Proteomic analysis of pulmonary fibrosis associated with lung cancer reveals dysregulation of the apoptotic pathway

Introduction

Pulmonary fibrosis (PF), especially idiopathic pulmonary fibrosis, is associated with a high risk of lung cancer (LC), but the mechanisms of carcinogenesis remains poorly understood. Combining Liquid Chromatography Mass Spectrometry (LC-MS/MS) and Mass Spectrometry Imaging (MSI), we compared the proteome of fibrotic areas from PF tissue samples with and without LC to identify altered signaling pathways.

Methods

34 Formalin Fixed Paraffin-Embedded (FFPE) surgical samples from patients with PF with LC (LC+, n = 17) and without LC (LC−, n = 17) were included. For each case, a representative FFPE block was selected for downstream analyses. After macrodissection of fibrotic areas, whole protein extracts were analyzed by LC-MS/MS. MSI was performed on tissue section after tryptic digestion, on selected regions of interest within fibrotic areas. In silico tryptic digestion of peptides of interest was further performed, and spatial localization of these peptides was visualized on ion maps.

Results

By LC-MS/MS analysis, we identified 72 out of 4901 proteins significantly differentially expressed between fibrotic areas of LC- and LC+ samples (P < 0.001). Pathway analysis suggested the involvement of oxidative stress metabolism and apoptosis, with significant downregulation of two mitochondrial proteins (BAX, Frataxin), known to activate apoptosis, in fibrotic areas adjacent to LC. After in silico tryptic digestion of BAX protein, MSI analysis revealed at least 2 unique peptides per protein that were significantly underexpressed in LC+ fibrotic areas. Complementary ion map analysis showed a specific expression of these mitochondrial peptides within metaplastic epithelial cells ligning honeycomb cysts.

Conclusion

Using an original proteomic approach integrating LC-MS/MS and MSI, we show that, in patients with PF, fibrotic areas have distinct proteomic profiles depending on the presence of LC. Fibrotic areas adjacent to LC exhibit a significant downregulation of pro-apoptotic mitochondrial proteins. Interestingly, spatial analysis by MSI show that these proteins are expressed by metaplastic epithelial cells within fibrotic areas, suggesting that dysregulation of apoptosis in these cells might be involved in lung carcinogenesis in patients with PF.