Pyroptosis is a newly recognized kind of programmed cell death, which has a double-edged role in the progression of malignancy and mechanisms of treatment. Inflammatory cytokines are secreted in pyroptosis and normal cells are stimulated, contributing to the progression of cancer . Meanwhile, pyroptosis can enhance cellular death in malignancy, which makes pyrolysis a possible therapeutic and prognostic target for malignancy . However, how the genes related to pyroptosis interact with each other and whether they have impact on the survival time in ccRCC remain unrevealed.
In this research, the mRNA expression of 52 currently known PRGs were first discovered in normal and ccRCC tissues. It was found that 44 of them were differentially expressed. What makes sense is that the 2 clusters in the consensus clustering analysis according to the pyroptosis-related DEGs showed significant differences in clinical characteristics. This implied that pyroptosis in cancer tissues was different in ccRCC, which resulted in different OS.
To further evaluate the prognostic value of the regulators related to pyroptosis, a 6-gene risk signature was constructed. OS in cases of alternative subgroups was different. The functional analyses presented that the DEGs between the high- and low-risk groups showed significant differences in immune-related pathways, consistent with our expectations. Pyroptosis could contribute to the accumulation of various inflammatory factors, which was also resulted from the activation of inflammasomes [6, 30]. Additionally, we also enriched Cytokine, NF−kappa B, IL-17, Ras and TNF signaling pathways which were closely associated with the development of RCC.
Meanwhile, the pathway activation and immune infiltration were compared between the HR group and the LR group, and the HR group showed increased activities of pathways related to immune responses and elevated levels of IIC in comparison with the LR group. Enhanced infiltration of immune cells was related to poor prognosis, which was consistent with previous studies. Another key result in our research was that the above 6 prognostic genes related to pyroptosis had a significant correlation with immune infiltration, which further proved the fact that pyroptosis had an essential part in the immune microenvironment of tumor.
The current study found a signature of 6 genes linked to pyroptosis (ELANE, AIM2, GSDMB, IL6, NIRP1, and NOD2) and discovered that it could play a role in predicting OS of ccRCC patients. AIM2 (Absent in melanoma 2) was first found in melanoma with decreased expression. AIM2 is a member of the IFN-inducible PYHIN (pyrin and HIN200 domain-containing) family, and works as a cytoplasmic senor for DNA which can connect with dsDNA (double-stranded DNA) . AIM2 can activate CASP-1 via junctional proteins regulated by ASC to facilitate secretion and maturation of IL-18 and IL-1β, thus promoting pyroptosis. Previous research demonstrated that AIM2 worked as a suppressor in multiple kinds of tumors such as prostate cancer , colon cancer , melanoma , melanoma , and breast cancer but as a promoter in NSCLC (non-small cell lung cancer) , OSCC (oral squamous cell carcinoma) , and HPV (human papillomavirus)-associated cervical cancer . Therefore, AIM2 might had different effects in different tumors. In this research, the expression of AIM2 was significantly increased in tumor tissues in comparison with normal ones. Furthermore, an increased expression level of AIM2 was closely related to poor survival and gene mutations in AIM2 could ameliorate infiltration of immune cells. Thus, it was suggested that AIM2 was more like a pro-oncogene. The molecular mechanisms of AIM2 in the development of ccRCC currently remained unrevealed and our findings in AIM2 might provide a new insight into further research.
