In the research, we first identified 1547 DEGs by analyzing the expression of every gene in 374 HCC patients and normal tissue, and we then took the intersection of the DEGs and 38 pyroptosis-related genes and discovered 3 DEGs (BAX, GSDMD, BAK1). Afterwards, By using the consensus clustering analysis and the three DEGs with a substantial difference, we created two clusters. By using Cox univariate analysis, LASSO Cox regression analysis, and well-validated results from the ICGC cohort, we created a 7-gene risk pattern to further assess the prognostic usefulness of the genes connected to pyroptosis. The function analysis suggested that metabolism related pathways were distinct between the low- and high-risk group. We also discovered that the high-risk group had fewer immune cells and immunological pathways that were infiltrated.
By releasing inflammatory chemicals, pyroptosis can prevent HCC cells from proliferating and migrating, thus raising cancer cell death is a HCC treatment strategy[28]. However, excessive pyroptosis activation causes persistent hepatocytes damage and instead leads to liver fibrosis and HCC[29, 30]. Therefore, New methods for preventing and treating HCC would be discovered by researching the pyroptosis balance in HCC. In our study, we framed a 7 pyroptosis-related gene pattern (BAK1, CHMP4C, APIP, CASP3, CASP8, IL1B, GZMA) and discovered that in patients with HCC, the pattern might predict the OS.
BAK1 is a pro-apoptotic Bcl-2 family member, activated BAK1 forms pores in outer mitochondrial membrane and leads to pro-apoptotic substance release and CASP3 cleavage[31]. In our study, there was a decrease in BAK1 in the low-risk group, which is purely related to earlier studies that reported a decrease in BAK mRNA associated with improved overall liver cancer survival[32]. Low expression of BAK1 was associated with longer progression-free survival (Pfs) and better prognosis in metastatic melanoma treated with chemotherapy[33]. BAK1 can interact with the tumor suppressor P53 after exposure to cell stress. Expression decrease of BAK1 corresponds with a better prognosis in a therapy scenario because apoptosis resistance is a characteristic of cancer.[34]. Caspase-3 (CASP3) is a classic apoptosis executor. Earlier studies identified caspase-1, caspase-4, caspase-5 and caspase-11 can induce pyroptosis via GSDMD cleavage [3]. Recently, caspase-3 was indentified as a GSDME-dependent pyroptosis executor, and switch TNF-induced apoptosis to pyroptosis [35]. In our study, CASP3 was decreased in low-risk group, inconsistent with this, activated caspase-3 and its effector cleaved GSDME, preventing gastric cancer cells from proliferating and migrating, esophageal cancer cells and lung cancer cells [36]. However, GSDME is not found in the majority of tumor cells and is expressed in a variety of normal organs. Therefore, chemotherapy drugs target pyroptosis is really risky to kill normal cells thus induce adverse effect. Earlier studies reported that bleomycin treated mice, Compared to GSDME deficient mice, WT mice experienced more severe lung inflammation and damage[36, 37], Cisplatin treated WT mice showed more severe crypts, villi damage, vascular injury and alveolar wall thickening than GSDME null mice [38, 39]. Therefore, Further research is required to determine if the role of CASP3-mediated pyroptosis in cancer is pro-cancer or pro-host. Caspase-8 (Casp8) is the initiation factor and inhibitor of apoptosis in vitro [40]. It was reported associated with embryonic development for that CASP8 null mice was embryonic lethality at 10.5 to 12.5 embryonic days [41]. Studies on overexpression demonstrated that inactive caspase-8 expression alone was sufficient to create ASC spots and cause cell pyrosis[42]. Viruses were found encoding apoptosis and necroptosis suppressors that inhibited CASP8[43], therefore, CASP8-induced pyroptosis may the counter-adaptation of host defense. Activation of CASP8 and CASP3 activated p38 and MAPK protein induced apoptosis and inhibited progression of human skin melanoma through high mobility group 1 (HMGB1)[44]. Furthermore, CASP8 siRNA injected intravitally into a mouse model of retinal ischemia reperfusion was able to significantly attenuate retinal damage and retinal ganglion cell loss via blockade of hypoxia inducible factor-1α (HIF-1α)[45]. Our research revealed that the high-risk HCC group had elevated CASP8.Our research revealed that the high-risk HCC group had elevated CASP8, further verification study may focus on the CASP8 role in HCC. Our study showed that IL1B was downregulated in low-risk HCC group, numerous studies have shown that interleukin-1 (IL1B) had a role in the growth, invasion, and proinflammatory microenvironment of HCC[46].Additionally, it has been shown that IL1B may encourage colorectal cancer cell proliferation and the EMT (epithelial-to-mesenchymal transition) phenotype[47]. And a recent meta-study revealed that individuals with HCC who had high expression of IL1B had a significantly poorer prognosis[48]. Chromatin Modified Protein 4C (CHMP4C) is expressed in both nucleus and cytoplasm and is a