Effect of Chronic Intermittent Hypoxia-induced HIF-1α/ATAD2 Expression on Lung Cancer Stemness

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.