Identification and validation of a prognostic signature of autophagy, apoptosis and pyroptosis-related genes for head and neck squamous cell carcinoma: to imply therapeutic choices of HPV negative patients

Abstract

Introduction: An effective tool is needed to predict the prognosis of head and neck squamous cell carcinoma (HNSCC). Human papillomavirus (HPV) positive HNSCC patients generally have a favorable survival and a promising responsiveness to radiotherapy, chemoradiotherapy and checkpoint blockades. However, HPV negative patients, the majority of HNSCC patients, have been largely overlooked. Cell death has been involved in the therapeutic resistance of cancers. To this end, we aimed to identify the association of autophagy, apoptosis and pyroptosis-related genes with the prognosis of HNSCC, and construct a prognostic signature to predict the prognosis for HNSCC, especially for HPV negative HNSCC.

Methods: Autophagy and apoptosis-related genes were obtained from Gene Set Enrichment Analysis (GSEA) website, and pyroptosis-related genes were obtained from GSEA and Gene Ontology (GO) database. We established the cell death index (CDI) based on RNA sequencing (RNA-seq) data and clinicopathological information from The Cancer Genome Atlas (TCGA) dataset. The prognostic value of CDI was verified by Kaplan-Meier, receiver operating characteristic (ROC) and univariate and multivariate Cox regression analyses in TCGA dataset, and validated with the datasets from Gene Expression Omnibus (GEO) and Qilu Hospital of Shandong University. We further assessed the immune microenvironment of patients with high and low CDI scores. Moreover, the expression of the signature genes in HNSCC cell lines were explored.

Results: We found that CDI was an independent prognostic indicator for overall survival (hazard ratio 3.80, 95% confidential interval: 2.70-5.40, P < 0.001). Furthermore, HNSCC patients with high CDI scores obtained increased overall survival post radiation indicating benefits from radiotherapy of this subgroup. On the other hand, HPV negative HNSCC patients with low CDI exhibited increased checkpoint gene expressions, an inflamed tumor microenvironment and an enriched immune response-related functions, suggesting the potential benefits from checkpoint immunotherapies of this subgroup. Moreover, we validated the baseline and induced expressions of above 16 genes in two HPV negative HNSCC cell lines, CAL27 and SCC-15.

Discussion: We established a prognostic signature and emphasized its implements in the therapeutic choices of HPV negative HNSCC patients, the majority and the poor outcome population of HNSCC.

Keywords: apoptosis; autophagy; head and neck squamous cell carcinoma; human papillomavirus; prognosis; pyroptosis; radiotherapy; tumor microenvironment.

Figure 1

Identification of overall survival associated autophagy, apoptosis and pyroptosis-related genes. (A) A heatmap illustrates a unsupervised hierarchical clustering of the expressions of the 48 overall survival associated genes identified by univariate Cox regression based on the Ward D2 method. The data was scaled by row using Z-score normalization. The clinicopathologic characteristics, including age, gender, smoking status, drinking status, tumor site, TNM stage, tumor grade, HPV status, as well as survival status are annotated. (B) The 16 overall survival associated autophagy, apoptosis and pyroptosis-related genes are shown respectively. (C) A correlation heatmap shows the coefficients of 16 autophagy, apoptosis and pyroptosis-related genes. The area of the pie represents the absolute value of the coefficients. Blue indicates a positive correlation and red is a negative correlation.

Figure 2

CDI is an independent prognostic indicator for overall survival of HNSCC. (A–D) Kaplan-Meier survival curves of high CDI and low CDI subgroups in TCGA dataset (A), GSE42743 dataset (B), GSE65858 dataset (C) and the dataset of Qilu Hospital of Shandong University (D). Log-rank test is used to assess the statistical significance. (E) ROC curves of CDI for the prediction of overall survival at one-year, three-year, five-year and ten-year. (F) ROC curves of CDI and clinicopathological factors for prediction of five-year overall survival. (G) Forest plots of the univariate and multivariate Cox regressions of CDI, indices of apoptosis, autophagy and pyroptosis and the clinicopathologic characteristics for overall survival. (H) A nomogram estimates the probabilities of one-year, three-year, five-year overall survival for HNSCC patients based on CDI and clinicopathologic characteristics. (I) Calibration plots of the nomogram in terms of agreement between the predicted and observed one-year, three-year and five-year overall survival. The close-ended vertical lines indicate the 95% CI, the dashed line represents the ideal performance of the nomogram. HNSCC: head and neck squamous cell carcinoma; CDI: cell death index; ROC: receiver operator characteristic curve; AUC: areas under the curve; TNM: tumor-node-metastasis; HPV: human papillomavirus; HR: hazard ratios; CI: confidential interval; C-index: Harrell’s concordance index using “Hmisc” package.