subgroup of endosomal sorting complexes responsible for transport [49]. its polymorphisms may induce tumorigenesis via sabotaging genome stability thus increasing cancer susceptibility[50]. Therefore, CHMP4C was suggested as susceptibility gene for ovarian cancer, it expressed highly in cervical cancer tissues with lower survival [51]. The results were in line with our calculation that CHMP4C was downregulated in the low-risk HCC group. Additionally, CHMP4C is a transcritional target of P53 to initialize its exosome secretion, therefore, it may be a potential cancer treatment target[52].Strongly expressed in skeletal muscle and the heart, PAF1 interacting protein (Apip) promotes a variety of cell death processes [53] . APIP was stably expressing in mice myoblast cells and suppressing cell death induced by hypoxia and etoposide[54]. Oxygen deficiency is an important factor in the pathogenesis of many human diseases and cancers [55]. In response to salmonellosis, recently discovered APIP reduces caspase-1-mediated cell coking via methionine repair[56]. Additionally, APIP was elevated in patients gastric cancer and cells originating from gastric cancer, and APIP suppression decreased proliferation and tumor development [57]. ranzyme A (GZMA) from cytotoxic lymphocytes (CTL) could cleave GSDMB to form membrane pore then induce extensive target cell pyroptosis which lead to CTL-mediated tumor clearance. GSDMB cleavable by Introducing GZMA in mouse cancer cells promoted tumor clearance. Additionally, GZMA could switch cells apoptosis into pyroptosis, results conformed with our results that GZMA was upregulated in low-risk group. In conclusion, 6 genes (BAK1, CHMP4C, APIP, CASP3, CASP8, IL1B) were downregulated in the pyroptosis gene pattern for low-risk HCC, while GZMA was upregulated for the group. The 7 genes signature for HCC prognosis needs further investigation.
Glycolysis inhibition inhibited LPS-induced IL1B mRNA production in macrophages and significantly increased production of Krebs circulating intermediate succinate [58]. NLR family, pyrin domain containing 3 (NLRP3) inflammasome can promote pyroptosis. Inhibition of glycolytic enzymes GAPDH and α- enolase can induce NLRP3 inflammasome formation, then reduce NADH synthesis while promoting mitochondrial ROS production[59]. According to reports, fatty acids like palmitate can promote NLRP3, which may explain why affects inflammation. During times of hunger, the NLRP3 inflammasome can be targeted by the ketone body of free fatty acids and beta-hydroxybutyrate (BHB) [60]. A recent research revealed resistance to the symptoms of the metabolic syndrome brought on by diet and kidney disease in NLRP3-deficient mice, suggesting that activation of inflammatory small bodies is associated with lipid metabolism [61]. Inflammation, including pyroptosis is involved in several biological process, further research focuses on the possible therapies for inflammation-related disorders and the influence of glycolysis and lipid metabolism on inflammasome activation.The important role of small inflammatory bodies in autoimmunity and infectious disease has been demonstrated in numerous studies, and their role in tumor progression remains elusive. Recent studies have revealed a dual function for inflammatory compounds in different tumor types, with inflammatory components conferring protection against colon cancer progression while promoting the progression of skin and breast cancer [62-64]. These findings imply that the dual function of inflammatory bodies in the development of tumors relies on both the kind of inflammatory body and the tumor. Since inflammasomes play a complex role in cancer, manipulating inflammasome pathways presents both new opportunities and difficult challenges. We therefore explored the gene signature related to pyroptosis for prognosis of HCC.
Pyroptosis is a kind of planned cell death that is accompanied by more pronounced inflammation.Therefore, we studied the immune cells and between the low- and high-risk groups in both the TCGA and ICGC cohorts via immune-related mechanisms. We discovered that the HCC low-risk group had higher levels of crucial anti-tumor immune cells like CD8+T cells, neutrophils, NK cells, Th cells (Tfh, Th1), and tumor-infiltrating lymphocytes (TILs), demonstrating that the anti-tumor immune cells infiltration was linked to a better prognosis through immune checkpoints upregulation[65]. Similar findings were confirmed in the ICGC cohort, our results were consistent with earlier studies. Notably, despite reports of Treg cells suppressing anti-tumor immunity, our results show no difference between the two groups. Treg was proved been highly resistant to programmed cell death 1 ligands 1 (PD-L1) induced apoptosis in HCC patients[66]. Although the mechanism of pyroptosis and apoptosis is different, they share certain similar properties. We assumed that Treg was activated to respond pyroptosis in two groups, but the up-regulated CD8+, NK cells, and TILs offset the antitumor inhibition by Treg in the low-risk group. Therefore, the uncertain role of Tregs in HCC required more exploration.
