The expression of AAGAB in breast cancer was significantly up-regulated
We screened the difference in AAGAB gene expression in the relevant cancer datasets from the TCGA databases, supplemented by the GTEx databases. The results demonstrated that the expression of AAGAB in BRCA, CESC and OV tissue was significantly higher than that in normal tissues (P <0.05) (Figure 1). 1104 breast cancer samples were included in this analysis and 403 normal breast tissues used for comparison. The expression of AAGAB in breast cancer was significantly up-regulated (p <0.001, log2FC = 1.163) relative to normal tissue (Figure 2).
For the purpose of analyzing the association between clinicopathologic variables and AAGAB expression, we divided breast cancer patients into AAGABhigh group and AAGABlow group based on the expression of AAGAB, in which performed chi-square analysis (Table 1). The pathological information of the patient includes: age, gender, ethnicity, pathological stage, TMN stage, PAM50 typing, typing, tumor purity, number of lymph nodes and histological type. The analysis shows that AAGAB expression was significantly associated with the patient's age, gender, race, ER status, PR status, N-stage, PAM50 classification and histological type (p<0.05). For N-stage group, which is the case of lymph node metastasis, 43.39% (223/514) of patients of AAGAB high expression were in N0, 56.79% (205/361) of patients were in N1, 51.66% (62/120) of patients were in N2, 52.63% (40/76) of patients were in N3. The results indicate that the expression of AAGAB was relevant to the lymph node metastasis of patients. Regarding age, AAGABhigh group under 60 and over 60 was 43.82% (248/566) and 57.06% (291/510), respectively. These results indicate that the expression of AAGAB may be related to age, AAGAB expression also had a significant relationship with histological type, AAGAB was expressed low in medullary carcinoma and metaplastic carcinoma.
Validation of elevated AAGAB protein level in breast cancer tissues
To assess AAGAB protein level in our paraffin-embedded tumor samples, we performed IHC staining and found that significant elevated AAGAB protein level in terms of density and intensity in breast cancer tissues compared to adjacent normal breast tissues (p=0.001, Figure 3)
Survival analysis and multivariate analysis
In order to determine the effect of AAGAB gene expression levels on breast cancer prognosis, Cox multiple regression analysis was carried out. According to the expression of AAGAB, the cases were divided into the groups with the high AAGAB expression and the low AAGAB expression, followed by making the survival analysis (Figure 4). The results illustrated that there was no significant difference in disease-free survival between the high and low AAGAB expression groups (Figure 4a), but a significant correlation was found for overall survival (p = 0.005, Figure 4b), indicated that breast cancer with AAGAB-high had a worse prognosis than that with AAGAB-low. Univariate analysis using logistic regression revealed that age, pathological stage, and number of lymph nodes were significantly associated with poor OS (all p<0.05) (Figure 5).
Association of AAGAB’s expression with tumor purity and immune infiltration
The above finding suggested that the expression of AAGAB may have an impact on the prognosis of breast cancer. The relationship between six types of immune infiltrating cells (including B cells, CD4 T cells, CD8 T cells, dendritic cells, macrophages and neutrophils) together with tumor purity and AAGAB expression was analyzed to determine whether AAGAB expression was related to the level of immune invasion in cancer (Figure 6). AAGAB expression level was positively significantly correlated with cell purity (cor=0.326, p=7.9E-28), CD8 T cells (cor=0.07, p=2.26E-02), and macrophages (cor=0.104, p=6.3E-04) of infiltration level. In addition, AAGAB expression level in breast cancer was negatively significantly correlated with CD4 T cells (cor=-202, p=2.18E-11) and dendritic cells (cor = -0.074, p = 1.5E-02). Whereas, the expression of AAGAB in breast cancer was independent of B cells (cor=0.012, p=7.07E-01) and neutrophils (cor= -0.033, p=72.76E-01). In breast cancer, AAGAB expression levels were markedly positively correlated with tumor purity, indicating its relative enrichment in tumor cells. These findings indicate that AAGAB can affect the prognosis of breast cancer by interacting with breast cancer immune infiltration.
GSEA identifies differentially enriched AAGAB-related signaling pathways
GSEA was conducted between high and low AAGAB expression gene sets to identify differentially enriched signaling pathways in breast cancer. Nom p-val<0.05, FDR q-val<0.25 are considered as significant. Two gene sets are significant at FDR<0.25 in phenotype high, 0 gene set is significant at FDR<0.25 in phenotype low. The Figure 7 shows that Intra Golgi Traffic (p<0.004), Peroxisomal Lipid Metabolism (p<0.006) are differentially activated signaling pathways in AAGAB high expression phenotype.