Background
Programmed cell death is an active and orderly form of cell death regulated by intracellular genes that plays an important role in the normal occurrence and development of the immune system [
1]. Pyroptosis is a new type of programmed cell death mediated by caspase-1, characterized by rapid rupture of the plasma membrane, followed by the release of cell contents and proinflammatory substances such as IL, thereby triggering an inflammatory cascade that leads to cell damage [
2]. With the extensive development of research on pyroptosis, its complex biological functions are beginning to emerge. As a new type of programmed inflammatory necrosis, pyroptosis is involved in the occurrence and development of various diseases [
3], including acute liver injury, rheumatoid arthritis, and acute kidney injury [
4‐
6]. In recent years, pyroptosis has been found to be involved in tumorigenesis and development [
7]. For example, human mesenchymal stem cells can induce pyroptosis by secreting interleukin-1β (IL-1β), which can lead to the death of breast cancer cells [
8]. Euxanthin can significantly inhibit the proliferation, migration and invasion of hepatoma cells, and this process plays a main role in the mechanism by which euxanthin activates the pyroptosis signalling pathway mediated by Caspase-1 [
9]. Simvastatin inhibits the proliferation and migration of non-small-cell lung carcinoma (NSCLC) cells. The mechanism may be related to the simvastatin-induced activation of the NLRP3 inflammatory body, caspase-1, IL-1β and interleukin-18 (IL-18), thereby inducing pyroapoptosis in NSCLC. Caspase-1 inhibitor attenuates the inhibitory effect of simvastatin on NSCLC [
10]. Conversely, activation of the NLRP3 inflammasome promotes the proliferation and migration of lung adenocarcinoma A549 cells, which is related to the ability of the NLRP3 inflammasome to mediate the release of IL-18 and IL-1β through caspase-1-dependent or caspase-1-independent pathways [
11]. Similarly, the NLRP1 inflammasome promotes melanoma growth by increasing Caspase-1 activity and promoting IL-1 β secretion [
12]. In addition, pyroptosis is involved in the immune regulation process in tumours. Elion et al. explored the role of the RIG-I-mediated innate immune response in breast cancer cells, which was partly attributed to the activation of pyroptosis-inducing inflammatory cytokines [
13]. Furthermore, Caspase 3/GSDME-dependent pyroptosis contributes to chemotherapy drug-induced nephrotoxicity. These results show that pyroptosis is involved in tumour progression and treatment, and better understanding the mechanism of pyroptosis will facilitate new approaches to tumour treatment.
In this study, we first performed a comprehensive analysis of molecular patterns, including somatic copy number alterations, mutations, deoxyribonucleic acid (DNA) methylation, pathway enrichment, the immune microenvironment, patient survival, effects on immunotherapy and drug resistance, across cancers. We also established a model of pyroptosis-related gene levels across 33 cancer types. Our results highlight the important role of pyroptosis in cancer and provide new insight into the functions of pyroptosis and therapy in cancer.
Methods
Datasets and source
We downloaded the following data from the University of California SANTA CRUZ (UCSC:
https://xenabrowser.net/datapages/): marked copy number segment, DNA methylation (Illumina human methylation 450), gene expression RNAseq (HTSeq), somatic mutation (SNPx and small INDELs), and survival data. The lists of cancer types are presented in Additional file
1: Table S1. The drug response data and genomic markers of sensitivity were downloaded from the Genomics of Drug Sensitivity in Cancer (
https://www.cancerrxgene.org/) and Cancer Therapeutics Response Portal (
http://portals.broadinstitute.org/ctrp/) identifying and targeting cancer dependencies with small molecules. Immune-associated data, including immune cells and immunophenoscores, were downloaded from ImmuCellAI (Immune Cell Abundance Identifier) (
http://bioinfo.life.hust.edu.cn/ImmuCellAI#!/). Three immunotherapy datasets (GSE13507: primary bladder cancer; GSE32894: urothelial carcinoma; GSE61676: non-squamous non-small cell lung cancer) were from the Group on Earth Observations (GEO) dataset (
https://www.ncbi.nlm.nih.gov/gds). Thirty-three pyroptosis-related genes were obtained from a previous publication
(Additional file
1: Table S2) [
14].
Differentially expressed gene analysis
To explore the expression differences in pyroptosis-related genes between cancerous and normal tissues, we performed differential expression analyses of 32 cancers using the “limma” R package [
15]. A |Log2-fold change (FC)| value > 1 and adjusted
P value < 0.05 were defined as threshold for indicating significant differential expression levels.
Somatic copy number alteration and mutation analysis
Data on mutation, fusion, amplification, homozygous and heterozygous deletion and amplification were included to evaluate the copy number alterations and mutations of pyroptosis genes. We defined over five percent of the genes as having high-frequency copy number alterations. Pearson’s correlation coefficient was used to evaluate the association between copy number alterations and mRNA expression. The R “maftool” package was employed to evaluate the overall mutation of pyroptosis genes in cancers.
