ΔNp63 Blocks Differentiation and Supports Cells Proliferation in Nasopharyngeal Carcinoma via Stablizing EGFR

【摘要】：Purpose Nasopharyngeal carcinoma(NPC) is a unique subtype of head and neck cancer rare throughout most of the world but prevalent in south China and southeast Asia. NPC is a malignancy arise from the nasopharynx stratified ciliated columnar epithelium or pseudostratified ciliated columnar epithelium, but NPC is widely regarded to be squamous in origin because of most of NPC exhibit various extent of squamous cell differentiation.That being said,the most majority of NPC cells lose columnar cell differentiation markers and express squamous cell markers. It's remain unclear how the ciliated columnar epithelium features was replaced by squamous-cell characters during NPC development. p63 is a member of the p53 family of transcription factors that plays a pivotal role in a wide range of biological processes,including stem/progenitor phenotypes, differentiation,adhesion,migration,invation,apoptosis and senescence. The ΔNp63α lacks the N-terminal transactivation domain is the predominant p63 isoform expressed in the basal and superbasal layer of nasopharynx epithelium(NPE) and overexpressed in most of NPC samples. Although the oncogenic role of ΔNp63α has been broadly reported, the role of ΔNp63α in NPC dedifferentiation is still a open question. We investigated cellular differentiation that are controlled by ΔNp63α in the basal-to-luminal switch of NPC development.Methods ΔNp63α was silenced by small interfering RNA in p63-positive NPC cells. Transcriptome profiles were performed by utilizing RNA-Seq. ChIP-Seq were performed to analyze p63 binding sites in NPC cells. The transcriptomic alteration induced by p63 silencing was compared with previous NPC transcriptome profiles(assession number GSE12452, GSE13597 and GSE15724). The effects of Np63 depletion on NPC differentiation were measured by a airliquid interface 3 D cultured assay. Cells proliferation were measured by cell growth curve, colony formation assay and cell cycle analysis. Cells migration and invasiveness were measured by wound-healing assay and transwell assays. Tumorigenicity were measured by nude mice tumor cell subcutaneous xenograft model. Proteins expression in tissue samples were detected by immunohistochemistry assay.Results In this study,we demonstrate that silecing ΔNp63α in NPC cells triggered a genes expression switch from basal to luminal type, resulting in induction of cells differentiation and reduction of cells proliferation and tumorigenicity. ChIP-Seq analysis revealed the basal-to-luminal genes expression switch triggered by ΔNp63 depletion is concomitant with genome wide H3 K4 me3, H3 K27 ace redistribution. Transcriptome profiles also revealed ΔNp63α controls near 50% differentially expressed genes during NPC development. Among these ΔNp63α promoted genes, TOP2 A, BUB1, BUB1 B, MAD2 L1, AURKA, CDC6, PCNA, CCNA2,CCNB1,CCNB2,CCND1,CDK1, and CDK4 expression were consistently reduced in ΔNp63 depletion cells, which were previously identified as hub genes during NPC development. We also demonstrated ΔNp63α maintain EGFR expression via transcriptional and post-tanslational regulation. Silencing ΔNp63α led to inhibition of EGFR transcription and triggered EGFR degradation. Immunohistochemistry assay revealed p63 protein level correlate with EGFR protein level in NPC patient samples.Conclusions Our works proved that ΔNp63α is a master factor in controlling NPC differentiation and cell proliferation. ΔNp63α could serve as a target for new therapy development.