In situ and functional analysis of Integrin-alpha 5 reveals its role in tumor progression in non-small cell lung cancer

[Background] Integrins are transmembrane receptors that mediate cell adhesion to adjacent cells and the extracellular matrix, thereby regulating fundamental cellular processes. Abnormal integrin expression has been linked to tumor progression and metastasis across multiple cancer types, primarily through their interactions with components of the tumor microenvironment. Integrin alpha-5 (ITGA5) typically forms a heterodimer with the commonly shared beta-1 subunit and functions as a receptor for fibronectin and fibrinogen. This study aimed to investigate the clinical and biological significance of ITGA5 in non-small cell lung cancer (NSCLC). [Methods] Survival analysis was based on RNA-seq data sets from NSCLC patient cohorts of the University of Tokyo Hospital (n = 100) and The Cancer Genome Atlas (TCGA; n = 986). Protein expression was analyzed by immunohistochemistry (IHC) in diagnostic tissue samples from NSCLC patients from The University of Tokyo Hospital (n = 20) and from the Uppsala University Hospital (Sweden; n = 312), which also included extensive mutation data. The biological relevance of ITGA5 was experimentally evaluated in a xenograft model with Calu-1 cells using an ITGA5 inhibitor (GLPG0187), and the effect of ITGA5 knockdown derived from the same cell line was analyzed by bulk RNA-seq analysis. [Results] Both RNA and protein-level survival analyses consistently revealed that high ITGA5 expression was associated with shorter survival across multiple cohorts (TCGA RNA: p=0.011; University of Tokyo Hospital RNA: p=0.025; University of Tokyo Hospital protein: p=0.038; Uppsala University Hospital protein: p=0.013). Notably, protein IHC analysis indicated a greater impact of ITGA5 in tumors than in stromal cells. ITGA5 inhibition suppressed tumor growth in vivo compared to controls. Bulk-RNA seq data showed that ITGA5 knockdown altered the expression of genes involved in integrin-mediated signaling. Genes such as FERMT2, SEMA7A, and CCN1/CCN2 showed decreased expression following knockdown, consistent with their roles in integrin activation, ECM remodeling, and pro-invasive signaling. In addition, this analysis showed downregulation of pathways related to inflammation and tumor immunity (e.g., CSF3, CXCL8, IL6ST), EMT (e.g., TGFBR1, CDH2, PXN), and mTOR signaling (e.g., PIK3R2, STAT3, HRAS). [Conclusion] Our study provides a comprehensive analysis of ITGA5 expression on RNA and protein levels in the clinical context. The results indicate an essential role of ITGA5 in promoting tumor progression and poor prognosis in lung cancer, providing the rationale for further studying ITGA5 as a target with biomarker and therapeutic potential for NSCLC patients with elevated ITGA5 expression.