DNA methylation analysis
DNA methylation (Illumina Human Methylation 450) data of 33 cancers were downloaded from the UCSC database. Some cancers do not have normal methylation data. The differential methylation of 14 cancerous tissues and normal tissues was determined using the Wilcoxon rank test. Genes were defined as hypomethylated or hypermethylated according to the adjusted P value (P < 0.05). The correlations between methylation and gene expression levels were evaluated using Spearman correlation. P < 0.05 was considered significant.
Establishment of the pyroptosis level model
To evaluate the pyroptosis level in cancer, we calculated the pyroptosis score using single sample gene set enrichment analysis (ssGSEA) in the R ‘performed gene set variation analysis (GSVA)’ package [
16]. The enrichment score was divided into positive and negative components. The normalized difference between positive and negative components was defined as the pyroptosis level. To evaluate the pathway enrichment of each sample, we performed GSVA and estimated the gene set enrichment of pyroptosis genes [
17]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set (c2.cp.kegg. v6.2. symbols) was downloaded from the GSEA database (
http://www.gsea-msigdb.org/gsea/index.jsp).
Survival analysis
We evaluated the effect of pyroptosis levels on cancer survival prognosis. The following four survival outcomes were included: overall survival (OS), disease-specific survival (DSS), progression-free interval (PFI), and disease-free interval (DFI). We calculated the hazard ratio of the pyroptosis score in each cancer using Cox regression. The pyroptosis score was categorized into high and low groups according to the median. Kaplan–Meier analysis was used to compare the survival curves of the high and low pyroptosis groups. P < 0.05 was considered significant.
Immune feature analysis
To investigate the association between pyroptosis and the immune microenvironment, we calculated the Pearson correlation coefficients between the pyroptosis score and immune parameters, including the immune score, stromal score, estimated score, and tumour purity.
Immune cells included B cells, T cells, myeloid dendritic cells, endothelial cells, NK cells, macrophages, haematopoietic stem cells, and immune cell subsets. Some immune-related pathways, matrix/metastasis-related pathways, and DNA damage repair pathways were also evaluated.
We also investigated the effect of the pyroptosis level on survival prognosis in three GEO datasets (GSE13507: primary bladder cancer, survival outcome: OS; GSE32894: urothelial carcinoma, survival outcome: PFS; GSE61676: non-squamous non-small cell lung cancer, survival outcome: OS).
Drug sensitivity analysis and pyroptosis level
To evaluate the association between the pyroptosis level and small-molecule drugs, we calculated the Pearson correlation coefficients between the pyroptosis score and drug sensitivity percentage calculated by the percent viability curve approach. We identified and targeted cancer dependencies with small molecules using the Genomics of Drug Sensitivity in Cancer and Cancer Therapeutics Response portal dataset.
Discussion
Pyroptosis is an inflammatory programmed cell death method that is characterized by the activation of inflammatory caspases (caspases 1, 4, 5 and 11) in inflammasomes and the secretion of inflammatory cytokines such as interleukin-1β and interleukin-18 [
20]. Researchers have found that GSDMA acts as an essential downstream substrate of inflammatory caspases, inducing pyroptosis by forming pores in the plasma membrane [
21]. Currently, the functions of pyroptosis in tumorigenesis and tumour treatment are being increasingly studied. However, compressive analysis and an understanding of the biological regulation of pyroptosis genes are lacking in cancers. In the present study, we integrated multiomics data and clinically relevant outcomes across 33 cancers from a public dataset and depicted the landscape of alterations and epigenetic and transcript levels of pyroptosis genes. We also evaluated the pyroptosis levels across cancers using ssGSEA and identified the correlations between the pyroptosis level and immune features, survival outcomes, immunotherapy, and drug sensitivity. Pyroptosis showed different genetic, epigenetic, and transcriptional patterns in different cancers, and differential effects of pyroptosis on survival and immune treatment were observed, especially in KIRC, LGG, GBM, PADD, and SKCM. An increased pyroptosis level has an adverse effect on the immunotherapeutic treatment of primary bladder cancer, urothelial carcinoma, and non-squamous non-small cell lung cancer, which means that the pyroptosis level should be considered in cancer immunotherapies.
