NLRP1 inhibits lung adenocarcinoma growth through mediating mitochondrial dysregulation in an inflammasome-independent manner

NLRP1, the first identified inflammasome-forming sensor, is thought to be involved in cancer, yet its definite function in lung adenocarcinoma (LUAD) remains unclear. Herein, we explored the expression and function of NLRP1 in LUAD. Decreased NLRP1 expression was identified in LUAD, which was associated with a poor prognosis. Overexpression of NLRP1 inhibited tumor growth in vitro and in vivo. Mechanically, this effect was observed regardless of inflammasome activation. Further studies revealed that overexpression of NLRP1 downregulated the phosphorylation of DRP1 and promoted mitochondrial fusion, which was mediated by inhibition of NF-κB activity. NF-κB agonist could neutralize the effect of NLRP1 on mitochondrial dynamics. In addition, LUAD sensitivity to cisplatin was enhanced by decreased mitochondrial fission resulting from up-regulated NLRP1. In conclusion, our findings demonstrated an unexpected role of NLRP1 in LUAD by modulating mitochondrial activities, which provides strong evidence for its potential in LUAD treatment.

NLRP1; Mitochondrial dysfunction; NF-κB signaling; Lung adenocarcinoma; Inflammasome

Authorship

Chen-jing Lin

Department of Central Laboratory, Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, ChinaShanghai Chest Hospital, Shanghai Jiao Tong University School of MedicineChinaShanghai, ChinaDepartment of Central Laboratory, Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

* These authors contributed equally to this work.

https://orcid.org/0000-0002-1852-9960

Guang-ang Tian

* These authors contributed equally to this work.

https://orcid.org/0000-0001-7047-0612

Wen-ya Zhao

https://orcid.org/0009-0005-2999-5616

Yi Tian

https://orcid.org/0009-0005-5990-1158

Yi-ru Liu

Department of Chemotherapy, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, ChinaThe First Affiliated Hospital of Wenzhou Medical UniversityChinaWenzhou, Zhejiang, ChinaDepartment of Chemotherapy, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

https://orcid.org/0009-0008-5603-7626

Dian-na Gu Correspondence: Ling Tian: <tl09168@hotmail.com> | Dian-na Gu <yinuo801@126.com>

https://orcid.org/0000-0002-2324-9920

Ling Tian Correspondence: Ling Tian: <tl09168@hotmail.com> | Dian-na Gu <yinuo801@126.com>

https://orcid.org/0000-0002-1058-3559

Correspondence:
Ling Tian: <tl09168@hotmail.com> | Dian-na Gu <yinuo801@126.com>

*
These authors contributed equally to this work.

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Figures

Figures (8)

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Figure 1
Decreased NLRP1 expression was correlated with poor clinical outcomes in lung adenocarcinoma (LUAD). A, Expression of NLRP family in LUAD and adjacent normal tissues. B, Expression level of NLRP1 in LUAD and adjacent non-tumor tissues from The Cancer Genome Atlas (TCGA) database was analyzed using GEPIA2. C, Expression analysis of NLRP1 in LUAD and adjacent normal tissues using 3 independent Gene Expression Omnibus (GEO) datasets. D, Immunostaining intensity score analysis of NLRP1 was analyzed using The Human Protein Atlas (THPA). E, Representative IHC images of the decreased NLRP1 expression in LUAD tissues compared with adjacent normal tissues. Scale bar, 100 μm. F, Association of NLRP1 expression with TNM stage was analyzed using GEPIA2. G and H, Prognostic value of NLRP1 in LUAD disease-free survival (DFS) and overall survival (OS) was analyzed using GEPIA2 (G) and K-M Plotter (H). T: Tumor tissue; N: Normal tissue; P: Paracancerous tissue. Data are reported as mean and SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Student's t-test.

