3.1 Expression of the GSDM family in breast cancer: we examined the mRNA levels of the GSDM family in breast and paracancerous tissues using the UALCAN database. The results revealed that, with the exception of GSDMA, which exhibited no significant difference in expression between breast and paracancerous (P=0.499), the other members of the GSDMS family displayed differential expression in both tissue types. Specifically, GSDMD was highly expressed in breast cancer while GSDMB, GSDMC,GSDME(DFNA5) and PJVK(DFNB59) were all expressed at low levels (Figure 1).
3.2 Transcriptional differences among molecular subtypes of breast cancer: To further investigate potential variations in mRNA expression levels among different molecular subtypes of breast cancer within the various members of the GSDM family, we analyzed their transcript levels using data from the UALCAN database (Figure 2). The expression levels of GSDMB and GSDMC are significantly upregulated in HER2-positive breast cancer, while they are lower in triple-negative and luminal breast cancer. (Figures 2b,c). The expression levels of GSDMD, GSDME (DFNA5), and PJVK (DFNB59) were significantly elevated in the luminal phenotype of breast cancer compared to Her2+ and triple-negative breast cancer subtypes (Figure 2d, 2e, 2f). These findings suggest a potential correlation between the expression patterns of the GSDMS family and molecular subtypes in breast cancer patients.
3.3 The expression of the GSDM family was analyzed in relation to clinical characteristics of breast cancer patients: In order to analyze the expression of GSDM family in clinical case characteristics of breast cancer patients, we summarized the results based on age (with a cut-off at 51 years old), presence or absence of lymph node metastasis, ER and PR expression status, Her2 status, occurrence of triple-negative breast cancer, and presence or absence of P53 mutation. Among these factors, GSDMA showed significantly higher expression in patients aged 51 years old or younger (p=0.0207). Similarly, GSDMB and GSDMC also showed significantly higher expression in this age group (p=0.0181 and p<0.0001 respectively) [Table 1]. There was no significant association between age and the expression levels of other members within the GSDM family. The expression levels of GSDMB (P<0.0001), GSDMC (P=0.0011), GSDMD (P=0.0113), and GSDME (P=0.0388) were significantly upregulated in breast cancer patients with lymph node metastasis, while PJVK showed high expression in breast cancer patients without lymph node metastasis (p<0.0001). Additionally, the expression of GSDMA remained unchanged regardless of the patient's lymph node status (P=0.1862). Moreover,In patients positive for ER and PR antibodies, the expression levels of GSDMD (P<0.0001) and PJVK (P<0.0001) were significantly upregulated. GSDMA (ER-P=0.0128, PR-P<0.0001), GSDMB (P<0.0001), GSDMC (P<0.0001), and GSDME (P<0.0001) exhibited elevated expression in ER and PR antibody-positive individuals.The expression levels of GSDMA (P=0.0034), GSDMB (P< 0.0001), and GSDMC (P< 0.0001) were significantly higher in HER2+ positive patients, whereas the expression levels of GSDMD (P< 0.0001) and PJVK (P< 0.0001) were markedly elevated in HER2- patients. No significant association was observed between the expression level of GSDME and HER2 status (P=0.3308).The expression of GSDMC (P<0.0001) and GSDME (P<0.0001) exhibited a positive correlation in patients with triple-negative breast cancer, whereas the expression of GSDMD (P<0.0001) and PJVK (P<0.0001) demonstrated an inverse association with triple-negative breast cancer. However, no significant correlation was observed between the expression of GSDMA (P=0.6576), GSDMB (P=0.6184), and triple-negative breast cancer.. Furthermore, patients harboring P53 mutations exhibited significantly elevated expressions of GSDMB and GSDMC (P<0.0001). In contrast, pronounced expression of PJVK was specifically observed in patients with wild-type P53. No significant association between other members of the GSDM family and P53 status was found.
3.4 Genetic alterations of GSDMS in patients with breast cancer:To explore the underlying factors contributing to differential expression of GSDMS among breast cancer patients, we employed cBioPortal (http://www.cbioportal.org), an online database, for analyzing genetic alterations associated with GSDMS. The findings are presented in Figure 3, demonstrating a predominant occurrence of missense mutations, deletion mutations, amplifications, and deep deletions within GSDMA-E and PJVK genes at alteration rates of approximately 10%, 11%, 19%, and 15% respectively.