GSDMB (Gasdermin B) was the gene most linked to RS in the prognostic model, prompting that GSDMB might be highly involved in ccRCC. According to previous research on human malignancies, it was found that GSDMB was upregulated in tumor tissues, including breast, uterine, gastric, and cervical cancers . It was demonstrated that GSDMB was located in amplicons, the genomic regions frequently amplified in cancer development . Thus, GSDMB might participate in cancer development and metastasis. GSDMB can be cleaved into two fragments by caspasa-1. One cleavage form is the N-terminal of GSDMB protein with a molecular weight of 20 kDa. Cell pyroptosis can be caused by the secretion of the N-terminal domain. On the contrary, the full-length N-terminal domain and C-terminal fragment would not cause cell pyroptosis [43, 44]. In general, GSDMB may be the downstream protein of pyroptosis pathway. The key is whether some factors can trigger the upstream mechanism of GSDMB and cause pyroptosis. At the same time, the specific mechanism of GSDMB in ccRCC is not clear. The increased level of GSDMB in ccRCC is related to poor prognosis. This finding may provide help for the study of tumor treatment targets.
ELANE (neutrophil elastase gene) is a main serine protease produced by neutrophils, which can activate proinflammatory cytokines including IL-1β, IL-18, and TNF-α[45, 46], which are regarded as promoters for pyroptosis. Kambara et al. presented that ELANE could cleave and activate GSDMD and subsequently induce pyroptosis in neutrophils. The ELANE expression level in the HR group was remarkably increased compared with the LR group, but paradoxically, the neutrophil infiltration score was incredibly higher compared with the LR group. These findings may be resulted from many complex factors driving the difference of gene expression between alternative tissues, particularly levels of genes and PRGs linked to inflammation, such as the proportion of infiltrated immune cells and the differentiation of ccRCC [48, 49]. Such factors might not influence application of PRG expression signature in diagnosing and predicting ccRCC prognosis. The relationship between the expression of pyroptosis genes and infiltration of immune cells, ccRCC differentiation status, and other factors requires further investigation, which may provide new enlightenment for predicting the diagnosis and prognosis of ccRCC.
NLRP1 (NLR family, pyrin domain containing1), a bipartite adaptor protein, is considered as apoptosis-associated speck-like protein with a ASC (caspase-recruitment domain). NLRP1 can promote the recruitment process of pro-caspase-1 to the complex of inflammasomes . GSDMD can be cleaved by active caspase-1, allowing the N-terminal domain of GSDMD to form pores in the plasma membrane, and then triggers the pyroptosis mechanism[10, 15, 51, 52]. NOD2 (nucleotide-binding oligomerization domain-containing protein 2), which can initiate NF-κB (nuclear factor-κB)-dependent and MAPK (mitogen-activated protein kinase)-dependent gene transcription. For macrophages, NOD2 could be used as its promoter to induce the activation of inflammatory bodies. In our study, NOD2 was highly expressed in the HR group, which might be one of the reasons why macrophages obtained higher scores in the HR group compared with the LR group. At the same time, the activation effect of NOD2 on NF-κB pathway might make this pathway more enriched in KEGG analysis. Interestingly, as one of the genes in the risk prognostic model, activated NF-kB pathway was able to increase the amount of mRNA for IL-6 . In advanced metastatic breast cancer cells, over-activated NF-kB promoted chromatin accessibility of the IL-6 promoter region and enhanced transcription of the IL-6 gene. Appropriate expression of IL-6 is of great importance for human immune defense, but sustained production of IL-6 has a pivotal role in the occurrence of multiple inflammation-related diseases and cancer. Whereas in our study, IL-6 was significantly highly expressed in the HR group, we speculated that NOD2 might act as an upstream initiator of IL-6 to promote IL-6 expression by activating the NF-kB pathway.
The purpose of our research was to categorize cases with ccRCC into different subtypes, screen DEGs, set up a model of prognosis, and connect pyroptosis with the prognosis. Though we conducted multi-angle and multi-omics validation, there were still limitations in this study. All the analyses were performed using the TCGA KIRC cohort, and GEO cohort was recommended to verify them. Additionally, in-vivo and in-vitro experiments are required to further verify our findings. Pyroptosis, particularly the mechanism in ccRCC, is not sufficiently investigated. We initially investigated the prognostic value of the 6 genes related to pyroptosis in the risk prognostic model, which provided theoretical support for future studies.