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Figure 2
Decreased NLRP1 expression was correlated with poor clinical outcomes in lung adenocarcinoma (LUAD). A and B, Expression level of NLRP1 in LUAD and adjacent non-tumor tissues from tissue microarray. Scale bar, 100 μm. C, Representative images for strong staining and weak staining of NLRP1 in LUAD. Scale bar, 100 μm. D, Overall survival (OS) analysis of LUAD patients divided by NLRP1 expression. T: Tumor tissue; P: Paracancerous tissue.

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Figure 3
NLRP1 overexpression inhibited lung adenocarcinoma (LUAD) cell proliferation in vitro. A, Cell viability was determined by CCK8 assay in PC-9 and H1299 cells with stable overexpression (OE) of NLRP1. B, Colony formation assay was performed in PC-9 cells and H1299 cells with NLRP1 overexpression and control. C, Cell apoptosis of PC-9 and H1299 cells with stable overexpression of NLRP1 was assessed by flow cytometry assay with Annexin V/7-AAD double staining. D, Cell cycle arrest was determined for PC-9 cells and H1299 cells with NLRP1 overexpression and control. Data are reported as mean and SD. *P<0.05, **P<0.01, ***P<0.001; Student's t-test.

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Figure 4
NLRP1 overexpression inhibited lung adenocarcinoma (LUAD) growth in vivo. A-C, Representative tumor images (A), growth curves (B), and weights (C) in BALB/c mice bearing PC-9 cells with or without NLRP1 overexpression (OE) (n=5). D, Representative IHC images of PCNA staining in BALB/c tumor-bearing mice. Scale bar, 100 μm. E, Representative IHC images of capase-1 staining in mice tumors. Scale bar, 100 μm. Data are reported as mean and SD. *P<0.05, **P<0.01; Student's t-test.

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Figure 5
NLRP1 overexpression (OE) did not activate inflammasome in lung adenocarcinoma (LUAD) cells. A, RT-qPCR was performed to assess relative mRNA expression of interleukin (IL)-1β in NLRP1 stably-transfected LUAD cells. B, RT-qPCR was performed to assess relative mRNA expression of IL-18 in NLRP1 stably-transfected LUAD cells. C, Western blot was used to detect the expression levels of GSDMD and GSDME, and the quantification of the bands is shown in the lower part. D, Cell supernatant was harvested for ELISA detection of secreted IL-1β. Data are reported as mean and SD. ***P<0.001; Student's t-test. ns: no significance.

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Figure 6
NLRP1 overexpression (OE) inhibited mitochondria function. A, Reactive oxygen species (ROS) levels were detected by flow cytometry assay with DCFH-DA fluorescent probe. B, Mitochondrial membrane potential was evaluated by flow cytometry analysis with JC-1 fluorescent probe. C, NLRP1-induced mitochondrial fusion in PC-9 and H1299 cells was detected by confocal microscopy. Scale bar, 20 μm. D, Expression levels of p-DRP1, DRP1, MFN1, MFN2, and OPA1 were analyzed by western blot, and the quantification of the bands is shown on the right. E, Co-localization of DRP1 with mitochondria was detected by confocal microscopy. Scale bar, 10 μm. Data are reported as mean and SD. *P<0.05, **P<0.01, ***P<0.001; Student's t-test.

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Figure 7
NLRP1 overexpression (OE) reduced NF-κB activity and mediated mitochondria fusion. A, DEseq2 analysis showed differentially expressed genes (DEGs) between control group and NLRP1 overexpression groups in PC-9 cells. B, Gene Set Enrichment Analysis (GSEA) revealed that NF-κB signaling was enriched in the control group but not in the NLRP1 overexpression group. C, NF-κB phosphorylation (p-p65) expression was detected by western blot in NLRP1 stable-transfected lung adenocarcinoma (LUAD) cells, and the quantification of the bands is shown on the right. D, NF-κB phosphorylation (p-p65) and DRP1 phosphorylation (Ser616) expression were detected by western blot, and the quantification of the bands is shown in the lower part. E, Mitochondrial morphology was detected by confocal microscopy in lung cancer cells treated with DMSO or NF-κB activator 1. Scale bar, 10 μm. Data are reported as mean and SD. *P<0.05; Student's t-test.

