System analysis based on the pyroptosis-related genes identifies GSDMC as a novel therapy target for pancreatic adenocarcinoma

Abstract

Background: Pancreatic adenocarcinoma (PAAD) is one of the most common malignant tumors of the digestive tract. Pyroptosis is a newly discovered programmed cell death that highly correlated with the prognosis of tumors. However, the prognostic value of pyroptosis in PAAD remains unclear.

Methods: A total of 178 pancreatic cancer PAAD samples and 167 normal samples were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. The "DESeq2" R package was used to identify differntially expressed pyroptosis-related genes between normal pancreatic samples and PAAD samples. The prognostic model was established in TCGA cohort based on univariate Cox and the least absolute shrinkage and selection operator (LASSO) Cox regression analyses, which was validated in test set from Gene Expression Omnibus (GEO) cohort. Univariate independent prognostic analysis and multivariate independent prognostic analysis were used to determine whether the risk score can be used as an independent prognostic factor to predict the clinicopathological features of PAAD patients. A nomogram was used to predict the survival probability of PAAD patients, which could help in clinical decision-making. The R package "pRRophetic" was applied to calculate the drug sensitivity of each samples from high- and low-risk group. Tumor immune infiltration was investigated using an ESTIMATE algorithm. Finally, the pro-tumor phenotype of GSDMC was explored in PANC-1 and CFPAC-1 cells.

Result: On the basis of univariate Cox and LASSO regression analyses, we constructed a risk model with identified five pyroptosis-related genes (IL18, CASP4, NLRP1, GSDMC, and NLRP2), which was validated in the test set. The PAAD samples were divided into high-risk and low-risk groups on the basis of the risk score's median. According to Kaplan Meier curve analysis, samples from high-risk groups had worse outcomes than those from low-risk groups. The time-dependent receiver operating characteristics (ROC) analysis revealed that the risk model could predict the prognosis of PAAD accurately. A nomogram accompanied by calibration curves was presented for predicting 1-, 2-, and 3-year survival in PAAD patients. More importantly, 4 small molecular compounds (A.443654, PD.173074, Epothilone. B, Lapatinib) were identified, which might be potential drugs for the treatment of PAAD patients. Finally, the depletion of GSDMC inhibits the proliferation, invasion, and migration of pancreatic adenocarcinoma cells.

Conclusion: In this study, we developed a pyroptosis-related prognostic model based on IL18, CASP4, NLRP1, NLRP2, and GSDMC , which may be helpful for clinicians to make clinical decisions for PAAD patients and provide valuable insights for individualized treatment. Our result suggest that GSDMC may promote the proliferation and migration of PAAD cell lines. These findings may provide new insights into the roles of pyroptosis-related genes in PAAD, and offer new therapeutic targets for the treatment of PAAD.

Keywords: Drug; GSDMC; Immune infiltration; Pancreatic adenocarcinoma; Prognostic model; Pyroptosis.

Fig. 1

The flowchart of our research process

Fig. 2

Differentially expressed pyroptosis-related genes between PAAD tissues and normal tissues. A Volcano plot indicates pyroptosis-related genes, with red dots indicating high expression and blue dots indicating low expression. B The protein–protein interaction (PPI) network shows the interaction of pyroptosis-related genes (interaction score = 0.4). C Heatmap of differentially expressed pyroptosis-related genes, with red indicating high expression, blue indicating low expression, n representing normal tissues, and t representing tumor tissues. D Boxplots of differentially expressed pyroptosis-related genes, with red boxes representing tumor groups and blue boxes representing normal groups. E Mutation analysis of differentially expressed pyroptosis-related genes in TCGA cohort

Fig. 3

Construction of a risk prognostic model based on pyroptosis-related genes in the TCGA cohort. A Univariate Cox regression analysis was performed for all pyroptosis-related genes. A value of P < 0.05 was considerated statistically significant. B Multivariate Cox regression analysis was performed on the genes derived from the univariate Cox regression analysis. C LASSO regression of the 5 OS-related genes. D Cross-validation for tuning the parameter selection in the LASSO regression

Fig. 4

Construction of risk model in TCGA cohort. A The patients were equally divided into two groups according to the threshold of the median risk score. Green represents the low-risk group. Red represents the high-risk group. B Kaplan Meier curves showing the overall survival of patients in the high-risk and low-risk groups. C Survival status of patients with PAAD in high and low risk groups. Green represents survival. Red represents death. D Heatmap showing the expression of the five pyroptosis-related genes. Pink represents the low-risk group. Bright blue represents the high-risk group. E The predictive efficiency of the risk score was verified by the ROC curve

Fig. 5

Nomogram to predict survival probability of pancreatic cancer patients. A Nomogram combining risk score with pathologic features. B–D Calibration plots for predicting 1 -, 2 -, 3-year OS of patients. D ROC curves for prediction of survival by the risk score and other variables (age, gender, stage, N stage, T stage)

Fig. 6

The screened drugs for PAAD treatment. IC 50 value of A.443654 (A), PD 173,074 (C), Epothilone. B (E), Lapatinib (G) in high-and low-risk patients with PAAD. The corresponding 3D structures are shown in (B), (D), (F) and (H), respectively

Fig. 7

Tumor Microenvironment and immune cell infiltration analysis. Violin plots represent the relationship of risk score with immune score (A), stromal score (B) and ESTIMATE score (C). (D) Relative proportion of immune cell infiltration in high-risk and low-risk group. Green represents the low-risk group. Red represents the high-risk group

Fig. 8

Knockdown of GSDMC inhibited PAAD cell proliferation and migration. A Western blot results showing the expression levels of indicated proteins in PANC-1 (left panel) or CFPAC-1 (right panel) cellline transfected with scrambled or two independent siRNA targeting GSDMC, respectively. B, C Cell viability was determined by CCK8 assay in the PANC-1 (B) and CFPAC-1 (C) cell lines transfected with either scrambled or two independent GSDMC siRNA targeting GSDMC, respectively. ***p < 0.001 by one-way ANOVA. D, F Colony formation assay of CFPAC-1 (D) and PANC-1 (F) cell lines with either scrambled or two independent siRNA targeting GSDMC, respectively. ***p < 0.001 by one-way ANOVA. E, H Edu assay to show the cell proliferation of PANC-1 (E) and CFPAC-1 (H) cell lines transfected with either scrambled or two independent siRNA targeting GSDMC, respectively. *p < 0.05 by one-way ANOVA. J, K Wound healing assay of PANC-1 (J) and CFPAC-1 (K) cell migration capability following transfected with scrambled or two independent siRNA targeting GSDMC, respectively. ***p < 0.001 by one-way ANOVA