The role of N-WASP and TXNIP in skin cancer cell proliferation

The ubiquitously-expressed neural Wiskott-Aldrich syndrome protein (N-WASP) regulates actin cytoskeleton remodelling. N-WASP messenger ribonucleic acid (mRNA) levels was found to be reduced in skin squamous cell carcinoma (SCC) samples compared to matched perilesional samples of 33 patients, suggesting it plays a role in skin carcinogenesis. The SCC cell line human skin squamous cell carcinoma 5 (HSC-5) was used to generate control HSC-5 (HSC-5CTR) cells HSC-5 cells overexpressing N-WASP (HSC-5N-WASP) cells. HSC-5N-WASP cells had increased epithelial cadherin (E-cadherin) and vinculin localizations but reduced cell migration, paxillin localization, cell proliferation and protein kinase B (AKT) signalling compared to HSC-5CTR cells. Ingenuity Pathway Analysis (IPA) comparative analysis of proteomics, protein microarray and RNA sequencing (RNA-Seq) data suggested that N-WASP probably regulates Integrin-mediated signalling, forkhead box protein class O1 (FOXO1)-mediated signalling to reduce cell proliferation in HSC-5 cells. Experimental validation found altered Integrin-mediated signalling, whereby HSC-5N-WASP cells had reduced signalling of focal adhesion kinase (FAK), proto-oncogene tyrosine-protein kinase Src (SRC) and growth factor receptor-bound protein 2 (GRB2), but increased signalling of son of sevenless homolog 1 (SOS1) compared to HSC-5CTR cells. It was also found that increased FOXO1 cytoplasmic translocation and degradation may be responsible for reduced HSC-5N-WASP cell proliferation. The activity of extracellular signal-regulated kinase 2 (ERK2) may be responsible for FOXO1 cytoplasmic translocation and degradation. Inhibition of ERK2 in HSC-5N-WASP cells restored cell proliferation rate, E-cadherin, vinculin and paxillin localizations, and cell migration to that of HSC-5CTR cells. Thioredoxin-interacting protein (TXNIP) is a FOXO1 target gene that negatively regulates the thioredoxin system and its ROS-scavenging activity. TXNIP levels were increased in HSC-5N-WASP cells compared to HSC-5CTR cells, suggesting cell proliferation is reduced due to increased reactive oxygen species (ROS) levels. TXNIP knockdown in HSC-5N-WASP cells restored cell proliferation rate, E-cadherin, vinculin and paxillin localizations, and cell migration to that of HSC-5CTR cells. These results suggest that in skin carcinogenesis, reduced N-WASP protein levels keep FOXO1 in the nucleus via reduced ERK2-dependent phosphorylation. TXNIP expression is repressed, allowing unhindered thioredoxin system-mediated ROS scavenging, increasing cell migration and proliferation via altered Integrin-mediated FAK-SRC-GRB2-SOS1 and AKT signalling pathways. These in vitro results could be translated into in vivo solutions for skin cancer treatment and prevention.Doctor of Philosoph