Functional analysis of the 5' flanking domain of the LOXL4 gene in head and neck squamous cell carcinoma cells.

Lysyl oxidases are a family of five copper-dependent amine oxidases including LOX, LOXL, LOXL2, LOXL3 and LOXL4. LOX and LOXL are essential for the assembly and maintenance of extracellular matrixes. LOXL2, LOXL3 and LOXL4, secreted and active enzymes, were also noted in association with diverse tumor types. We have recently reported overexpression of the LOXL4 mRNA and protein and a close relation of LOXL4 with the pathogenesis of head and neck squamous cell carcinomas (HNSCC). In this study, we analyzed the organization of the LOXL4 gene and addressed the regulatory mechanisms responsible for the overexpression. We demonstrated de novo transcription of the LOXL4 gene in HNSCC, but not in normal squamous epithelial cells. Analysis of the consecutive promoter region spanning positions -960 to -1 identified binding sites for several transcription factors. Promoter constructs containing selected specific promoter regions and consensus binding sites exhibited significantly increased reporter gene activity in HNSCC cells, but not in normal epithelial cells in transient coexpression experiments. The activity profiles of some of these constructs were similar in both cell types indicating that elements of the basic transcriptional regulatory mechanisms remained intact in HNSCC cells. DNA-binding experiments demonstrated that nuclear extracts from HNSCC cells have increased binding activity to the TATA (-25) and the SP1 (-181) sites compared to normal epithelial cells, suggesting that these transcription factors are involved in the upregulation of LOXL4 gene expression in HNSCC.