TCGA data mining suggested that high GSDMB expression in bladder cancer indicated an excellent prognosis


 Background. Researchers have demonstrated that GSDMB is highly expressed in cancer tissues and located in amplicons, genomic regions that are often amplified during cancer development. We evaluated the role of GSDMB in bladder cancer using publicly available data.Methods. The relationship between clinical correlation features and GSDMB were analyzed with the Wilcoxon rank sum test (2 groups), Kruskal-Wallis test (multiple groups) and logistic regression. Clinicopathologic characteristics associated with overall survival in TCGA patients using Cox regression and the Kaplan-Meier method. Gene Set Enrichment Analysis (GSEA) was performed using TCGA data set. Results. GSDMB was differentially expressed between the tumor group and the normal group and was highly expressed in the tumor group. Reduced GSDMB expression in BLCA as significantly associated with grade (OR = 4.55, High Grade vs. Low Grade), clinical stage (OR = 2.34, Ⅱvs. Ⅳ), status (OR = 0.51 survival vs. death), all p-values <0.05. Kaplan-Meier survival analysis showed that BLCA with GSDMB- low had a worse prognosis than that with GSDMB-high (p = 0.001). The univariate analysis revealed that GSDMB- low correlated significantly with a poor overall survival (OS) (HR: 0.93; 95%CI: 0.89-0,98; p = 0.006). The multivariate analysis revealed that GSDMB remained independently associated with overall survival, with a HR of 0.711 (CI: 0.55-0.92; p = 0.009). GSEA show that focal adhesion, pathways in cancer, small cell lung cancer, renal cell carcinoma, bladder cancer and other cancer related pathways are differentially enriched in GSDMB low expression phenotype. Conclusions. High GSDMB mRNA expression is an independent risk factor for excellent prognosis in bladder cancer.


Introduction
GasderminB(GSDMB) is a member of the Gasdermin(GSDM) family and can be regulated by different intramolecular domain interaction mechanisms for lipid binding and pore forming activity [1]. According to previous studies on human cancers, GSDMB is highly expressed not only in healthy tissues (such as lymphocytes, esophagus, stomach, liver, colon, skin epithelium and gastrointestinal tract), but also in gastric cancer, uterine cancer, cervical cancer, breast cancer and other cancer tissues [2,3]. Studies have shown that GSDMB is located in an amplicon, an area of the genome that is often ampli ed during cancer development. Therefore, GSDMB may play a role in tumor progression and metastasis [1].
Bladder cancer (BC) is a frequently occurring and highly recurrent, fatal malignancy of the genitourinary system, the most common of which is bladder urothelial carcinoma (BUC) [4]. The diagnosis and follow-up of bladder cancer mainly rely on cystoscopy and other means, but this kind of examination will bring some pain to patients, and some patients will interrupt the follow-up due to their intolerance [5]. GSDMB has been found to have signi cant expression in a variety of tumors. However, the correlation between GSDMB and the prognosis of bladder cancer Bladder Urothelial Carcinoma BLCA has not been previously reported. Therefore, this study attempted to evaluate the prognostic value of GSDMB expression in human BLCA patients using the data obtained from TCGA (The Cancer Genome Atlas; https://cancergenome.nih.gov/). In order to further understand the biological pathways of the GSDMB regulatory network related to the pathogenesis of BLCA, WE conducted GSEA.
Presently, we demonstrated the effect of GSDMB on prognosis of BLCA, increased GSDMB expression associated with excellent survival in bladder cancer. GSEA show that focal adhesion, pathways in cancer, small cell lung cancer, renal cell carcinoma, bladder cancer and other cancer related pathways are differentially enriched in GSDMB low expression phenotype.

Materials And Methods
2.1. RNA-sequencing patient data and bioinformatics analysis.
The gene expression data (433 cases, normal: tumor=19:414, Work ow Type: HTSeq Counts) and corresponding clinical information were downloaded from TCGA o cial website. The expression of GSDMB was extracted from the downloaded data, and the data of the normal group and the tumor group were used to draw a scatter plot by applying R (v. 4.0.2) (Fig. 1A). As can be seen from gure, GSDMB expression level in the tumor group was signi cantly higher than that in the normal group (P =0.008).
In tumor and adjacent nontumor tissues of the 434 samples, the expression difference was further analyzed and plotted (P =0.022, Fig. 1B). We reached the same conclusion that GSDMB expression was signi cantly higher in the tumor group than that in the control group.
Normal BLCA samples were excluded, the samples were divided into the high-expression group and the low-expression group by the median value of GSDMB expression, and the Kaplan-Meier survival curve analysis showed signi cant difference in the overall survival rate of the groups (p=0.001, Fig. 2A).

Gene set enrichment analysis
Gene set enrichment analysis (Gene set enrichment analysis, GSEA) is a kind of calculation method, use the software of the GSEA 4.0.3 to determine whether a priori de ned set of genes in a state between two organisms show consistency difference was statistically signi cant. [6]. A list was rst created based on the correlation of all genes with GSDMB expression, and the samples were then divided into the high GSDMB group and the low GSDMB group to identify signi cant differences in survival. The expression level of GSDMB was used as phenotypic marker, and 1000 genome sequences were performed for each analysis.
Nominal phosphorus values and standardized enrichment scores (NES) were used to classify enrichment pathways in each phenotype. Gene sets with a false discovery rate (FDR) <0.25 and a normal P-value<0.05 were considered signi cant. GSEA show that focal adhesion, pathways in cancer, small cell lung cancer, renal cell carcinoma, bladder cancer and other cancer related pathways are differentially enriched in GSDMB low expression phenotype.

