PDAC is one of the most malignant digestive tract tumors, whose mortality and morbidity are almost the same and the 5-year overall survival rate is only about 9% . At present, surgical treatment is still the radical treatment for PDAC. However, due to the hidden onset of pancreatic cancer, most of PDAC cannot be removed by surgery at the time of discovery. The lack of effective tumor biomarkers to evaluate the prognosis of PDAC and the difficulty in developing personalized treatment plan have resulted in a low survival rate [44, 45]. Tumorigenesis is associated with a variety of factors, including activation of proto-oncogenes and anticancer genes, TME, oxidative stress, and chronic inflammatory stimuli. Activation of pyroptosis leads to the release of the inflammatory mediators IL-1 and IL-18, which can contribute to the development of cancer in a number of ways. For another, pyroptosis can promote tumor cell death, making it a potential prognostic marker and therapeutic target for cancer. Therefore, PRGs play different roles in the occurrence and progression of different cancers. For example, pyroptosis inhibits the progression of hepatocellular carcinoma, colorectal cancer and gastric cancer [46–51], but it promotes the proliferation and metastasis of breast cancer cells . However, the role of PRGs in PDAC has not been clarified. Therefore, in this study, we aimed to discover a novel prognostic marker related to pyroptosis through data analysis and mechanism exploration to provide potential approaches in the treatment of PDAC.
Firstly, we obtained the mRNA expression levels of 33 currently known PRGs in TCGA-PAAD samples. In order to further evaluate the prognostic value of these PRGs, we constructed a risk score model based on NLRP1 and CASP4 gene signature through univariate Cox analysis and LASSO regression analysis, and then verified their good predictive performance in external datasets. Patients in the high-risk group fared worse when grouped by risk score, and the same conclusion was obtained from external data, further demonstrating the specificity and accuracy of this PRGs signature in distinguishing different prognostic PDAC. Based on epidemiological studies, previous cohort studies and expert consensus [53–60],We included a variety of clinical pathological factors that may affect OS in PDAC patients and the result showed that risk score was found to be an independent prognostic factor, further proving the great influence of PRGs on the prognosis of PDAC. However, the above factors have no obvious effect on the OS of PDAC, which may be due to the lack of patient data, as only 43 patients were included with complete data.
Previous studies have shown that NLRP1 is considered a tumor suppressor gene. NLRP1 is one of inflammasome sensors, the activator of which induces the proteasome-mediated destruction of the N-terminal fragment and liberates the C-terminal fragment to form an inflammasome . Inflammasome represents a group of protein complexes that induce inflammation and pyroptosis, and its abnormal and chronic activation is the pathological basis for many common inflammatory diseases and tumorigenesis . Studies have indicated that NLRP1 mediates the production of IL-18 to help prevent colorectal cancer associated with colitis . Targeting the activation of NLRP1 in epidermal keratinocytes represented a potential therapeutic strategy for NLRP1-dependent inflammatory skin disease and cancer [63, 64]. The prognostic significance of CASP4 overexpression in cancers remains controversial. For example, the clinical cohort study of Shibamoto et al. showed that CASP4 may play a role as a tumor suppressor gene in esophageal cancer and as a potential biomarker for predicting esophageal cancer prognosis [65, 66]. However, silencing CASP4 gene inhibited the migration, adhesion, and invasion of epithelial cancer cells . Terlizzi et al. reached a similar conclusion in non-small cell lung cancer (NSCLC), which means that CASP4 overexpression was associated with poor prognosis in NSCLC patients . Meng et al. found that CASP4 was highly expressed in renal clear cell carcinoma based on TCGA data, suggesting poor prognosis, and was associated with tumor drug resistance . In our study, high expression of CASP4 may be associated with poor prognosis in PDAC patients, while NLRP1 is thought to play a tumor suppressor role, and similar expression differences were found on protein levels. Moreover, CCK8 and transwell assay suggested that CASP4 may accelerate the progression of PDAC by promoting proliferation and migration of pancreatic cancer cells, while NLRP1 has been found to have tumor suppressive effect in vitro. It is noteworthy that CASP4 is commonly known as a cell pyroptosis gene, but it has been found to promote cancer in some experimental and clinical studies, the mechanism of which has not been explored. In