An HDAC9-associated immune-related signature predicts bladder cancer prognosis

Abstract

Background: The close relationship between histone deacetylase 9 (HDAC9) and immunity has attracted attention. We constructed an immune signature for HDAC9, a vital epigenetic modification, to predict the survival status and treatment benefits in bladder cancer (BC).

Methods: An exhaustive analysis of HDAC9 and immunology via the tumor and immune system interaction database (TISIDB) was performed, and an immune prognostic risk signature was developed based on genes enriched in the top five immune-related pathways under high HDAC9 status. Comprehensive analysis of survival curves and Cox regression were used to estimate the effectiveness of the risk signature. The relationship between immunological characteristics and the risk score was evaluated, and the mechanisms were also explored.

Results: In the TISIDB, HDAC9 was closely related to various immunological characteristics. The risk signature was obtained based on genes related to prognosis enriched in the top five immune-related pathways under high HDAC9 status. The survival rate of the high-risk BC patients was poor. The risk score was closely related to multiple immunological characteristics, drug sensitivity, immunotherapy benefits and biofunctions.

Conclusion: An immune-related prognostic signature established for HDAC9 expression status could independently predict the prognosis of BC patients. The use of this signature could help clinicians make personalized treatment decisions.

Fig 1. Preliminary exploration of HDAC9.

According to TCGA data, low expression of HDAC9 was found in tumor samples (A). According to HPA data, lower expression of HDAC9 was found in tumor samples (B) than in normal samples (C). There was a significant difference between HDAC9 expression and immune subtype (D). It was worth noting that the significance level of the relationship between HDAC9 expression and immune subtypes in the BC ranked second among all tumors (E). HDAC9, histone deacetylase 9; TCGA, The Cancer Genome Atlas; HPA, Human Protein Atlas; BC, bladder cancer; C1, wound healing; C2, IFN-gamma dominant; C3, inflammatory; C4, lymphocyte depleted; C6, TGF-b dominant; ***, P < 0.001.

Fig 2. Establishment of the risk score.

In the TCGA cohort, BC patients were distributed into different risk groups (A). Survival status of BC patients in different risk groups (B). The survival rate of the high-risk BC patients was worse than that of the low-risk patients (C). AUCs of the ROC curves were shown (D). In the verification cohort of the GEO, BC patients were distributed into different risk groups (E). Survival status of BC patients in different risk groups (F). The survival rate of the high-risk BC patients was worse than that of the low-risk patients (G). AUCs of the ROC curves were shown (H) TCGA, the Cancer Genome Atlas; GEO, Gene Expression Omnibus; AUC, area under the ROC curve; OS, overall survival; ROC, receiver operating characteristics; BC, bladder cancer.

Fig 3. Independent prognostic analysis of the risk score.

In the TCGA cohort, the results of univariate (A) and multivariate Cox analyses (B) showed that the risk score could be used as an independent prognostic factor for BC. In the GEO cohort, the results of univariate (C) and multivariate Cox analyses (D) also showed that the risk score could be used as an independent prognostic factor for BC. TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus; BC, bladder cancer.

Fig 4. Establishment of the nomogram in TCGA.

The results of the nomogram (A) and the calibration curve appropriately predicted 1-year (B), 3-year (C) and 5-year (D) OS for BC patients in TCGA. TCGA, The Cancer Genome Atlas; OS, overall survival; BC, bladder cancer.

Fig 5. Establishment of the nomogram in GEO.

The results of the nomogram (A) and the calibration curve appropriately predicted 1-year (B), 3-year (C) and 5-year (D) OS for BC patients in GEO. GEO, Gene Expression Omnibus; OS, overall survival; BC, bladder cancer.

Fig 6. Comparison between signatures.

By comparing with other signatures constructed through only prognostic-related genes (A-C) and other risk factors (D-F), our risk signature was found to be superior in performance.

Fig 7. The risk score was associated with infiltration of various immune cells.

The results of CIBERSORT indicated enrichment of signatures of M0 (A) and M2 macrophages (B) and neutrophils (C) in high-risk BC patients. Conversely, signatures of CD8 T cells (D), CD4 memory activated T cells (E), activated dendritic cells (F), follicular helper T cells (G) and plasma cells (H) were enriched in low-risk BC patients. CIBERSORT, cell type identification by estimating relative subsets of RNA transcripts; BC, bladder cancer; *, P <0.05; **, P < 0.01; ***, P < 0.001.

Fig 8. The risk score was significantly related to the TME based on the ESTIMATE algorithm.

The stromal score was positively correlated with the risk score (A), and tumor purity was negatively correlated with the risk score (B). There was no significant difference between the risk score and immune score (C). TME, tumor microenvironment.

Fig 9. Stromal cells in BC patients based on the xCell algorithm.

Results indicated that enrichment of signatures of eight stromal cells [adipocytes (A), chondrocytes (B), endothelial cells (C), fibroblasts (D), ly endothelial cells (E), MSCs (F), myocytes (G) and smooth muscle (H)] from the TME in the high-risk group. BC, bladder cancer; MSCs, mesenchymal stem cells; ly endothelial cells, lymphatic endothelial cells; TME, tumor microenvironment; P. adj, P. adjust.

Fig 10. Risk score and drug sensitivity.

Cisplatin (A) and docetaxel (B) exhibited higher IC50 values in low-risk patients; thus, the high-risk group was more sensitive to cisplatin and docetaxel. Methotrexate (C) and gemcitabine (D) exhibited a higher IC50 in high-risk BC patients; thus, low-risk BC patients were more sensitive to methotrexate and gemcitabine. However, there were no significant differences between other chemotherapy drugs (paclitaxel and doxorubicin) and the risk score (E-F). The CR/PR rate in low-risk patients was higher than that in high-risk patients (G), indicating more significant immunotherapy benefits for low-risk patients. IC50, half-maximal inhibitory concentration; SD, stable disease; PD, progressive disease; CR, complete response; PR, partial response; BC, bladder cancer; *, P <0.05; ****, P < 0.0001.