3.5 Prognostic value of GSDM family mRNA expression in patients with breast cancer:We employed Kaplan-Meier analysis to evaluate the prognostic significance of each member within the GSDMS family in breast cancer patients, encompassing overall survival (OS) and disease-free survival (RFS). Results: As shown in Figure 4A,high expressionlevels of GSDMB and GSDMC were negatively correlated with overall survival (OS) among breast cancer patients. Conversely, high expression levels of GSDMD and PJVK demonstrated a positive association with overall survival (OS), while no significant correlation was observed between GSDMA or GSDME expression and overall survival. As illustrated in Figure 4B, high expressions of GSDMB, GSDMC, GSDMD, and PJVK were positively associated with the recurrence-free survival rate (RFS) in breast cancer patients. On the other hand, high levels of expressed GSDME displayed a negative correlation with the disease-free survival rate among breast cancer patients; however, no significant association was found between RFS and the expression level of GSDMA. The aforementioned data indicate that elevated expressions of both GSDMD and PJVK are positively associated with both OS and RFS in breast cancer patients. Therefore, these two markers may serve as valuable prognostic indicators for assessing the prognosis of individuals diagnosed with breast cancer.
3.6 RFS prognostic value of GSDM family mRNA expression levels in breast cancer subtypes:In order to investigate the association between mRNA expression levels of GSDMS family and RFS prognostic value in breast cancer patients with different subtypes, we further analyzed the corresponding survival curve. The subtypes were classified based on the 2011 St. Gallen criteria [28], including basal-like, luminal A, luminal B, and HER2+. As shown in Table 2, within these subclasses, high expressions of GSDMB, GSDMD, and PJVK were positively correlated with improved RFS prognosis. Specifically in Luminal A subtype, high expressions of GSDMB, GSDMC and PJVK showed a positive correlation with RFS prognosis. In Luminal B subtype, high expressions of GSDMC,GSDME,and PJVK were also associated with better RFS prognosis; however,in HER2+ subtype,the high expressions of GSDMB and PJVK indicated a positive correlation with RFS prognosis while higher expression level of GSDMC was negatively correlated.
3.7 The relationship between the GSDM family and immune cells in breast cancer patients has been investigated in several studies [29,30]. In this study, we utilized the Timer2.0 online database to further analyze this relationship. Our findings revealed a positive correlation between the expression of GSDMA and B cells, macrophages, dendritic cells, and neutrophils (Figure 5A). Similarly, GSDMB expression showed a positive correlation with CD4+ T cells, B cells, and dendritic cells but a negative correlation with CD8+ T cells and macrophages (Figure 5B). Additionally, GSDMC expression demonstrated a positive correlation with CD4+ T cells, dendritic cells, and neutrophils while exhibiting a negative correlation with B cells (Figure 5C);The expression of GSDMD exhibited a positive correlation with CD4+ T cells, B cells, and dendritic cells, while it demonstrated a negative correlation with CD8+ T cells, macrophages, and neutrophils (Figure 5D). The expression of GSDME showed a positive correlation with CD4+ T cells, CD8+ T cells, macrophages, dendritic cells, and neutrophils; however, it displayed a negative correlation with B cells (Figure 5E). PJVK expression was positively associated with CD4+ T cells but negatively associated with B cells and dendritic cells (Figure 5F).
3.8 Methylation levels of GSDM family in breast cancer patients: The negative correlation between gene expression and DNA hypermethylation levels has been demonstrated [31,32]. To investigate the methylation level of GSDMS in breast cancer patients, we utilized the DiseaseMeth database online for analyzing the methylation status of GSDMS in breast cancer . The results are presented in Figure 6. Notably, the methylation levels of GSDMC, GSDMD, and PJVK were lower in breast cancer compared to paracancerous; conversely, higher methylation levels were observed for GSDMA and GSDME in breast cancer when compared to normal tissues. Our previous findings have demonstrated a significant upregulation of GSDMD in breast cancer, while GSDME exhibits low expression levels in these tissues. The observed methylation patterns for both GSDMD and GSDME are consistent with their respective expression trends, suggesting a potential influence of methylation on the regulation of thei rexpressionlevels.