order to explore the potential mechanism of CASP4 regulating tumor progression, we compared DEGs in the high and low CASP4 expression groups and found that in addition to significant differences in programmed cell death, more DEGs were enriched in P53 signaling pathway and nucleotide metabolism pathway. In addition, Michela Terlizzi analyzed changes in lipid metabolism characteristics in CASP4-positive NSCLC and found increased palmitic acid and malonic acid in tissues of CASP4-positive patients, which are important for fatty acid biosynthesis and elongation [70, 71]. KRAS and P53 mutations, the most common mutation in pancreatic cancer, can change normal metabolic pathways and initiate metabolic reprogramming by activating transcription factors and enhancing enzyme activity . Moreover, recent studies have found that KRAS and P53 play a synergistic role mediated by transcription factors in promoting pancreatic cancer metastasis . By grouping pancreatic cancer samples in TCGA, we found that the expression level of CASP4 was higher in both KRAS mutation samples and P53 samples than in wild-type samples. Therefore, it is reasonable to speculate that CASP4 may be one of the factors in the synergistic regulatory network of KRAS and P53 and promoted the biosynthesis of fatty acids in pancreatic cancer and reserves productive substrates for the proliferation and migration of tumor cells in addition to the occurrence of pyroptosis. Our experiment results suggested that CASP4 knockdown in PANC-1 cells significantly reduces the number of lipid droplets, and the expression of key enzymes and transcription factors involved in fatty acid synthesis (ACC and SREBF-2), which was the first in vitro study of CASP4 regulation of pancreatic cancer lipid metabolism. This will pave the way for further exploration of CASP4 gene function in the future.
Functional analysis showed that the DEGs between the low-risk and high-risk groups were closely related to antigen presentation, gene transcription, cleavage of pyroptosis-related proteins and some classical cancer pathways. Due to the fact that pyroptosis is usually associated with the release of pro-inflammatory factors and activation of the immune system, we compared the abundance of TIICs in the high and low risk groups and found that there were more macrophage M1 and Tfh in the high-risk group. Survival analysis also showed that infiltration of macrophage M1 predicted poor prognosis. Macrophages are the most abundant cells in tumor stroma and have strong plasticity and play a variety of functions in the TME. Among them, macrophage M1 have the ability to kill tumor cells, while the other part, tumor-related macrophages, usually manifested as macrophage M2, show anti-inflammatory and tumor-promoting effects . Tfh plays an important role in promoting differentiation of B cell and inducing antibody responses in humoral immunity and immune-related inflammatory diseases, including infection, autoimmune diseases and cancer. Tfh induces the formation of ectopic lymphoid structures at tumor sites and recruits CD8 + T cells, macrophages and natural killer (NK) cells involved in anti-tumor immunity to suppress tumor growth . Wu et al. also found higher levels of Tfh infiltration in the high-risk group when predicting prognosis in PDAC patients . The analysis of the infiltration of TIICs further explained the mechanism of the effect for pyroptosis on the occurrence and development of cancer, and provided a glimmer of hope for the immunotherapy of PDAC.
We tried to apply the results of this study into clinical practice, not only establishing a prognostic risk model, but also exploring the correlation between PRGs and tumor drug resistance. Through drug sensitivity analysis, CASP4 was significantly related with the inhibitors of AKT and MEK. This further suggested that CASP4 may be involved in tumor cell lipid metabolism through AKT and MAPK signaling pathways, which also provided evidence for the regulatory relationship between CASP4 and KRAS mutation. Our research focuses on predicting the diagnostic, therapeutic and prognostic value of PRGs in PDAC from the perspective of bioinformatics and in vitro experiments, and exploring the regulation of CASP4 on lipid metabolism in pancreatic cancer for the first time in vitro. However, we have to say that this study is a retrospective study with some limitations. Therefore, we call for a prospective study with a larger sample size to verify the clinical application of PRGs in personalized management of PDAC patients. Besides, this study concluded that there was a lack of a series of in-depth experimental verification, such as the regulatory mechanism between CASP4 and KRAS/MEK signaling pathway and genes specifically regulated by CASP4 in regulating lipid metabolism. As a continuation of future research, we will supplement it in future research.