The molecular mechanism of pyroptosis in tumorigenesis and development remains unclear. However, the correlations of pyroptosis with some functions and pathways may provide some important clues. The GSVA results indicated that the pyroptosis genes were mainly enriched in two components. The first was inflammation-related pathways, such as the IL6/JAK/STAT3 signalling, inflammatory response, IL2/STAT5 signalling, TNF-alpha signalling via NFKB, and KRAS signalling pathways, which have been proven to be associated with tumours. The inflammatory response can promote the occurrence and progression of tumours [
22]. Sustained oxidative stress during the process of chronic inflammation leads to DNA damage and inhibition of DNA damage repair, resulting in inactivation of tumour suppressor genes. Inflammatory cells and inflammatory factors in the microenvironment can induce the expression of a variety of cytokines [
23]. These inflammatory cells, cytokines and their downstream products promote the occurrence, development, and metastasis of cancer via various mechanisms, such as by inhibiting apoptosis, promoting angiogenesis and inducing immune tolerance [
24]. The inflammatory response is the primary characteristic of pyroptosis. The pyroptosis process depends on the activation of Caspase-1, which importantly functions to mediate the cleavage of the interleukin-1 β precursor into active IL-1β. IL-1β can recruit and activate other immune cells and induce the synthesis of chemokines, inflammatory factors, adhesion molecules, etc., eventually causing the cascade effect, which amplifies the inflammatory response and leads to a severe inflammatory response [
25]. We also found that pyroptosis genes were enriched in immune-related pathways such as the allograft rejection, complement, and interferon alpha and gamma response pathways. The GSEA results indicated that several immune-related pathways, such as the innate immune system, adaptive immune system, and cytokine signalling in the immune system, were associated with multiple cancers. Pyroptosis, as a form of programmed cell death, is an important natural immune response of the body. A previous study reported that the induction of pyroptosis in tumours induced high antitumour immune activity and promoted tumour clearance [
26,
27]. However, our results indicated that pyroptosis had a favourable effect on survival outcomes in BRCA, KICH, MESO, SARC, SKCM, STAD, TCHA and BLCA, while pyroptosis exerted adverse prognostic effects in ESCA, GBM, HNSC, KIRC, LAML, LGG, LUSC, PADD, THYM, UCES, UCS and UVM. Previous studies have also reported that pyroptosis inhibits the progression of breast, liver, ovarian, stomach, and colon cancers and promotes the progression of melanoma. NLRP3 inflammasome-mediated pyroptosis promotes the progression of lung adenocarcinoma but inhibits the progression of non-small-cell lung cancer, while GSDMD-mediated pyroptosis promotes the progression of non-small-cell lung cancer [
28]. Our results validated the dual roles of pyroptosis in cancers. In addition, tumours at the same location, such as the thyroid, kidneys, and lungs, with the same pyroptosis level have different prognostic patterns. Furthermore, we analysed the survival outcomes of patients subjected to immunotherapy and found that elevated pyroptosis levels were correlated with an adverse prognosis in primary bladder cancer, urothelial carcinoma, and non-squamous non-small-cell lung cancer. Pyroptosis does not always exert antitumour immune effects.
We further analysed the correlation between drug sensitivity and pyroptosis gene expression. A previous study reported that GSDMD is activated by the cleavage of Caspase-3 and induces pyroptosis in response to tumour chemotherapy drugs [
29]. Human neuroblastoma SH-SY5Y cells and human malignant melanoma MeWo cells have high GSDME expression levels. Under the action of chemotherapy drugs such as topotecan, etoposide, and cisplatin, the cells undergo obvious pyroptosis rather than apoptosis [
30,
31]. Our results indicated that GSDME was positively associated with multiple molecular drugs. GSDME, as a tumour suppressor gene, is expected to be the target of a new clinical treatment direction. NLRP1 expression showed a negative association with drug sensitivity, proving its tumour promotional role. A previous study reported that NLRP1 promotes melanoma growth by enhancing inflammasome activation and suppressing apoptotic pathways [
12]. These results highlight the dual roles of pyroptosis in cancers.
Our study has several advantages over previous studies on certain cancers. First, previous studies focused on the development and validation of prognostic models involving certain cancer types [
32‐
35]. Our studies aimed to assess pyroptosis levels in cancer as a whole, and our model of pyroptosis levels is thus more universal. Second, previous studies tended to assess only one survival outcome (usually OS), and our study assessed four survival outcomes (OS, DSS, PFI, and DFI) in all kinds of cancers. Third, we first assessed the association of the pyroptosis level with immunotherapeutic responses based on available data, and this association has never been reported for certain cancers. Finally, our results depicted a landscape of gene dysregulation, signalling pathways, immune therapy, model assessment, and clinical relevance in cancers, which has important significance for guidelines and practice.
Some study limitations should be addressed. The effect of the pyroptosis level on immunotherapy in several cancers was not assessed due to a lack of available immunotherapy data. In addition, this study did not explore the molecular mechanisms of pyroptosis in cancers. Future studies should validate the effect of the pyroptosis level on immunotherapy in more cancers, and the potential molecular mechanisms should also be explored to identify potential treatment targets in certain cancers.
Conclusions
In the present study, we performed a comprehensive analysis of pyroptosis and genes regulating pyroptosis in cancers. Our study found that (1) aberrantly expressed pyroptosis genes are mainly attributed to CAN frequency and differences in DNA methylation levels in cancer. (2) Moreover, the established pyroptosis level model based on the ssGSEA method uncovers the dual roles of pyroptosis in different cancers, and (3) the pyroptosis level is associated with clinical prognosis in multiple cancers, especially LGG, GBM, KIRC, PAAD, SKCM, UVM, BLCA, COAD, THYM, and SARC. (4) The dual role of pyroptosis also affects the immunotherapeutic efficacy in several cancers, including bladder cancer, urothelial carcinoma, and non-squamous non-small cell lung cancer, and (5) six pyroptosis genes (AIM2, CASP3, CASP4, NLRC4, NLRP6, and TNF) are closely correlated with drug sensitivity across cancers and may be considered therapeutic targets in cancer. Our comprehensive analysis highlighted the possibility of pyroptosis-based cancer therapeutic strategies.
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