Effect of chronic intermittent hypoxia-induced HIF-1α/ATAD2 expression on lung cancer stemness

Abstract

Background: Obstructive sleep apnea is associated with increased lung cancer incidence and mortality. Cancer stem cells (CSCs) are characterized by their self-renewing ability, which contributes to metastasis, recurrence, and drug resistance. ATPase family AAA domain-containing protein 2 (ATAD2) induces malignancy in different types of tumors. However, a correlation between ATAD2 expression and CSCs in lung cancer has not yet been reported.

Methods: The relative messenger RNA (mRNA) levels of ATAD2, CD44, CD133, and hypoxia-inducible factor (HIF)-1α were determined using reverse-transcription quantitative polymerase chain reaction. ATAD2 protein levels were determined using Western blotting. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assays were performed to analyze the proliferation of lung cancer cells. Transwell migration and invasion assays were performed to evaluate cell migration and invasion, respectively. Tumor sphere formation analysis was used to determine tumor spheroid capacity. The link between ATAD2 and HIF-1α was verified using a dual-luciferase reporter assay. Immunofluorescence staining was performed to assess mitochondrial reactive oxygen species (mtROS) production. Flow cytometry analysis was conducted to determine the CD133 and CD44 positive cell ratio.

Results: We evaluated the relative expression of ATAD2 in four lung cancer cell lines (A549, SPC-A1, H460, and H1299 cells) and found increased mRNA and protein levels of ATAD2 in lung cancer samples. ATAD2 overexpression was a poor prognostic factor for lung cancer patients. Loss of ATAD2 reduced lung cancer cell viability and proliferation. Additionally, ATAD2 knockdown repressed lung cancer cell migration, invasion, stem-cell-like properties, and mtROS production. Chronic intermittent hypoxia (CIH)-induced HIF-1α expression significantly activated ATAD2 during lung cancer progression.

Conclusions: This study found that CIH induced HIF-1α expression, which acts as a transcriptional activator of ATAD2. The present study also suggests a novel mechanism by which the integrity of CIH-triggered HIF-1α/ATAD2 may determine lung cancer aggressiveness via the interplay of mtROS and stemness in lung cancer cells.

Keywords: ATAD2; CIH; HIF-1α; Lung cancer stem cells.

Fig. 1

ATAD2 expression is upregulated in LUAD tissues and cells. A Overexpression of ATAD2 mRNA is observed in LUAD tissues compared with normal samples [gene expression profiling interactive analysis (GEPIA)]. B ATAD2 mRNA expression is significantly related to the cancer stage of the patient (GEPIA). C Prognostic feature of mRNA expression of ATAD2 in LUAD patients (Kaplan–Meier plot). Overall survival (OS) curve comparing patients with high (red) and low (black) ATAD2 expression in lung cancer plotted using the Kaplan–Meier method with a threshold of p-value < 0.05. D mRNA expression of ATAD2 is increased in lung cancer cells compared with normal lung epithelial HBE cells. E, F Protein expression of ATAD2 is increased in the tissues of lung cancer patients. *p < 0.05

Fig. 2

Effects of ATAD2 on lung cancer cell viability and proliferation. A Validation of siRNA knockdown efficiency in A549 and SPC-A1 cells as determined using RT-qPCR. B, C Cell counting kit (CCK)-8 proliferation assay in siRNA-negative control (NC) or siRNA-ATAD2-transfected A549 and SPC-A1 cells. D 5-Ethynyl-2′-deoxyuridine (EdU) proliferation assay using siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. Scale bar = 100 μm. E Colony-formation proliferation assay in siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. Error bars indicate mean ± standard deviation (SD). All experiments performed in triplicate. *p < 0.05

Fig. 3

Effects of ATAD2 knockdown on lung cancer cell migration, invasion, and stemness. A, B Transwell migration assay using siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. C, D Transwell invasion assay using siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. E, F Tumor sphere formation assay using siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. Scale bar = 100 μm. G, H CD133 and CD44 mRNA levels in siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. Error bars indicate mean ± SD. All experiments performed in triplicate. *p < 0.05

Fig. 4

Effect of decreased ATAD2 levels on mtROS production in lung cancer cells. A ATAD2 mRNA expression in lung cancer cells transfected with the ATAD2 overexpression plasmid. B Cellular mtROS accumulation in A549 and SPC-A1 cells assessed using flow cytometry analysis. C, D Nuclei are stained with Hoechst (blue), and mitochondria ROS are stained using MitoSOX-red in A549 and SPCA1 cells. Merged panels demonstrate the number of MitoSOX-red positive cells among the total cells. Scale bar = 100 μm. Error bars indicate mean ± SD. All experiments performed in triplicate. *p < 0.05

Fig. 5

CIH-induced HIF-1α activated ATAD2 during lung cancer progression. A Gene correlation between ATAD2 and HIF-1α in LUAD determined by consulting http://timer.comp-genomics.org/. B, C Analysis of ATAD2 promoter activity. Cells were transfected with the HIF-1α-FLAG or control plasmid for 24 h. A549 and SPC-A1 cells were transfected with an ATAD2 promoter plasmid or a plasmid containing a mutated ATAD2 promoter sequence for another 24 h. D HIF-1α, ATAD2, CD133, and CD44 mRNA expression levels in A549 cells incubated under normoxia, CIH, or CIH combined with HIF-1α. E, F Flow cytometry analysis of A549 cells transfected with siRNA-NC or siRNA-ATAD2 under normoxia or CIH. Error bars indicate mean ± SD. All experiments performed in triplicate. *p < 0.05