Thumbnail

Figure 8
NLRP1 enhanced lung adenocarcinoma (LUAD) sensitivity to cisplatin by inhibiting DRP1 activity. A, Genomics of Drug Sensitivity in Cancer (GDSC) database was used to predict the drug sensitivity of NLRP1. B, NLRP1 overexpression (OE) enhanced cisplatin sensitivity according to CCK8 assay. C NLRP1 overexpression increased 5-fluorouracil and erlotinib sensitivity. D, Reactive oxygen species levels were detected by DCFH-DA fluorescent probe. E and F, Mitochondrial membrane potential was evaluated by flow cytometry assay with JC-1 fluorescent probe. G, Mitochondrial morphology was detected by confocal microscopy. Scale bar, 10 μm. H, The phosphorylation level of DRP1(Ser616) in control and NLRP1-overexpressing cancer cells after cisplatin treatment was detected by western blot, and the quantification of the bands is shown on the right. Data are reported as mean and SD. *P<0.05, ***P<0.001; Student's t-test.

Figure 1 Decreased NLRP1 expression was correlated with poor clinical outcomes in lung adenocarcinoma (LUAD). A, Expression of NLRP family in LUAD and adjacent normal tissues. B, Expression level of NLRP1 in LUAD and adjacent non-tumor tissues from The Cancer Genome Atlas (TCGA) database was analyzed using GEPIA2. C, Expression analysis of NLRP1 in LUAD and adjacent normal tissues using 3 independent Gene Expression Omnibus (GEO) datasets. D, Immunostaining intensity score analysis of NLRP1 was analyzed using The Human Protein Atlas (THPA). E, Representative IHC images of the decreased NLRP1 expression in LUAD tissues compared with adjacent normal tissues. Scale bar, 100 μm. F, Association of NLRP1 expression with TNM stage was analyzed using GEPIA2. G and H, Prognostic value of NLRP1 in LUAD disease-free survival (DFS) and overall survival (OS) was analyzed using GEPIA2 (G) and K-M Plotter (H). T: Tumor tissue; N: Normal tissue; P: Paracancerous tissue. Data are reported as mean and SD. P<0.05, P<0.01, P<0.001, ****P<0.0001; Student's t-test.

Figure 2 Decreased NLRP1 expression was correlated with poor clinical outcomes in lung adenocarcinoma (LUAD). A and B, Expression level of NLRP1 in LUAD and adjacent non-tumor tissues from tissue microarray. Scale bar, 100 μm. C, Representative images for strong staining and weak staining of NLRP1 in LUAD. Scale bar, 100 μm. D, Overall survival (OS) analysis of LUAD patients divided by NLRP1 expression. T: Tumor tissue; P: Paracancerous tissue.

Figure 3 NLRP1 overexpression inhibited lung adenocarcinoma (LUAD) cell proliferation in vitro. A, Cell viability was determined by CCK8 assay in PC-9 and H1299 cells with stable overexpression (OE) of NLRP1. B, Colony formation assay was performed in PC-9 cells and H1299 cells with NLRP1 overexpression and control. C, Cell apoptosis of PC-9 and H1299 cells with stable overexpression of NLRP1 was assessed by flow cytometry assay with Annexin V/7-AAD double staining. D, Cell cycle arrest was determined for PC-9 cells and H1299 cells with NLRP1 overexpression and control. Data are reported as mean and SD. P<0.05, P<0.01, P<0.001; Student's t-test.

Figure 4 NLRP1 overexpression inhibited lung adenocarcinoma (LUAD) growth in vivo. A-C, Representative tumor images (A), growth curves (B), and weights (C) in BALB/c mice bearing PC-9 cells with or without NLRP1 overexpression (OE) (n=5). D, Representative IHC images of PCNA staining in BALB/c tumor-bearing mice. Scale bar, 100 μm. E, Representative IHC images of capase-1 staining in mice tumors. Scale bar, 100 μm. Data are reported as mean and SD. *P<0.05, **P<0.01; Student's t-test.

