Identification of differentially expressed ARGs in HNSCC.
As a first step toward understanding the prognostic value of autophagy in HNSCC, we screened the differentially expressed genes related to autophagy in HNSCC. As presented by the Venn diagram, among 10,342 differentiated expression genes in HNSCC, there were 104 autophagy-related genes (ARGs), including 69 up-regulated and 35 down-regulated genes (Fig. 1A).
To further figure out the biological functions of these 104 genes, we performed Gene Ontology (GO) enrichment analysis. Results showed that the differentially expressed ARGs were mainly enriched in biological processes (BP) of macroautophagy/autophagy (Fig. 1B). In cellular components (CC), the top enriched terms were autophagosome assembly (Fig. 1C). In molecular functions (MF), the top ranked function terms were cysteine-type endopeptidase activities and ubiquitin-like protein ligase bindings (Fig. 1D).
Construction of the prognostic models using ARGs in HNSCC.
We assessed the prognostic value of above 104 ARGs using univariate Cox regression, and found 21 ARGs to be significantly associated with the prognosis of HNSCC patients. Based on these 21 ARGs, we used LASSO Cox regression analysis (“lars” package and “glmnet” package in R software) to screen out 14 prognosis-related genes. Next, we employed the multivariate Cox regression analysis to finally identify 9 most prognostically valuable ARGs, including ATG4A, RAF1, BAG3, RAB11A, MAP2K7, STK11, ATIC, FADD and USP10. The risk scores were then calculated based on the gene expression levels and the associated Cox regression coefficients, as risk score = expr MAP2K7*-0.1020 + expr STK11*-0.0592 + expr FADD*0.0109 + expr USP10*0.0248 + expr RAB11A* 0.0149 + expr RAF1*-0.0496 + expr ATIC *0.0328 + expr ATG4A*0.0989 + expr BAG3*0.0087 (Fig. 2A-B).
Then we classified HNSCC patients into high- and low-risk score groups based on the median of the OS-related prediction signature. The Kaplan-Meier survival curves clearly showed that the low-risk group had lower mortality and better prognosis than that in the high-risk score group (p < 0.001) (Fig. 2C). Additionally, the ROC curves for the overall survival (OS) were applied to reveal the predictive performance of the 9-gene based risk signature. The AUC values of ROC for 1-, 3-, and 5- years were 0.685, 0.78, and 0.749, respectively, indicating an excellent predictive value of the signature (Fig. 2D).
Identification of the ARGs prognostic signature in HNSCC.
To further evaluate the ARGs prognostic signature, we established the risk factor association map by the "ggplot2" package and the "pheatmap" package in R software, and analyzed the distribution of risk scores, survival time and correlated ARG expression for HNSCC patients. As shown in the association map, the risk scores of patients increased in parallel with decreased survival time and increased numbers of death (Fig. 3A-B).
Next, we analyzed the expression profiles of the 9 ARGs including ATG4A, RAF1, BAG3, RAB11A, MAP2K7, STK11, ATIC, FADD and USP10. It’s noted that MAP2K7, STK11 and RAF1 were highly expressed in the group of low-risk scores, whereas genes including RAB11A, FADD, BAG3 and USP10 were in the group of high-risk scores (Fig. 3C).
To further confirm the effect of the 9 ARGs on the prognosis of HNSCC, we performed Kaplan-Meier logarithm test to assess the OS difference between the different ARGs expressions. Results showed that low expression levels of MAP2K7, STK11 and RAF1 were tightly correlated with the poor survival time in HNSCC patients, whereas low expression levels of RAB11A, FADD, BAG3 and USP10 were closely associated with better prognosis (Fig. 4). All the survival analysis results were in accordance with the gene expression patterns with different risk-scores.
Establishment of the nomogram prognostic model in HNSCC.
To provide better prognosis for HNSCC patients based on our risk score model, we conducted a nomogram to predict the OS of 1-, 3-, and 5-years using five prognostic factors, including gender, stage, grade, age, and risk scores (Fig. 5). The calibration curves showed that the predicted calibration curves were close to the standard curves, suggesting that the nomogram could be reliable in the prediction of OS in HNSCC patients.