Function of AXL and molecular mechanisms in regulation of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignant tumor with unique geographical and ethnic distribution characteristics. NPC is mostly found in south China and Southeast Asia, and its treatment mainly depends on radiotherapy and chemotherapy. However, NPC is usually found in the late stage, and local recurrence and distant metastasis are common, leading to poor prognosis. The receptor tyrosine kinase AXL is up-regulated in various tumors and it is involved in tumor proliferation, migration, invasion, and other processes, which are associated with poor prognosis of tumors. This study aims to detect the expression of AXL in NPC cell lines and tissues, and to investigate its biological function of AXL and the underlying molecular mechanisms in regulation of NPC.

The expression levels of AXL in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed by GSE68799, GSE12452, and GSE53819 data sets based on Gene Expression Omnibus (GEO) database. The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between AXL and prognosis of head and neck squamous cell carcinoma (HNSC). The indicators of prognosis included overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS), and progression-free interval (PFI). Western blotting assay was used to detect the AXL protein expression levels in normal nasopharyngeal epithelial cell line and NPC cell lines. Immunohistochemical method was used to detect AXL expression levels in normal nasopharyngeal epithelial tissues and NPC tissues. Cell lines with stable AXL knockdown were established by infecting 5-8F and Fadu cells with lentivirus interference vector, and cell lines with stable AXL overexpression were established by infecting C666-1 and HK-1 cells with lentivirus expression vector. Real-time PCR and Western blotting were used to detect the efficiency of knockdown and overexpression in stable cell lines. The effects of AXL knockdown or overexpression on proliferation, migration, and invasion of NPC cells were detected by CCK-8, plate colony formation, and Transwell assays, and the effect of AXL knockdown on tumor growth in nude mice was detected by subcutaneous tumor formation assay. The sequence of AXL upstream 2.0 kb promoter region was obtained by UCSC online database. The PROMO online database was used to predict AXL transcription factors with 0% fault tolerance, and the JASPAR online database was used to predict the binding sites of ETS1 to AXL. Real-time PCR and Western blotting were used to detect the effect of ETS1 on AXL protein and mRNA expression. The AXL upstream 2.0 kb promoter region was divided into 8 fragments, each of which was 250 bp in length. Primers were designed for 8 fragments. The binding of ETS1 to AXL promoter region was detected by chromatin immuno-precipitation (ChIP) assay to determine the direct regulatory relationship between ETS1 and AXL. Rescue assay was used to determine whether ETS1 affected the proliferation, migration, and invasion of NPC cells through AXL.

Bioinformatics analysis showed that AXL was highly expressed in NPC tissues (P<0.05), and AXL expression was positively correlated with OS, DFI, DSS, and PFI in HNSC patients. Western blotting and immunohistochemical results showed that AXL was highly expressed in NPC cell lines and tissues compared with the normal nasopharyngeal epithelial cell line and tissues. Real-time PCR and Western blotting results showed that knockdown and overexpression efficiency in the stable cell lines met the requirements of subsequent experiments. The results of CCK-8, plate colony formation, Transwell assays and subcutaneous tumor formation in nude mice showed that down-regulation of AXL significantly inhibited the proliferation, migration, invasion of NPC cells and tumor growth (all P<0.05), and the up-regulation of AXL significantly promoted the proliferation, migration, and invasion of NPC cells (all P<0.05).As predicted by PROMO and JASPAR online databases, ETS1 was a transcription factor of AXL and had multiple binding sites in the AXL promoter region. Real-time PCR and Western blotting results showed that knockdown or overexpression of ETS1 down-regulated or up-regulated AXL protein and mRNA expression levels. ChIP assay result showed that ETS1 bound to AXL promoter region and directly regulate AXL expression. Rescue assay showed that AXL rescued the effects of ETS1 on proliferation, migration and invasion of NPC cells (P<0.05).

AXL is highly expressed in NPC cell lines and tissues, which can promote the malignant progression of NPC, and its expression is regulated by transcription factor ETS1.