High GSDMB expression in bladder cancer
GSDMB expression in bladder cancer and normal tissues was compared (Fig. 1A), and the results indicated that GSDMB expression was elevated in bladder cancer (P=0.008). In the tumor and adjacent nontumor tissues, GSDMB expression in the tumor group was higher than that in the control group (P =0.022) (Fig. 1B).

High Expression of GSDMB in BLCA Is Related to Excellent Overall Survival
The Kaplan-Meier survival curve analysis showed signi cant difference in the overall survival rate of the groups (p=0.001, Fig.   2A). BLCA with GSDMB-low had a worse prognosis than that with GSDMB-high. The 5-year survival rate of patients in the high-GSDMB expression group (52.4%) was higher than that of patients in the low-GSDMB expression group (38.1%).

Association with GSDMB expression and clinicopathologic variables.
A total of 433 BLCA samples with GSDMB expression data across all patient characteristics were analyzed from TCGA. As shown in Fig. 2. ( B-F), Wilcoxon rank sum test(2 groups) or Kruskal-Wallis test(multiple groups) was used to analyze the clinical correlation indicate increased expression of GSDMB correlated signi cantly with the survival status(p=1.793e-6), histological grade(p=9.667e-5), clinical stage (p=0.002), T classi cation (p=0.002),M classi cation (p=0.042).Univariate analysis using logistic regression revealed that GSDMB expression as a categorical dependent variable was associated with poor prognostic clinicopathologic characteristics (  Fig. 3A). Univariate and multivariate Cox analysis indicated that GSDMB expression was an independent risk factor for OS in bladder cancer.

Discussion
Bladder cancer (BLCA) is the ninth most common cancer worldwide [7]. According to the degree of in ltration, bladder cancer can be divided into non-muscular invasive bladder cancer and muscular invasive bladder cancer [8]. Non-muscular invasive bladder cancer accounts for about 75% of bladder cancers, 50% of which are low-level. The main shape of the tumor is micropapillary [9].
The 5-year survival rate is 90%, but the recurrence rate is high [10,11]. Therefore, early diagnosis of BLCA can reduce mortality and improve quality of life. Most current bladder etiology studies focus on genetic changes [12]. Oncogenes are mutated forms of proto-oncogenes that encode growth factors and receptor proteins necessary for normal cell growth [1]. Mutations in protooncogenes can lead to uncontrolled cell division and bladder cancer recurrence and progression. The oncogenes associated with bladder cancer include HER-2, H-RAS, Bcl-2, FGFR3, C-MYC, C-Erbb-2, MDM2, CDC91L1, etc. [10][11][12][13][14][15][16] Hypermethylation of SYK, CAGE-1 and other genes has been associated with the progression of bladder cancer [10,11]. In addition, the occurrence of bladder cancer also includes the ampli cation or overexpression of normal genes encoding growth factors or their receptors [16,17], for example, the overexpression of EGEF can increase the aggressiveness and metastasis of bladder cancer [18].
It has been found that GSDMB is highly expressed in various malignant tumors such as gastric cancer, liver cancer and cervical cancer. In different types of cancer in different ways to participate in or affect the occurrence, development, metastasis, drug response and so on [2,19,20]. However, the speci c function and mechanism of GSDMB in tumorigenesis, progression and metastasis are still unclear [21]. GSDMB expression in bladder cancer Bladder Urothelial Carcinoma BLCA has not been previously reported, We evaluated the role of GSDMB in bladder cancer using publicly available data from The Cancer Genome Atlas (TCGA).
In our study, bioinformatics analysis was performed on data from bladder cancer in TCGA, and the results showed that decreased GSDMB expression in BLCA was associated with clinicopathological characteristics, survival time and poor prognosis. The Kaplan-Meier curve of OS also showed that low GSDMB expression was associated with poor prognosis in bladder cancer patients. Subsequent univariate and multivariate Cox analysis indicated that GSDMB mRNA expression level in bladder cancer patients may be an independent biomarker for prognosis of bladder cancer. In order to further study the function of GSDMB in BLCA, TCGA data was used for GSEA. The results showed that many cancer-related pathways (e.g., bladder, kidney, prostate, pancreas, Table 4) were enriched with the GSDMB low-expression phenotype. GSDMB may be a potential prognostic marker and therapeutic target in BLCA. metastases, and GSDMB is also used as a new marker [3,20,[22][23][24][25][26]. In addition, GSDMB may be a potential therapeutic target for cancer therapy [24]. Considering the role of GSDMB in cytotoxic lymphocyte-mediated tumor clearance [20], this analysis may provide guidance for future immunotherapy approaches for cancer. At present, the research of GSDMB in cancer is still unclear, and there is still a large space for the research on the therapeutic targets for the treatment of cancer [27].

Conclusion
Our analysis showed that GSDMB expression was up-regulated in patients with bladder cancer, and increased GSDMB expression was associated with clinical progression, and was an independent risk factor for OS in patients with bladder cancer. Our ndings suggest that GSDMB may be a useful biomarker for prognosis of bladder cancer. The correlation between GSDMB mRNA expression and GSDMB protein expression was not assessed in this report. The speci c biological effects of GSDMB need to be veri ed by further relevant experiments.

Declarations Ethics approval and consent to participate
The data in this paper are from public databases, and do not involve animal or human trials, and do not involve approval by ethics committees.

Consent for publication
All authors agree to be published

Availability of data and materials
The data that support the ndings of this study are available in TCGA (http: //cancergenome.nih.gov/).

Competing interests
The authors declare that they have no competing interests.