Inhibition of cell survival and invasion by Tanshinone IIA via FTH1: A key therapeutic target and biomarker in head and neck squamous cell carcinoma.

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a worldwide public health problem; its incidence is increasing and it is now the sixth most common cancer type worldwide. As indicated by existing studies, ferroptosis contributes to HNSCC progression and Tanshinone IIA (TanIIA) may exert therapeutic effects via affecting ferroptosis. However, the underlying mechanisms have remained to be clarified. Therefore, the main aim of the present study was to screen and investigate the key genes in regulating ferroptosis of the human hypopharynx squamous carcinoma cell line FaDu and further elucidate the mechanism of action of TanIIA. A list of ferroptosis-related genes was obtained from the FerrDb database. RNA-sequencing expression (level 3) profiles and corresponding clinical information (cases, n=502; normal controls, n=44) were downloaded from The Cancer Genome Atlas dataset for HNSCC (https://portal.gdc.com). The limma package in R software was used to study the differentially expressed mRNAs. Adjusted P<0.05 and Log2(fold change) >1 or <-1 were defined as the threshold for the differential expression of mRNAs. The ClusterProfiler package (version 3.18.0) in R was employed to analyze the Gene Ontology functional terms associated with potential targets and perform a Kyoto Encyclopedia of Genes and Genomes pathway analysis. The R package ggplot2 was used to draw the boxplot and the pheatmap package was used to draw the heatmap. The DEG-related protein-protein interaction network was built with the Search Tool for the Retrieval of Interacting Genes and proteins database and then the visualization was performed using Cytoscape. Ferritin heavy chain 1 (FTH1), transferrin (TF) and TF receptor were screened out using a Wayne diagram, which was drawn by the Venn Diagram package in R. Kaplan-Meier survival analysis and the log-rank test were used to compare differences in survival between the groups. The receiver operating characteristic (v 0.4) (ROC) curve analysis was used to compare the predictive accuracy of mRNAs. FTH1 was screened out and the expression results were verified using The Human Protein Atlas data. Immunohistochemistry and immunofluorescence were used to localize FTH1 expression in FaDu cells. Furthermore, Cell Counting Kit-8 and Transwell assays were used to detect the cell survival and invasion ability, respectively. Furthermore, western blot analysis was performed to analyze protein expression. The results of the present study indicated that three validated ferroptosis marker genes were differentially expressed in HNSCC, among which FTH1 was significantly associated with poorer survival. TanIIA was demonstrated to significantly affect FaDu cell survival and invasiveness and markedly attenuate FTH1 expression. To conclude, the ferroptosis gene FTH1 is highly expressed in HNSCC and TanIIA significantly inhibited HNSCC, partially by suppressing FTH1.