Diagnostic and Prognostic implications of AAGAB expression in human breast cancer

Background: Alpha and gamma adaptin binding protein p34 (AAGAB) was previously reported as a novel on-treatment biomarker can improve prediction of response to neoadjuvant chemotherapy in breast cancer. However, the expression and prognostic value of AAGAB in breast cancer is unknown, the function of AAGAB in breast cancer remains to be elucidated. Methods: Herein we investigated the role of AAGAB in human breast cancer from the TCGA database, immunohistochemistry, Gene set enrichment analysis (GSEA) and immune inltration analysis. Results: Increased AABAB expression in breast cancer was signicantly associated with age, gender, race, ER status, PR status, N-stage, PAM50 classication and histological type (all p-values<0.05). Kaplan-Meier survival analysis showed that breast cancer patients with AAGAB-high had a worse prognosis than that with AAGAB-low (p=0.005). Univariate analysis using logistic regression revealed that age, pathological stage, and number of lymph nodes were signicantly associated with poor overall survival (OS) (all p <0.05). AAGAB expression level was signicantly correlated with cell purity, CD8 T cells, macrophages, CD4 T cells and dendritic cells. Functional annotations indicated that AAGAB is involved in the most signicant signaling pathways including intra Golgi trac and peroxisomal lipid metabolism pathways. Conclusions: Our study revealed that elevated AAGAB expression was signicantly correlated with aggressive progression, poor survival in breast cancer patients. AAGAB may serve as a new biomarker and potential treatment target in breast cancer. stage, PAM50 typing, typing, tumor purity, number of lymph nodes and histological type. The analysis shows that AAGAB expression was signicantly associated with the patient's age, gender, race, ER status, PR status, N-stage, PAM50 classication 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.


Background
Breast cancer is a malignant tumor occurring in mammary epithelial tissue, which proliferating out of control via the action of various carcinogenic factors. According to the latest data from the International Agency for Research on Cancer (IARC) survey in 2018, incidence (24.2%) and mortality (15.0%) of breast cancer occupy the top positions among women cancer worldwide [1]. Over the last few decades, the incidence of breast cancer has been increasing year by year in China. The implementation of neoadjuvant treatment and the popularization of screening can signi cantly reduce the mortality of breast cancer [2].
However, the mortality rate from breast cancer has not decreased signi cantly in China, especially in the vast rural areas [3]. So far, scientists have not found the exact cause of breast cancer, but many risk factors have found associated with breast cancer.
As a highly heterogeneous disease, patients' response to treatment and prognosis are different even if the clinical stage and pathological grade are the same [4]. Biomarkers are now widely used for early cancer screening and prognosis assessment. Common biomarkers for breast cancer include estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (Her2) [5]. Despite the advances in screening and diagnosis techniques in recent years, which have greatly improved the survival rate of breast cancer patients, it remains one of the major diseases with the highest female mortality rates [6]. At present, most biomarkers that predict prognosis of breast cancer lack speci city, thus, it is clinically important to discover new biomarkers to enhance prognosis and individualized treatment.
AAGAB encodes the alpha and gamma adaptin binding protein p34, which interacts with the gammaadaptin and alpha-adaptin subunits of complexes involved in clathrin-coated vesicle tra cking [7,8]. Previous studies have found that heterozygous loss mutations in AAGAB are associated with palmoplantar keratodermas and Punctate palmoplantar keratoderma type I, which based on hyperproliferation within the punctate lesions [9]. AAGAB maybe involving in endocytic recycling of growth factor receptors such as EGFR, which can result in increasing cell division [10]. There was no close correlation between AAGAB expression and tumor in previous studies. Richard et al. found that AAGAB is a novel on-treatment biomarker can improve prediction of response to neoadjuvant chemotherapy in breast cancer [11]. However, the expression and functions of AAGAB in breast cancer are unknown, the role of AAGAB in breast cancer need to be further con rmed.
This study attempts to evaluate the diagnostic and prognostic value of AAGAB expression in human breast cancer. GSEA was performed to gain the biological pathways involved in breast cancer pathogenesis related to AAGAB. According to our informational analysis, we indicated that the signi cant diagnostic and prognostic value of AAGAB for breast cancer, AAGAB may serve as a new biomarker and potential treatment target for patients with breast cancer.

TCGA and GTEx databases
The Cancer Genome Atlas (TCGA), supervised by the National Cancer Institute and the American Human Genome Institute, utilizes high-throughput genomic analysis technology to analyze gene mutations, which have improved the ability to prevent, diagnose and treat cancer [12].

Immunohistochemistry (IHC)
Para n-embedded tumor sections of breast cancer (from Tongji Medical College of HUST) were incubated overnight at 4°C with rabbit anti-AAGAB polyclonal antibody (FLJ11506, from Novus) that was diluted 1:200 in TBST containing 1% NGS. After washing, the sections were conjugated with goat antirabbit horseradish peroxidase (HRP) antibody (from Novus) at 1:5000 dilution for 30 min at room temperature, Subsequently, the sections were stained with 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin, as well as dehydrated through a graded ethanol series and sealed with neutral gum. Finally, the sections were observed using a light microscope (Olympus).

Immune in ltration analysis
To examine the interactions between tumor in ltrating immune cells and the expression of AAGAB, we performed integrated repository tool for tumor-immune system interactions (TIMER, http://cistrome.shinyapps.io/timer/), which contains 10,897 samples from diverse cancer types available in the TCGA database [13,14].
Gene set enrichment analysis GSEA is a computational method to determine whether an a priori de ned gene sets is signi cant between two groups [15,16]. We used MSigDB database C2 collection to perform GSEA. For the purpose of analysis, we divided breast cancer patients into AAGAB high group and AAGAB low group based on the expression level of AAGAB. Gene set permutations were 1000 times for each evaluation. The nominal p value and normalized enrichment score (NES) were used to sort the enriched pathways of gene sets.

