Abstract
Lung adenocarcinoma (LUAD) develops through a stepwise progression from pre-cancerous lesions, including atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), and minimally invasive adenocarcinoma (MIA). Understanding the molecular dynamics underlying these transitions is critical for early detection and therapeutic interventions. A comprehensive proteomic analysis was performed on pre-cancerous and early LUAD samples using label-free quantitative mass spectrometry. Immunohistochemistry (IHC) and double fluorescence staining were applied to validate protein expression and mt-dsRNA localization. Trajectory inference was utilized to model dynamic proteomic changes during lesion progression. Mt-dsRNA levels were significantly elevated in pre-cancerous lesions, peaking in AIS. Double fluorescence staining revealed partial co-localization with the mitochondrial marker TOMM20, suggesting mitochondrial origin. Differential γ-H2AX staining patterns, with nuclear positivity in AAH and cytoplasmic positivity in AIS and MIA, indicated stage-specific dynamics of the DNA damage response. Upregulation of dsRNA sensors, including RIG-I and MDA5, and dsRNA-binding proteins (dsRBPs) such as ADAR1 and HNRNPA2/B1, highlighted a complex regulatory feedback network for both oncogenic and anti-tumorigenic effects. Notably, in dsRNA-IP assay, ASPH was enriched across all stages including LUAD, while TMED9 and HNRNPA2/B1 were specific to pre-cancerous lesions, reflecting their stage-dependent roles in tumor transformation. Finally, Trajectory analysis identified distinct proteomic shifts, with AAH lesions exhibiting high progression scores resembling AIS and MIA, underscoring their malignant potential. This study reveals the multifaceted roles of mt-dsRNA and its associated proteins in pre-cancerous lesions, providing insights into immune activation, stress adaptation, and early carcinogenesis. These findings establish a framework for developing biomarkers and targeted therapies aimed at preventing the transition to invasive LUAD.