NAT10 as a Potential Prognostic Biomarker and Therapeutic Target for HNSCC

Abstract Background. Increasing evidence has demonstrated the critical roles of mRNA modification regulators on multiple types of cancers. However, it is still poorly known about the prognostic and therapeutic value of mRNA modification regulators in HNSCC.Methods. The gene expression profile of 36 mRNA modification regulators and their corresponding clinical data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Stepwise regression in R with both directions was used to construct a model for the prognosis of HNSCC. Univariate Cox regression survival analysis was performed to find the most significant risk gene. Gene set enrichment analysis (GSEA) was applied to identify the cancer-associated pathways with NAT10. Immunohistochemistry (IHC) staining was performed to evaluate the expression of NAT10 in formalin fixed paraffin-embedded (FFPE) samples of HNSCC. Univariate and multivariate Cox regression survival analysis performed to identify the independent risk factors associated with the OS of patients with HNSCC. HNSCC cell lines (Cal-27, FaDu, and Detroit-562) were transfected with short interfering RNA (siRNA) targeting NAT10 or treated with Remodelin, a small-molecule inhibitor of NAT10. Knockdown efficiency of siRNA was assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. In addition, CCK-8 assay, scratch assay and transwell assay were used to examine the proliferation, migration, and invasion abilities of the three HNSCC cell lines after NAT10 was inhibited genetically and pharmaceutically. Cell cycle and cell apoptosis assays were performed by flow cytometry. Finally, the therapeutic value of Remodelin in HNSCC was evaluated via a patient-derived xenograft (PDX) model. The statistical analysis was performed with SPSS 23.0.Results. A risk prediction model containing ten mRNA modification regulators was constructed and showed prognostic value in HNSCC. NAT10 was further identified as a key risk gene and independent prognostic factor in TCGA HNSCC dataset. The GSEA analysis suggested that high NAT10 expression was associated with MYC, E2F, G2M checkpoint, mTORC1, DNA repair and oxidative phosphorylation pathways. NAT10 protein expression was significantly up-regulated in tumour cells compared to normal epithelial cells in FFPE samples and increased NAT10 protein expression was correlated with poor overall survival of 267 HNSCC patients. Genetic depletion of NAT10 using siRNA or chemical inhibition of NAT10 using Remodelin resulted in reduced cell proliferation, migration and invasion abilities in Cal-27, FaDu and Detroit-562 cells. Knockdown of NAT10 using siRNA significantly increased cell cycle arrest in S/G2-phase. Remodelin significantly inhibited tumour growth and tumour cell proliferation in the PDX model of HNSCC.Conclusions. NAT10 could be a potential prognostic marker and a therapeutic target for HNSCC.