Multiomics analysis revealed the mechanisms related to the enhancement of proliferation, metastasis and EGFR-TKI resistance in EGFR-mutant LUAD with ARID1A deficiency

Abstract

Introduction: Dysregulated ARID1A expression is frequently detected in lung adenocarcinoma (LUAD) and mediates significant changes in cancer behaviors and a poor prognosis. ARID1A deficiency in LUAD enhances proliferation and metastasis, which could be induced by activation of the Akt signaling pathway. However, no further exploration of the mechanisms has been performed.

Methods: Lentivirus was used for the establishment of the ARID1A knockdown (ARID1A-KD) cell line. MTS and migration/invasion assays were used to examine changes in cell behaviors. RNA-seq and proteomics methods were applied. ARID1A expression in tissue samples was determined by IHC. R software was used to construct a nomogram.

Results: ARID1A KD significantly promoted the cell cycle and accelerated cell division. In addition, ARID1A KD increased the phosphorylation level of a series of oncogenic proteins, such as EGFR, ErbB2 and RAF1, activated the corresponding pathways and resulted in disease progression. In addition, the bypass activation of the ErbB pathway, the activation of the VEGF pathway and the expression level changes in epithelial-mesenchymal transformation biomarkers induced by ARID1A KD contributed to the insensitivity to EGFR-TKIs. The relationship between ARID1A and the sensitivity to EGFR-TKIs was also determined using tissue samples from LUAD patients.

Conclusion: Loss of ARID1A expression influences the cell cycle, accelerates cell division, and promotes metastasis. EGFR-mutant LUAD patients with low ARID1A expression had poor overall survival. In addition, low ARID1A expression was associated with a poor prognosis in EGFR-mutant LUAD patients who received first-generation EGFR-TKI treatment. Video abstract.

Keywords: ARID1A; Cell cycle; EGFR-TKI resistance; EGFR-mutant LUAD; Multiomics analysis.

Fig. 1

ARID1A serves as a protective factor in lung adenocarcinoma with or without EGFR mutations. A Lung adenocarcinoma patients harboring ARID1A mutations had poorer overall survival than the wild-type group. B–D Online database analysis revealed that low ARID1A expression was associated with poor overall survival in LUAD patients. E Low ARID1A expression was associated with a poor prognosis in EGFR-mutant lung adenocarcinoma patients in the TCGA database. F Verification of the relative expression of target proteins after the construction of ARID1A knockdown EGFR-mutant lung adenocarcinoma cell lines

Fig. 2

ARID1A knockdown significantly enhanced the proliferation, migration and invasion of EGFR mutant lung adenocarcinoma cells. A Proliferative examination using MTS experiments; B Migration and invasion examination using Transwell assays. C Volcano plots and heatmap of differentially expressed proteins revealed by mass spectrometry. D–E Enrichment analysis for differentially expressed proteins revealed by mass spectrometry for total proteins and phosphorylated proteins

Fig. 3

Multiomics analysis revealed the underlying mechanisms of the enhancement of proliferation and metastasis induced by ARID1A knockdown. A GSEA based on differentially expressed genes revealed by RNA-seq sequencing. B–C. Enrichment analysis based on differentially expressed proteins revealed by mass spectrometry for total proteins and phosphorylated proteins. D Relative expression level of proliferative biomarkers revealed by RNA-seq sequencing and mass spectrometry

Fig. 4

ARID1A knockdown promoted the cell cycle of EGFR-mutant lung adenocarcinoma and increased the cell proliferation rate. A–C Proteins enriched in the cell cycle-related pathways revealed by mass spectrometry (all listed proteins were found to be upregulated in ARID1A knockdown cells with statistical significance). D Relative expression levels of RB-E2F pathway-related proteins revealed by RNA-seq sequencing and mass spectrometry. E Relative expression levels of cell cycle proteins, including cyclins, cyclin-dependent kinase and cell division cycle proteins, revealed by mass spectrometry

Fig. 5

The enhancement of metastasis in ARID1A knockdown EGFR-mutant lung adenocarcinoma is a process involving multiple factors and multiple mechanisms. A–D Proteins enriched in metastasis-related pathways revealed by mass spectrometry (all listed proteins were upregulated in ARID1A knockdown cells with statistical significance). E Relative expression levels of phosphorylated ErbB family members. F Relative expression level of phosphorylated RAF1. G Relative expression level of phosphorylated AKT2. H Relative expression levels of phosphorylated proteins belonging to the MAPK pathway. I Relative expression levels of phosphorylated metastasis-related proteins and EMT biomarkers

Fig. 6

ARID1A expression serves as a novel biomarker for resistance to first-generation EGFR-TKIs in EGFR-mutant lung adenocarcinoma patients. A Underlying mechanisms based on differentially expressed proteins revealed by mass spectrometry for phosphorylated proteins, which might be associated with resistance to EGFR-TKIs. B Representative images of IHC staining of ARID1A in EGFR-mutant lung adenocarcinoma. C Progression-free survival after treatment with first-generation EGFR-TKIs in all enrolled patients and patients who received gefitinib (divided by ARID1A expression). D ROC curve for ARID1A expression in predicting the progression-free survival of patients treated with first-generation EGFR-TKIs. E The construction and verification of a nomogram based on ARID1A expression and ECOG score for EGFR-mutant lung adenocarcinoma patients who received first-generation EGFR-TKIs. F Drug selection based on the NCI-60 database for non-small cell lung cancer patients with ARID1A deficiency