Abstract

Introduction: Lung squamous cell carcinoma (SCC) is associated with a high mortality rate because of its assemblage of DNA mutations causing poor therapeutic responses. Therefore, elucidating the molecular events that underpin the pathobiology of lung SCC is essential to the development of effective therapies. Methods: We induced lung SCC in N-nitroso-tris-chloroethylurea (NTCU)-treated mice and RNA sequenced the resultant lung tumours. Cleaned sequence reads were aligned to the Mus musculus genome (GRCm38/mm10) using Spliced Transcripts Alignment to a Reference (STAR). Then, DESeq2, an R package, was used to normalise per-gene read counts before Gene Set Enrichment Analysis (GSEA). The functional impact of single nucleotide polymorphisms (SNPs) was assessed using the Protein Variation Effect Analyzer (PROVEAN) and Sorting Intolerant From Tolerant (SIFT). Pathogenic DNA variants were defined as having SIFT and PROVEAN scores <0.05 and -2.5, respectively. The final list of SNP-affected genes was analysed using g:Profiler to pinpoint overrepresented pathways. Adjusted p-values <0.05 were considered statistically significant. Results: The transcriptomic analysis revealed that the top five pathways potentially driving lung SCC development were cholesterol biosynthesis, keratinization, activation of gene expression by SREBF (SREBP), formation of the cornified envelope, and neutrophil degranulation. These pathways are plausibly involved in cancer development; for instance, cholesterol is an activator of tumorigenic signaling pathways and is required for increased membrane synthesis during cell proliferation. However, the association of the pathways with lung SCC has not been extensively reported. The SNP analysis revealed ‘homophilic cell adhesion via plasma membrane adhesion molecules’ as the most enriched gene ontology. The dysregulation of homophilic cell adhesion is a well-known contributor to cancer cell migration and metastasis. Conclusion: Our combinatorial analysis of differentially expressed genes and deleterious SNPs revealed novel, interacting biological pathways that may contribute to lung SCC development. These pathways are potential targets for effective lung SCC therapy.

Keywords: Lung squamous cell carcinoma (SCC), RNA sequencing, Single Nucleotide Polymorphism (SNP), Cholesterol, Homophilic cell adhesion