Statistical analysis
All statistical analyses were performed using R (v3.4.3). The association between clinical pathologic features and AAGAB levels were analyzed with the Wilcoxon signed-rank test and logistic regression. To further assess the prognostic signi cance of AAGAB in breast cancer, Kaplan-Meier analysis and multivariate Cox regression analysis were performed. The cut-off value of AAGAB expression was determined by its median value.

Results
The expression of AAGAB in breast cancer was signi cantly 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 signi cantly 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 signi cantly up-regulated (p <0.001, log 2 FC = 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 AAGAB high group and AAGAB low 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 signi cantly associated with the patient's age, gender, race, ER status, PR status, N-stage, PAM50 classi cation 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, AAGAB high 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 signi cant 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 para n-embedded tumor samples, we performed IHC staining and found that signi cant 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 signi cant difference in disease-free survival between the high and low AAGAB expression groups (Figure 4a), but a signi cant 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 signi cantly associated with poor OS (all p<0.05) ( Figure 5).

Association of AAGAB's expression with tumor purity and immune in ltration
The above nding suggested that the expression of AAGAB may have an impact on the prognosis of breast cancer. The relationship between six types of immune in ltrating 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 signi cantly 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 in ltration level. In addition, AAGAB expression level in breast cancer was negatively signi cantly 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 ndings indicate that AAGAB can affect the prognosis of breast cancer by interacting with breast cancer immune in ltration.
GSEA identi es 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 signi cant. Two gene sets are signi cant at FDR<0.25 in phenotype high, 0 gene set is signi cant at FDR<0.25 in phenotype low. The Figure 7 shows that Intra Golgi Tra c (p<0.004), Peroxisomal Lipid Metabolism (p<0.006) are differentially activated signaling pathways in AAGAB high expression phenotype.

Discussion
Despite the advances in screening and diagnosis techniques in recent years, which have greatly improved the survival rate of breast cancer patients, it remains one of the major diseases with the highest female mortality rates. At present, most biomarkers that predict prognosis of breast cancer lack speci city, thus, it is clinically important to discover new biomarkers to enhance prognosis and individualized treatment.
AAGAB encodes the alpha-and gamma-adaptin binding protein p34, which interacts with the gammaadaptin and alpha-adaptin subunits of complexes involved in clathrin-coated vesicle tra cking. Previous studies have found that heterozygous loss mutations in AAGAB are associated with punctate palmoplantar keratoderma, which based on hyperproliferation within the punctate lesions. AAGAB maybe involving in endocytic recycling of growth factor receptors such as EGFR, which can result in increasing cell division. The level of AAGAB was found to be prognostic of response in renal cancers and in thyroid cancers from the TCGA database. AAGAB was identi ed as a novel on-treatment biomarker for accurate prediction of pathological complete response (pCR) and reaction in patients treated with neoadjuvant chemotherapy. However, the exact role of AAGAB in breast cancer is currently unclear and warrants further investigation.
Herein we investigated the role of AAGAB in human breast cancer from the TCGA database, immunohistochemistry, GSEA and Immune in ltration analysis. Increased AABAB expression in breast cancer was signi cantly associated with age, gender, race, ER status, PR status, N-stage, PAM50 classi cation and histological type (all p-values<0.05). Kaplan-Meier survival analysis showed that breast cancer patients with AAGAB-high had a worse prognosis than that with AAGAB-low (p=0.005). Univariate analysis using logistic regression revealed that age, pathological stage, and number of lymph nodes were signi cantly associated with poor overall survival (OS) (all p <0.05). AAGAB expression level was signi cantly correlated with cell purity, CD8 T cells, macrophages, CD4 T cells and dendritic cells, indicate that AAGAB can affect the prognosis of breast cancer by interacting with breast cancer immune in ltration.
Functional annotations indicated that AAGAB is involved in the most signi cant signaling pathways including intra Golgi tra c and peroxisomal lipid metabolism pathways. The underlying mechanisms between AAGAB and carcinogenesis of breast cancer remain unclear, elevated AAGAB maybe increase endocytic recycling of EGFR, which induce the signaling and division of breast cancer cells, however, future research is required to explore the detail mechanisms of AAGAB in breast cancer.
Our study revealed that elevated AAGAB expression was signi cantly correlated with aggressive progression, poor survival in breast cancer patients. AAGAB may serve as a new biomarker and potential treatment target in breast cancer. Figure 1 The expression pro le of AAGAB in corresponding cancers from TCGA datasets. The cancer names marked in red represent AAGAB is signi cantly up-regulated, in blue represent AAGAB is signi cantly down-regulated, in black represent AAGAB is not signi cantly different. The difference threshold is |log2FC|≥1, P-value ≤0.05.

Figure 2
The expression of AAGAB in breast cancer tissues and normal tissues from TCGA database. The expression of AAGAB in breast cancer tissues was signi cantly up-regulated as compared to normal breast tissues (p<0.001, log2FC=1.163).  Disease-free survival curve and overall survival curve related to gene expression. there was no signi cant difference in disease-free survival between the high and low AAGAB expression groups (A), but a signi cant correlation was found for overall survival (p = 0.005, B).

Figure 5
Univariate regression analysis of clinical information in TCGA database. Univariate analysis using logistic regression revealed that age, pathological stage, and number of lymph nodes were signi cantly associated with poor OS (all p<0.05).

Figure 6
Association of AAGAB's expression with tumor purity and immune in ltration. AAGAB expression level was positively signi cantly correlated with cell purity, CD8 T cells and macrophages, was positively signi cantly correlated with CD4 T cells and dendritic cells.