CASP5 associated with PANoptosis promotes tumorigenesis and progression of clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) is a globally severe cancer with an unfavorable prognosis. PANoptosis, a form of cell death regulated by PANoptosomes, plays a role in numerous cancer types. However, the specific roles of genes associated with PANoptosis in the development and advancement of ccRCC remain unclear. Our study developed a risk model utilizing three PANoptosis-associated genes (Caspase 4 (CASP4), TLR3, and CASP5). This model demonstrated a high degree of precision in predicting the prognosis for patients with ccRCC. ccRCC patients in the high-risk group had the strongest immune cell activity, experiencing immune evasion, and might potentially derive advantages from treatment involving combined immune checkpoint inhibitors. CASP5 was highly expressed in ccRCC tissues by RT-qPCR, western blotting, and immunofluorescence. Stable CASP5 knockdown cell lines were constructed by lentivirus in vitro transfection technique. Reducing CASP5 level suppressed the growth, migration, and invasion of ccRCC cells, while encouraging cell apoptosis. In addition, the results of in vivo tumorigenesis experiments showed that down-regulating CASP5 expression inhibited the tumorigenic ability of 786-O cells. Together, the innovative risk model using PANoptosis-associated genes effectively forecasts the tumor microenvironment and survival rates for ccRCC, offering a novel approach to the early, precise diagnosis of ccRCC and the advancement of personalized treatment strategies.

Keywords: CASP5; Clear cell renal cell carcinoma; Immune infiltration landscape; PANoptosis; Risk model.

Fig. 1

Construction and identification of prognostic models in ccRCC. (A) Histogram of differentially expressed genes in tumor tissues compared to normal tissues, with |Log₂ (Foldchange)| > 1 and p-value < 0.05. (B) The Venn diagram showed the overlap between DEGs and PANoptoosis-related genes. (C) A volcano diagram of 16 PANoptosis-related DEGs in ccRCC patients from entire cohort. (D) Forest plot of the 16 PANoptosis-related DEGs associated with OS based on univariate Cox regression analysis. (E) Cross-validation using LASSO-Cox regression in entire cohort. (F) Coefficient profiles in LASSO-Cox regression model. (G) Kaplan–Meier analysis for OS curves of patients from entire cohorts in low- or high-risk subgroups. (H) Time-dependent ROC curves for predicting 1-, 3-, and 5-year OS in patients from entire cohort

Fig. 2

Construction and identification of nomogram model. (A) The landscape of three PANoptosis-related DEGs (CASP4, TLR3, and CASP5), risk model, and clinicopathological features in ccRCC patients from entire cohort. (B) The relationship between risk score, clinical features and OS were analyzed by univariate and multivariate Cox regression. (C) The corresponding nomogram was constructed based on the risk model and clinical characteristics, which predicted 1-, 3-, and 5-year OS in ccRCC patients from entire cohort. (D) The calibration plots showed the comparison between predicted and actual OS for 1-, 3-, and 5-year survival probabilities in ccRCC patients from entire cohort

Fig. 3

Evaluation of immune infiltration landscape in risk model. (A) Heatmap represents the relationship between immune infiltration landscape and risk group. (B) Differences of the stromal score, immune score, ESTIMATE score, and tumor purity in low- and high-risk groups from entire cohort. (C) The proportion of 22 immune cells in low- and high-risk groups was analyzed by CIBERSORT. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

Estimation of immunotherapy and chemotherapy responses. (A) The relative expression of eight classical ICPs (CTLA4, LAG3, PD1, TIGIT, CD80, CD86, CD28, GZMB) in low- and high-risk groups. (B) Heatmap showing correlations between risk score and eight ICPs. Blue represents the positive correlation. (C) The relative expression of immunotherapy response with PD1-blocker, CTLA4-blocker, and CTLA4-PD1-blocker in low- and high-risk groups. (D) Drug sensitivity analysis for low- and high-risk groups. ****p < 0.0001

Fig. 5

Comparison of CASP5 between healthy control and ccRCC patients. (A) The relative mRNA level of CASP5 between normal and tumor in ccRCC patients from entire cohort. (B) The K-M curve analysis between high- and low-expression of CASP5. (C) Evaluation of the prognostic utility of CASP5 using ROC curves. (D) The differential expression of CASP5 in normal and tumor in ccRCC patients from GEO databases. (E) The relative mRNA level of CASP5 in normal, benign, low grade, and high grade among ccRCC patients from the GSE68417 database. (F) The relative mRNA level of CASP5 in normal, low stage, and high stage among ccRCC patients from the GSE71963 database. (G) Expression of CASP5 protein and mRNA in tumor (T) tissues and adjacent normal (N) tissues were analyzed by western blotting and RT-qPCR. (H) The expression of CASP5 protein in tumor tissues and adjacent normal tissues was analyzed by immunofluorescence. Scale bar: 100 μm

Fig. 6

The effect of CASP5 on the occurrence and development of ccRCC. (A) RT-qPCR was used to verify CASP5 knockdown in 786-O and 769-P cells. (B) Western blotting was used to verify CASP5 knockdown. (C) CCK-8 assay was used to evaluate the effect of CASP5 knockdown on cell proliferation. (D) Cell colony formation assay showed that downregulation of CASP5 inhibited the proliferation of 786-O and 769-P cells. (E) Transwell assay was used to evaluate the effect of CASP5 knockdown on migration and invasion of 786-O and 769-P cells, scale bar: 500 μm. (F) The migration ability in different groups were detected by wound healing, scale bar: 500 μm. (G) The representative images of the apoptosis rate in each group were detected by flow cytometry

Fig. 7

Knockdown CASP5 inhibited tumorigenicity of ccRCC in vivo. (A) Negative control (NC) and sh-CASP5 786-O cells were respectively injected into nude mice to detect tumor formation in vivo. (B) The visual characteristics of tumors were compared between NC and sh-CASP5 groups. (C) The tumor weights of NC and sh-CASP5 groups. (D) The protein and mRNA levels of CASP5 in tumor tissues of NC and sh-CASP5 groups were analyzed by western blotting and RT-qPCR assays. (E) The representative images of CASP5 in NC and sh-CASP5 groups were detected by immunofluorescence. (F) The representative images of Ki67 in NC and sh-CASP5 groups were detected by immunofluorescence. (G) TUNEL assay was used to evaluate the apoptosis rate of tumor tissues in NC and sh-CASP5 groups. Scale bar: 100 μm