Introduction
Whey-acidic-protein (WAP) four-disulfide core domain protein 2 (WFDC2) is a small, secretory glycoprotein that is characterised by possession of two cysteine-rich protein domains. In healthy tissues, WFDC2 is most abundantly expressed in the trachea and oral cavity, yet its function at these sites is unknown. Structural similarities to other family members suggests that WFDC2 may play a role in host defence, thus antimicrobial and anti-protease functions have been hypothesised. WFDC2 expression is exacerbated in ovarian cancer and as a result it is used clinically as a serum biomarker. WFDC2 is also reported to be upregulated in other malignancies, including lung cancer, in addition to benign diseases such as cystic fibrosis and renal fibrosis. It is unclear whether WFDC2 mediates tumorigenic effects. The aim of this study was to determine the function of WFDC2, with the primary objective of understanding whether it is involved in tumour progression.
Materials and methods
Recombinant human and murine WFDC2 were synthesised by gene cloning and transfection into HEK293 cells. WFDC2 protein was purified from conditioned media and utilised for protease inhibition and bacteria assays. The N-glycosylation site of human WFDC2 was elucidated via site-directed mutagenesis and enzymatic cleavage. The WFDC2 expression of lung and oral cancer-derived cell lines was analysed by RT-qPCR, ELISA and Western blotting. An oral cancer cell line was utilised for CRISPR gene editing to silence endogenous WFDC2 gene expression. Successful gene silencing was confirmed via sequencing, RT-PCR, RT-qPCR, Western blotting and ELISA. Changes in cell behaviour between wild-type and CRISPR edited cells were analysed via standard in vitro cell behaviour assays. WT and heterozygous Wfdc2-knockout mice were studied via H&E staining and immunohistochemistry. Micro-CT scans of WT and homozygous knockout mice embryos at E14.5 and E18.5 were compared to elucidate differences in phenotype.
Results
Recombinant human and murine WFDC2 were unable to inhibit the growth of any of the bacterial strains tested, nor were they able to inhibit protease activity. CRISPR edited cells showed a significant reduction in their capacity to invade Matrigel-coated Transwells compared to controls. Homozygous Wfdc2-knockout mice died shortly after birth as a result of respiratory distress while heterozygous animals survived to adulthood and had no obvious histological phenotype. Micro-CT analysis showed that the trachea and bronchi of homozygote embryos were constricted at E14.5 and E18.5.
Conclusions
WFDC2 is not a host defence protein and is instead involved in development of the tracheal and bronchial lumina during embryogenesis. WFDC2 is involved in tumorigenesis by promoting cancer cell invasion. This suggests that WFDC2 is a prognostic biomarker and could represent an interesting therapeutic target. The role of WFDC2 in development requires further analysis.
