EGFL6, a potential novel ligand of EGFR, plays roles in cancer metastasis by inducing cancer cell epithelial-mesenchymal transition in nasopharyngeal carcinoma

Abstract

Since the vast majority of Nasopharyngeal carcinoma (NPC) is sensitive to radiotherapy and chemotherapy, the overall survival rate of stage I NPC is nearly 100%. Clinical outcome for loco-regional advanced NPC, which make up more than 75% of NPC cases, is however, far from satisfactory. The conclusion that, distant metastasis is the main failure for NPC treatment, has being made by nearly all the clinical trials in NPC. But the mechanism beneath NPC metastasis is seldom investigated. To understand more about NPC metastasis and search for potential therapeutic target, we start from cDNA microarray comparing gene expression level between metastatic tumors and corresponding primary tumors from NPC patients. Up-regulated genes were selected and validated by qPCR. Secondary screening was performed by examining the correlation between gene expression level and distant metastasis free survival (DMFS) rate. EGFL6 was found an independent prognosis index for NPC DMFS (p=0.023, n=110). We speculated that EGFL6 might play important roles in NPC metastasis for its clinical association and its background as a critical secretory protein normally function on embryonic development and restrict expressed in certain follicle stem cells including vibrissa and stromal cells in adult.

Functional studies found that EGFL6 promoted cell migration, invasion, and especially the formation of aggressive morphology in the matrix-gel based three-dimensional (3D) culture. With the overexpression of secreting EGFL6, CNE2 cells formed follicle-like structure with more aggressive leading edge, which invaded deep into the matrigel instead of forming single layer epithelial structure. EGFL6 overexpressed SUNE1 cell formed tissues breakthrough the restriction of its regular sphere structure, and migrated for more spaces. In vivo study also showed the capacity of EGFL6 in promoting tumorigenesis in subcutaneous injection assay and increasing number and volume of metastatic lymph nodes in the mice tail vain injection experiment.

Mechanism study showed that EGFL6 was a potential novel ligand of EGFR. EGFL6 bound EGFR on cell membrane and caused its phosphorylation at site tyrosine 1068, which activated EGFR and leaded to its internalization. C-src was subsequently phosphorylated at Tyrosine 416. MAPK/ERK signaling cascade was also activated. The activation of MAPK/ERK signaling pathway, together with the phosphorylation of c-src, regulated the elevation of Twist1, a critical epithelial-mesenchymal transition (EMT) transcriptional factor, which contributed to NPC cells invasion and metastasis by inducing EMT. EGFL6 functions as a secretory protein deposited on ECM mainly by cancer cells or a small population of stromal cells with a dose-dependent way, peak at around 50ng/ml.

In conclusion, EGFL6 was found a potential novel ligand of EGFR, which plays roles in NPC metastasis through establishing invasive and distant metastatic niche via paracrine and autocrine with a dose-dependent manner.