E2F7 promotes lung adenocarcinoma progression by affecting phosphorylation and stabilization of β-catenin

Abstract – Background: – E2F transcription factor 7 (E2F7) has been implicated in the tumorigenesis and progression of multiple cancer types; however, the molecular mechanisms through which E2F7 regulates malignant phenotypes in cancer cells remain largely undefined. In this study, we investigated the biological functions and underlying mechanisms of E2F7 in lung adenocarcinoma (LUAD). Methods: – E2F7 expression in LUAD was analyzed using The Cancer Genome Atlas (TCGA) datasets and further validated in clinical specimens via quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. The effects of E2F7 on cancer cell self‑renewal and epithelial–mesenchymal transition (EMT) were assessed using sphere formation and Transwell assays, respectively. In vivo tumorigenicity and metastasis were evaluated using xenograft models combined with extreme limiting dilution analysis to assess tumor-initiating capacity. Wnt/β‑catenin pathway activity was measured using T-cell factor optimal promoter luciferase reporter plasmid/far-from optimal promoter luciferase reporter plasmid (TOP/FOP) flash reporter assays. β‑Catenin expression, stability, and ubiquitination were examined via western blotting, cycloheximide chase assays, and ubiquitination assays. Protein–protein interactions among E2F7, β‑catenin, and glycogen synthase kinase 3 beta (GSK3β) were verified through co‑immunoprecipitation (Co‑IP), glutathione S‑transferase (GST) pull‑down, and immunofluorescence assays. Truncated mutants were generated to map the functional binding domains of E2F7. In vitro immunoprecipitation and kinase assays were further performed to confirm that E2F7 regulates GSK3β autophosphorylation and β‑catenin phosphorylation. Results: – Bioinformatic analyses revealed that E2F7 was significantly upregulated in LUAD tissues, and elevated E2F7 expression correlated with poor patient prognosis. Functional assays demonstrated that E2F7 promoted LUAD cell self‑renewal and EMT. Mechanistically, cytoplasmic E2F7 directly associated with β‑catenin through its DNA‑binding domain (DBD) and PHA03247 domain. E2F7 modulated β‑catenin phosphorylation at Ser675 and Ser33/37/T41, thereby inhibiting ubiquitin‑mediated degradation and enhancing β‑catenin protein stability. Furthermore, E2F7 interacted with GSK3β and suppressed its autophosphorylation at Tyr216, concomitant with reduced β-catenin phosphorylation at Ser33/37/T41 and its accumulation. Conclusion: – Collectively, these findings indicate that E2F7 drives LUAD malignant progression through regulation of the GSK3β/β‑catenin signaling axis and stabilization of β‑catenin. This study unveils a novel oncogenic mechanism of E2F7 in LUAD and identifies E2F7 as a promising therapeutic target for clinical intervention in LUAD.