Cancer Res Treat.  2020 Jul;52(3):867-885. 10.4143/crt.2019.606.

Caspase Recruitment Domain Containing Protein 9 Suppresses Non-Small Cell Lung Cancer Proliferation and Invasion via Inhibiting MAPK/p38 Pathway

• Pan L
• Tan Y
• Wang B
• Qiu W
• Yin Y
• Ge H
• Zhu H

• KMID: 2504466
• DOI: http://doi.org/10.4143/crt.2019.606

Abstract

Purpose
Caspase recruitment domain containing protein 9 (CARD9) has been demonstrated to be a pro-tumor factor in various cancers. However, our previous study found a significant decrease of CARD9 in malignant pleural effusion compared with benign pleural effusion. So we investigated the role of CARD9 in non-small cell lung cancer (NSCLC) and its working mechanism.
Materials and Methods
Immunohistochemistry, western blot, and quantitative real-time polymerase chain reaction were used to detect the expression of CARD9 in specimens of NSCLC patients. The Cancer Genome Atlas (TCGA) databasewas also used to analyze the expression of CARD9 in NSCLC and its predicting value for prognosis. Immunofluorescence was used for CARD9 cellular location. Cell growth assay, clonal formation assay, wound healing assay, matrigel invasion assay, and flow cytometry were used to test cell proliferation, migration, invasion, apoptosis, and cycle progression of NSCLC cells with CARD9 knockdown or CARD9 overexpression. Co-immunoprecipitation was used to identify the interaction between CARD9 and B-cell lymphoma 10 (BCL10). SB203580 was used to inhibit p38 activation.
Results
CARD9 was decreased in NSCLC tissues compared with normal tissues; low CARD9 expression was associated with poor survival. CARD9 was expressed both in tumor cells and macrophages. Downregulation of CARD9 in NSCLC cells enhanced the abilities of proliferation, invasion and migration via activated MAPK/p38 signaling, while overexpression of CARD9 presented antitumor effects. BCL10 was identified to interact with CARD9.
Conclusion
We demonstrate that CARD9 is an independent prognostic factor in NSCLC patients and inhibits proliferation, migration, and invasion by suppressing MAPK/p38 pathway in NSCLC cells.

Keyword

CARD9; Non-small cell lung cancer; Prognosis; Targeted therapy

Figure

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  Fig. 1. Caspase recruitment domain containing protein 9 (CARD9) was decreased at the level of mRNA and protein in non-small cell lung cancer (NSCLC) tissues. (A, B) CARD9 mRNA level between tumor tissues and normal tissue in lung adenocarcinoma and lung squamous cell carcinoma based on The Cancer Genome Atlas database lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) projects. (C) Expression of CARD9 in LUAD and LUSC tissues and paired adjacent normal lung tissues by immunohistochemistry. (D) Comparison of immunohistochemistry score of CARD9 expression in 94 NSCLC tissues and paired adjacent normal lung tissues. tissues. (E) CARD9 expression in tumor tissues (T) and normal lung tissues (N) by western blot. (F) CARD9 mRNA level of 20 pairs of tumor tissues and normal lung tissues by real-time quantitative reverse transcription polymerase chain reaction. (G) Immunofluorescence scanning of tumor tissues from NSCLC patient. Anti-CD68 was for staining macrophages and anti–epithelial cell adhesion molecule (EpCAM) for tumor cells. White arrows showed the CARD9-expressing macrophages and tumor cells. Scale bar=10 μm. Values are presented as mean±standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 2. Decreased expression of caspase recruitment domain containing protein 9 (CARD9) correlated with poor prognosis. (A, B) Overall survival (OS) of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) from The Cancer Genome Atlas database. (C, D) The OS and progression-free survival (PFS) of our clinical cohort of non-small cell lung cancer patients. (E, F) The OS and PFS of our clinical cohort of LUAD patients.

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  Fig. 3. Downregulated caspase recruitment domain containing protein 9 (CARD9) promoted non-small cell lung cancer (NSCLC) migration and invasion. (A, B) Baseline of CARD9 in human bronchial epithelioid cells (HBE), human leukemic monocyte (THP-1), BEAS-2B cells (human normal lung epithelial cells), and NSCLC cell lines (H1299, A549, 95C, 95D, and PC9) by western blot. (C) Western blot of CARD9, E-cadherin, N-cadherin, transforming growth factor β (TGF-β), and vimentin in CARD9-overexpressing H1299 and PC9, CARD9-knockdown PC9, and their control cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal reference. Numbers under the bank presented relative expression of protein evaluated by Image J. (D) Reverse transcription polymerase chain reaction was used to determine successful cells transfection. (E, F) Migration and invasion abilities of CARD9-overexpressing H1299 and PC9 cells, and CARD9-knockdown PC9 cells compared with their control cells. Scale bar=50 μm. Experiments were performed in triplicate and values are represented as mean±standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 4. Caspase recruitment domain containing protein 9 (CARD9) suppressed cells proliferation but supported cell apoptosis. (A, B) Cell growth assay and clonal formation assay of CARD9-overexpressing H1299 and PC9 cells, PC9 shCARD9, and their control cells. (C, D) Analysis of cell circle progression and apoptosis of CARD9-overexpressing H1299 and PC9 cells, CARD9-knockdown PC9 and their control cells. (E) Western blot of CARD9, cleaved-caspase-3, caspase-3, and cyclin D1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal reference. Numbers under the bank presenting relative expression of protein evaluated by Image J. Experiments were performed in triplicate and values are represented as mean±standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 5. Caspase recruitment domain containing protein 9 (CARD9) interacted with BCL10 and inhibited p-p38 activation. (A, B) Western blot of nuclear factor κB signaling and mitogen-activated protein kinases (MAPK) signaling in CARD9-overexpressing H1299 and PC9 cells, CARD9-knockdown PC9 and their control cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal reference. Numbers under the bank presenting relative expression of protein evaluated by Image J. (C) The interaction between CARD9 and BCL10 was confirmed by coimmunoprecipitation in PC9 cells and H1299 cells. IB, immunoblot. (D) Interacting protein network of CARD9 was searched from String database.

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  Fig. 6. Caspase recruitment domain containing protein 9 (CARD9) suppressed non-small cell lung cancer proliferation and promoted apoptosis via inhibition of mitogen-activated protein kinase/p38 activation. (A) Western blot of p-p38 in PC9 shCARD9 and PC9 shControl cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal reference. Numbers under the bank presenting relative expression of protein evaluated by Image J. (B, C) Cell growth assay and clonal formation assay of SB203580-treated PC9 shCARD9 and PC9 shControl cells and their counterparts. ***The comparison between SB203580-treated PC9 shCARD9 and PC9 shCARD9 (p < 0.001); ns, the comparison between SB203580-treated PC9 shControl and PC9 shControl. DMSO, dimethyl sulfoxide. (D, E) Invasion and migration abilities of SB203580-treated PC9 shCARD9 and PC9 shControl cells and their counterparts. Scale bars=50 μm. (F, G) Analysis of apoptosis and cell circle progression in SB203580-treated PC9 shCARD9 and PC9 shControl cells and their counterparts. Experiments were performed in triplicate and values are represented as mean±standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

Reference

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