Figure 5 NLRP1 overexpression (OE) did not activate inflammasome in lung adenocarcinoma (LUAD) cells. A, RT-qPCR was performed to assess relative mRNA expression of interleukin (IL)-1β in NLRP1 stably-transfected LUAD cells. B, RT-qPCR was performed to assess relative mRNA expression of IL-18 in NLRP1 stably-transfected LUAD cells. C, Western blot was used to detect the expression levels of GSDMD and GSDME, and the quantification of the bands is shown in the lower part. D, Cell supernatant was harvested for ELISA detection of secreted IL-1β. Data are reported as mean and SD. ***P<0.001; Student's t-test. ns: no significance.

Figure 6 NLRP1 overexpression (OE) inhibited mitochondria function. A, Reactive oxygen species (ROS) levels were detected by flow cytometry assay with DCFH-DA fluorescent probe. B, Mitochondrial membrane potential was evaluated by flow cytometry analysis with JC-1 fluorescent probe. C, NLRP1-induced mitochondrial fusion in PC-9 and H1299 cells was detected by confocal microscopy. Scale bar, 20 μm. D, Expression levels of p-DRP1, DRP1, MFN1, MFN2, and OPA1 were analyzed by western blot, and the quantification of the bands is shown on the right. E, Co-localization of DRP1 with mitochondria was detected by confocal microscopy. Scale bar, 10 μm. Data are reported as mean and SD. P<0.05, P<0.01, P<0.001; Student's t-test.

Figure 7 NLRP1 overexpression (OE) reduced NF-κB activity and mediated mitochondria fusion. A, DEseq2 analysis showed differentially expressed genes (DEGs) between control group and NLRP1 overexpression groups in PC-9 cells. B, Gene Set Enrichment Analysis (GSEA) revealed that NF-κB signaling was enriched in the control group but not in the NLRP1 overexpression group. C, NF-κB phosphorylation (p-p65) expression was detected by western blot in NLRP1 stable-transfected lung adenocarcinoma (LUAD) cells, and the quantification of the bands is shown on the right. D, NF-κB phosphorylation (p-p65) and DRP1 phosphorylation (Ser616) expression were detected by western blot, and the quantification of the bands is shown in the lower part. E, Mitochondrial morphology was detected by confocal microscopy in lung cancer cells treated with DMSO or NF-κB activator 1. Scale bar, 10 μm. Data are reported as mean and SD. *P<0.05; Student's t-test.

Figure 8 NLRP1 enhanced lung adenocarcinoma (LUAD) sensitivity to cisplatin by inhibiting DRP1 activity. A, Genomics of Drug Sensitivity in Cancer (GDSC) database was used to predict the drug sensitivity of NLRP1. B, NLRP1 overexpression (OE) enhanced cisplatin sensitivity according to CCK8 assay. C NLRP1 overexpression increased 5-fluorouracil and erlotinib sensitivity. D, Reactive oxygen species levels were detected by DCFH-DA fluorescent probe. E and F, Mitochondrial membrane potential was evaluated by flow cytometry assay with JC-1 fluorescent probe. G, Mitochondrial morphology was detected by confocal microscopy. Scale bar, 10 μm. H, The phosphorylation level of DRP1(Ser616) in control and NLRP1-overexpressing cancer cells after cisplatin treatment was detected by western blot, and the quantification of the bands is shown on the right. Data are reported as mean and SD. *P<0.05, ***P<0.001; Student's t-test.

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NLRP1 inhibits lung adenocarcinoma growth through mediating mitochondrial dysregulation in an inflammasome-independent manner

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[1] Correspondence:
Ling Tian: <tl09168@hotmail.com> | Dian-na Gu <yinuo801@126.com>