Background Aberrations of EGFR signalling drive cancer development. In squamous cell lung cancer (SqCLC), EGFR is overexpressed. LRIG1 is a negative regulator of EGFR and patient pre-invasive SqCLC samples show LRIG1 loss, suggesting involvement in early disease pathogenesis. In skin and gut homeostasis, LRIG1 regulates stem cells. In the upper airway, basal cells act as stem cells and are the putative origin of SqCLC. We hypothesise LRIG1 has a key role in airway homeostasis and its loss promotes pre-invasive SqCLC development.
Methods Lrig1 EGFP-ires-CreERT2 mice were used to delineate airway LRIG1 expression. Flow sorted LRIG1-positive and -negative murine basal cells were used in 2D and 3D colony-forming, spheroid and proliferation assays. A murine SqCLC model was set up through application of N-Nitrosotris-(2-chloroethyl)urea (NTCU). Pre-invasive lesions and tumour development were compared between wild-type (WT), heterozygous and LRIG1-knockout (KO) animals. Human basal cells obtained from bronchoscopy were sorted according to LRIG1 expression and used directly in colony-forming assays or maintained in primary culture to assess the effect of shRNA knockdown of LRIG1. LRIG1-knockdown cells were assessed in colony-forming and proliferation assays, and differentiation and invasion were assessed using organotypic models.
Results LRIG1 is expressed by 40% of airway basal cells. LRIG1-expressing murine basal cells exhibit increased colony-forming capacity (p = 0.0286), spheroid formation (p = 0.0043) and proliferation (p = 0.0043) compared with LRIG1-negative cells. Similarly, LRIG1-expressing human airway basal cells isolated from endobronchial brush biopsy samples exhibit increased colony-forming capacity (p = 0.0469). Topical application of NTCU to mice recapitulates the development of human pre-invasive and SCLC lesions after 23 weeks. Results show lesions in LRIG1-KO mice to be larger than those of WT animals. Knock down of LRIG1 in cultured human airway basal cells alters cell phenotype, leading to an increased colony-forming efficiency and greater proliferation at cell confluence.
Conclusions LRIG1 has an important role in stem cell homeostasis of the human and murine airway epithelium. Loss of LRIG1 promotes pre-cancerous lesion development in a murine SqCLC mouse model and behaviour of human epithelial cells in culture, indicating a potential target for chemoprevention of SqCLC in humans.