Figure 3

CDI shows stronger prognostic effects on HPV negative HNSCC patients and associates with survival benefits from radiotherapy. (A) Beeswarm plot shows the differences of CDI distribution between HPV positive and HPV negative patients in TCGA cohort. The statistical significance is assessed by Wilcoxon test. (B) Kaplan-Meier survival curves illustrate the overall survival probabilities of HNSCC patients stratified according to CDI scores and HPV status (**** P < 0.0001, log-rank test). (C, D) Kaplan-Meier survival curves illustrate the overall survival probabilities of patients with different CDI scores receiving chemoradiotherapy, radiotherapy alone or neither of them in HNSCC patients (C) and HPV negative HNSCC subgroup (D) (** P < 0.01, log-rank test). HNSCC: head and neck squamous cell carcinoma; CDI: cell death index; HPV: human papillomavirus; RT: radiotherapy; CT: chemotherapy. ** P < 0.01, **** P < 0.0001.

Figure 4

The immune landscape between high and low CDI HNSCC in HPV negative subgroup. (A) Boxplots illustrate the immune scores and the microenvironment scores of high and low CDI subgroups in HPV negative HNSCC assessed using XCELL. (B) Boxplots of relative proportions of infiltrating immune cells in high and low CDI subgroups in HPV negative HNSCC assessed using CIBERSORT. (C) Boxplots of the expressions of immune checkpoint genes in different CDI subgroups in HPV negative HNSCC. HNSCC: head and neck squamous cell carcinoma; CDI: cell death index; HPV: human papillomavirus. Statistical significance is assessed by Wilcoxon test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Functional enrichments of HPV negative HNSCC patients with different CDI scores. (A) A volcano plot of differentially expressed genes between the subgroups of different CDI scores of HPV negative HNSCC. (B, C) The top 10 enriched GO biological processes of up-regulated genes in high CDI subgroup (B) and low CDI subgroup (C). (D, E) The top 10 enriched KEGG pathways of up-regulated genes in high CDI subgroup (D) and low CDI subgroup (E). HNSCC: head and neck squamous cell carcinoma; CDI: cell death index; HPV: human papillomavirus; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 6

Validation of the expression of 16 overall survival associated genes in two HPV negative HNSCC cell lines. (A) The transcripts of the 16 overall survival associated genes in CAL27 and SCC-15 cell lines detected by qRT-PCR. (B) Cell proliferation after chloroquine treatment for 24 h in CAL27 and SCC-15 cell lines assessed by CCK8 assay. (C, D) Cell apoptosis post chloroquine treatment for 24 h in CAL27 and SCC-15 cell lines indicated by annexin V and PI using flow cytometry. (E) The inductions of autophagy-related proteins (LC3 and p62) and apoptosis-related proteins (bcl-2 and caspase-3). (F) The transcripts of 14 overall survival associated genes post chloroquine treatment in CAL27 and SCC-15 cell lines. HNSCC: head and neck squamous cell carcinoma; HPV: human papillomavirus; CQ: chloroquine. Each bar represents the mean ± SD. Statistical significance is assessed by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Radiation enhances the expression of VDAC1 in HPV negative HNSCC cell lines. (A) Boxplots of the expressions of 16 overall survival associated genes between radiation sensitive and resistant subgroups in HPV negative HNSCC. (B) Cell proliferation after chloroquine treatment for 24 h, following radiation in CAL27 and SCC-15 cell lines. (C, D) Cell apoptosis post chloroquine and/or radiation treatments in CAL27 and SCC-15 cell lines indicated by annexin V and PI using flow cytometry. (E) VDAC1 transcript post chloroquine and/or radiation treatments in CAL27 and SCC-15 cell lines. HNSCC: head and neck squamous cell carcinoma; HPV: human papillomavirus; CQ: chloroquine. Each bar represents the mean ± SD. Statistical significance is assessed by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.