DDIT4 promotes malignancy of head and neck squamous cell carcinoma.

Abstract

This study investigated the cancer-promoting effect of ferroptosis regulator DNA damage-inducible transcript 4 (DDIT4) and its relevant mechanisms. Vital ferroptosis-related genes were identified using bioinformatic methods on the basis of data collected from TCGA and seven other online databases. Cell Counting Kit-8 (CCK8), colony formation, wound-healing and transwell assays, and western blot analysis were conducted for verifying the biological role of DDIT4 in vitro. The immune score and tumor purity were calculated using R package "estimate." The relationship was identified between DDIT4 expression and immune cell infiltration using ssGSEA and CIBERSORT algorithms. R package "Seurat" was used to perform unsupervised clustering of the single cells, and "SingleR" was utilized for annotation. R package "STUtility" was employed to plot the spatial expression of DDIT4. For trajectory analysis, monocle was used to predict cell differentiation and demonstrate the expression of DDIT4 at each state. Here, DDIT4 overexpression was observed in Head and Neck Squamous Cell Carcinoma (HNSCC) cohort, and DDIT4 upregulation showed a positive correlation with larger tumor size, lymph node metastasis, more advanced TNM stage and higher tumor mutational burden (TMB). Moreover, DDIT4 knockdown could markedly inhibit the proliferation, colony formation, invasion and migration of HNSCC cells, as well as suppress the expression of HIF-1a, VEGF and vimentin. In comparison, DDIT4 overexpression showed a negative correlation with immune score and infiltrations of several immune cells. DDIT4 played crucial roles in the differentiation of CAFs and T cells. Collectively, this study demonstrates that DDIT4 contributes a critical role in HNSCC progression. The positive feedback regulation between DDIT4 and HIF-1a may be a potential target for HNSCC treatment.