The inflammation-promoting pathway and type-I IFN response pathway were significantly increased in the low-risk group. CD8+ T cell-mediated anti-tumor response which we already observed above, could lead cytokines production such as INF-γ[67], Our findings showed that positive antiviral immunity was a protective factor for HCC, which was in line with that most of HCC developed from viral hepatitis. With the exception of the MHC-I and APC pathways, there were absolutely no similarities between the two groups, and all immunological pathways were down-regulated in the high-risk group, suggesting no significant differences between high-risk groups. Hepatic macrophages lead to clearance of pathogens, collection of leukocytes, and presentation of antigen to lymphocytes present in the vasculature [68]. The above HCC immune signatures and its microenvironment can provide an HCC immune profile for the immune therapy.
Among these small molecules, according to reports, Paclitaxel, Docetaxel, Ivermectin, Dactinomycin, walrycin A, Sodium Selenite have anti-cancer effects. Paclitaxel is a natural anticancer agent that induces autophagy, stabilizes microtubulin polymerization, and causes apoptosis and mitotic arrest [68, 69]. A microtubule depolymerization inhibitor with anticancer action called docetaxel (RP-56976) stops the cell cycle and induces death at the G2/M phase[68, 69]. Both paclitaxel and docetaxel were found to have anticancer effects, which shows the reliability of our screening and facilitates our late-stage drug trials. Actinomycin D (actinomycin) is an autophagy activator, inhibits DNA repair, transcription and replication, and actinomycin D inhibits the proliferating cell nuclear antigen (PCNA), adhesion kinase (FAK), and Raf protein expression levels.[70, 71]. Actinomycin D increases the extracellular signal-regulating kinase (Erk) that mediates cell cycle arrest [72]. Ivermectin (MK-933), a comprehensive antiparasitic drug, is a particular inhibitor of nuclear input mediated by impα / β1. Ivermectin inhibits bovine herpes virus 1 (BoHV-1) from replicating and blocks the BoHV-1 DNA polymerase. from entering the nucleus and possesses strong antiviral properties against the dengue and HIV-1 viruses. In addition, it is a chemical that positively regulates P2X4 and the α7 neuronal nicotine cholinergic receptors (NACHRs) [73-75]. Ivermectin, meanwhile, is a potential therapy for SARS-CoV-2 and COVID-19 [74]. A novel family of powerful tiny molecules known as walrycins targets the RR WalR and inhibits this crucial signaling pathway to kill bacterial cells[76].Sodium Selenite is an anticancer agent with anti-tumor properties in acute lymphoblastic leukemia (ALL), relieves breast cancer-associated lymphedema independent of antioxidant defenses, and reduces lung adenocarcinoma progression by inhibiting sox2-mediated dryness [77-79]. By triggering the ERK1/2 pathway, 1,2-dithiol-3-thione (D3T) shields PC12 cells against the apoptosis-inducing effects of A1-42 [80]. In pancreatic beta cells, The ROS-dependent autophagic cell death that sodium arsenite causes [81]. Thalidomide has been demonstrated to target cereblon (CRBN), a component of the cullin-4 ubiquitin E3 ligase complex CUL4-RBX1-DDB1, and to have immunomodulatory, anti-inflammatory, and antiangiogenic anticancer effects [82]. The molecules described above are of great value and are very useful for our experiments. Of these selected molecules, we hope to find combinations that could play an important role in future experiments.
According to the findings mentioned above, pyroptosis may be a potential target for HCC therapy. Here we performed a systematic study and clustered 374 HCC patients according to pyroptosis-related genes, then identified HCC patients into two types of pyroptosis-related subtypes and explored the prognostic value, In the TCGA and ICGC datasets, we discovered 7 pyroptosis-related genes as a standalone risk factor for HCC prognosis. And We talked about connections between pyroptosis and the microenvironment of the tumor immune system. In summary, we provided a pyroptosis genetc signature for HCC prognosis, and paved a way for further research on pyroptosis and HCC.