in our previous nasopharyngeal carcinoma gene expression profiles (n = 24) (D). GSEA was performed using GSEA 4.0.3 (http://www.broadinstitute.org/gsea/). Western blotting of AK4 protein expression in NP69 immortalized nasopharyngeal epithelial cells and eleven cultured NPC cell lines (E). Representative images of immunohistochemical staining for AK4 in normal nasopharyngeal epithelial biopsies, low and high AK4 expression in NPC tissues, and metastatic tissues of patients with NPC (F, G). Representative images of immunohistochemical staining of AK4 in chemosensitive and chemoresistant NPC tissues from our previous study (H). GSEA plot showing that AK4 expression is positively correlated with metastasis-activated gene signatures (WINNEPENNINCKX_MELANOMA_METASTASIS_UP) and inversely correlated with metastasis-suppressed gene signatures (WINNEPENNINCKX_MELANOMA_METASTASIS_DN) in our previous NPC gene expression profiles (n = 24) (I).

Fig. 2. AK4 confers resistance to taxol-induced apoptosis in vitro.

A Western blot analysis of ectopic AK4 overexpression in NPC cell lines (A). Western blot analysis of AK4 knockdown using specific shRNAs (B). CCK-8 assay for the viability of AK4-overexpressing (C) and AK4-knockdown (D) cells after 72 h of treatment with the indicated concentrations of taxol. Flow cytometry analysis of the percentage of apoptotic cells (Annexin V⁺/PI cells and Annexin V⁺/PI⁺ cells) in the indicated cell lines after 72 h of treatment with taxol (E, F).

Fig. 3. AK4 promotes the migration, invasion, and EMT of NPC cells in vitro.

AK4 overexpression promotes 6-10B and SUNE2 cell migration and invasion as determined using wound healing and transwell invasion assays (A, B). AK4 knockdown inhibits S18 and 5–8 F cell migration and invasion as determined using wound healing and transwell invasion assays (C, D). Gene set enrichment analysis (GSEA) plot showing AK4 expression is positively correlated with EMT-activated gene signatures (HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION) in published gene expression profiles of 31 cases of NPC (NCBI/GEO/GSE12452) (E). Western blot analysis for E-cadherin and vimentin in 6-10B-vector, 6-10B-AK4, SUNE2-vector, SUNE2-AK4, S18-vector, S18-AK4-KD#1 and S18-AK4-KD#2, and 5-8F-AK4-KD#1 and 5–8 F -AK4-KD#2 cells; α-tubulin was used as the loading control (F). Data are represented as the mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001; Student’s t test.

Fig. 4. AK4 promotes chemoresistance and metastasis in vivo.

Tumors formed by AK4-transduced SUNE2 cells were larger than the vector control tumors. Conversely, tumors formed by AK4-silenced 5–8 F cells were smaller than those formed by the vector cells (A). Tumor volume growth curve (B). Representative images of immunofluorescence staining of TUNEL-stained cells in the indicated tumors (C). SUNE2-vector, SUNE2-AK4, 5–8F-NC, and 5–8 F shAK4#1 cells (1 × 10⁶ cells in 100 µl PBS) were injected into the tail vein of mice. Mice in the AK4 group displayed a significantly higher number of metastatic lung nodules than mice in the control group and vice versa. Representative images and quantification of metastatic nodules in the lungs of mice (D, E). Data are represented as the mean ± SEM. ** p < 0.01, *** p < 0.001; Student’s t test.

Fig. 5. Transcriptome analysis reveals that IL-1β is a downstream target affected by AK4.

Heat-map analysis of 41 downregulated genes screened out between S18 NC versus S18 shAK4#1, S18 shAK4#2 cells and 5–8 F NC versus 5–8 F shAK4#1, 5–8 F shAK4#2 cells (A). Kyoto Encyclopedia of Genes and Genomes pathway determined the MAPK signaling pathway, NOD-like receptor signaling pathway, and cytokine receptor interaction gene sets, which were significantly enriched in DEGs regulated by AK4 (B). Venn diagram analysis shows that IL-1β is a key molecule in the above three signaling pathways (C). The mRNA expression level of IL-1β in AK4 knockdown cells was significantly decreased (D). The expression levels of IL-1β in the conditioned medium of AK4 knockdown cells was significantly decreased (E). Data are represented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t test.

Fig. 6. AK4 promotes NPC cell metastasis by regulating IL-1β secretion.

The promoting effect of IL-1β on NPC migration increased with the increase in concentration (A). Inhibition of IL-1β signaling using IL-1Ra partially abrogated AK4-promoted migration, invasion, and resistance to taxol-induced apoptosis in vitro (B–E). IL-1Ra inhibited lung metastasis in the vector control cells and AK4 overexpressing cells (D). Survival analysis showed that the 5-year DMFS, PFS, and OS rates of patients with NPC and higher serum IL-1β levels were significantly lower than those of patients with NPC and lower IL-1β concentration (E). Data are represented as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; Student’s t test.

Fig. 7. AK4 induces IL-1β secretion by activating the NLRP3 signaling pathway.

Western blot revealed that AK4 overexpression upregulated the expression of NLRP3, caspase-1, and IL-1β, and AK4 knockdown in cells reduced the expression of NLRP3, caspase-1, and IL-1β (A). AK4 overexpression enhanced the interaction between NLRP3 and ASC (B). Western blot showed that NLRP3-targeting siRNAs were transfected into 6-10B cells stably overexpressing AK4 (C). NLRP3 knockdown abrogated AK4-mediated IL-1β release and promotion of migration and invasion (D, E). Data are represented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Student’s t test.

Fig. 8. AK4 regulates NLRP3 signaling by binding to NNT.

AK4 overexpression increased ROS levels and vice versa (A, B). AK4 overexpression suppressed intracellular NADPH/NADP+ levels and vice versa (C, D). Representative mass spectrometry plots and sequences of peptides from AK4 (E). Immunoprecipitation assay showing the interaction between AK4 and NNT (F). Immunofluorescence staining revealed that endogenous AK4 co-localized with NNT in the cytoplasm of NPC cells (G). Multiplex immunofluorescence staining revealed that AK4 co-localized with NNT in NPC biopsies (I). G scale bar: 10 μm.