The demographic and clinicopathological characteristics of HRNBC patients
144 HRNBC patients were studied, and the demographic and clinicopathological characteristics was showed in the table 1. All biopsies were collected from women from 24 to 86 years of age at the time of surgery, garnering an average age of 50.4 years old. The time of follow-up ranged from 27 to 130 months, with the average of 75 months. 113 (78.5%) and 31 (21.5%) patients had tumors of TNM stages I-II and III, respectively. 30 (20.8%) and 114 (79.2%) patients had T classification of T1 and T2-T4, respectively. In total, 57 (39.6%) patients were devoid of lymph node invasion. 48(33.3%) and 96 (66.7%) patients had well + moderate cell differentiation and poor cell differentiation of IDC, respectively. 50(34.7%) patients had HER-2 positive. During the follow-up period, 29(20.1%), 19(10.5%), 10(6.9%), 16(11.1%) and 3 (2.1%) patients had Lung, Bone, Liver, Chest wall and Brain metastasis, respectively.
Expression of Btbd7 in HRNBC and benign breast lesions and relationships with clinicopathological parameters
Immunohistochemistry (IHC) analysis was performed to investigate Btbd7 expression in HRNBC and benign breast lesions tissues. Positive immunohistochemical staining of Btbd7 was mainly observed in the cytoplasm of the cells. Benign breast lesions cells exhibited negative or weak staining when compared with the staining of HRNBC cells (Figure 1 A). The total positive rate of Btbd7 expression in TNBC and Her-2+ patients was 64.9% (61/94) and 70% (35/50), respectively, which was higher than that in benign breast lesions tissues (20%) (6/30) (Figure 1 B, p < 0.001). Increased Btbd7 expression in HRNBC was significantly associated with larger tumour volume and poorer TNM stage (Figure 1 B p < 0.05). No significant association, however, could be described for the expression of Btbd7 and other clinicopathological factors in HRNBC.
The data from TCGA showed that in HRNBC patients and HER-2 overexpression subgroup breast cancer, high BTBD7 mRNA expression tended to have shorter DFS (P < 0.001) than low expression (Figure 1C-D). However, the DFS for patients with TNBC were insufficient to adequately analyse prognosis.
As shown in the Kaplan-Meier survival curves in Figure 1E-F, the median estimated DFS time was 93.5 ± 5.0 months for HRNBC patients with low Btbd7 expression. For HRNBC patients with high Btbd7 expression, however, the DFS time was only 72.7 ± 5.8 months (P = 0.003). What's more, the median estimated OS time was 98.4±3.8 months for HRNBC patients with low Btbd7 expression, which is significant longer than those with high Btbd7 expression (92.2 ± 5.2 months, P=0.028).
In order to analyse the function of Btbd7 in TNBC and HER-2+ patients, respectively, we had the subgroup analysis of these two molecular subtyping.
In the TNBC subgroup analysis, the patients with high Btbd7 expression had significant shorter DFS (P=0.049) and OS (P=0.048) than those with low Btbd7 expression (Figure 2 G-H). As shown in the figure 2 A-J, among the patients aged less than 50 years and TNM stages 1–2 and well + moderately differentiated IDC, a higher Btbd7 expression level was associated with shorter DFS time. As shown in the figure 2 K-T, among the patients age >50 years and with lymph node metastasis, a higher Btbd7 expression level was associated with shorter OS time.
In the HER-2+ subgroup analysis, the patients with high Btbd7 expression had significant shorter DFS (P=0.026) than those with low Btbd7 expression (Figure 2 I). No significant differences, however, were identified between the median estimated survival time between HER-2+ patients with low and high Btbd7 expression (P=0.338) (Figure 1 J). As shown in the figure 3 A-J, among the patients aged more than 50 years, tumour diameter more than 2cm, with lymph node metastasis and poor differentiated invasive ductal carcinoma, a higher Btbd7 expression level was associated with shorter DFS time. However, as shown in the figure 3 K-T, a higher Btbd7 expression level was not associated with shorter OS time among any subgroup.
Tumour recurrence and metastasis were the main cause of death in breast cancer patients. In our study, we found that high Btbd7 expression was significantly associated with high lung metastases (P = 0.001，r = 0.282) while no association could be linked between Btbd7 expression and bone, liver, brain metastatic and chest wall recurrence (P > 0.05) (Table 2).
Correlation between Btbd7 expression and Slug expression in HRNBC
As Slug is recognised to be an important regulatory factor in EMT, in order to verify the level of influence of Btbd7 on EMT, a correlation analyses on Btbd7 and Slug was performed. The staining of Slug was observed mainly in the cytoplasm and in the cell nucleus (Figure 4 A).
The data from TCGA showed that in HRNBC patients, high SLUG mRNA expression tended to have shorter DFS (P =0.014) than low expression (Figure 4B). Our IHC experiment also showed that high Slug protein expression in the HRNBC patients’ tissue was associated with shorter DFS (P=0.001) and OS (P=0.003) time (Figure 4C-D), which indicated that SLUG was also the prognostic indicator in HRNBC patients. As shown in Figure 4E, patients with high expression of Btbd7 subsequently exhibited high Slug expression (P = 0.001); The rate of high Slug expression in patients with high Btbd7 was 72.9% (70/96), while the rate was 41.7% (20/48) in patients with low Btbd7. A scatter diagram was further performed to identify the correlation between these two markers. The linear correlation coefficient was calculated to be 0.304 with a p value of less than 0.001. As such, the expression of Btbd7 positively correlated with Slug expression. (Figure 4F)
Considering the prognostic significance of Btbd7 and Slug, we generated ROC curves to assess the predictive value on 3-year, and 5-year recurrence-free survival rate. AUC combining Btbd7 and Slug was 74.5% for 3 years, and73.5% for 5 years, which was more accurate than prediction using pT, pN, and Btbd7 or Slug alone (Figure 4G-H).
Univariate and multivariate analysis
Furthermore, the factors potentially affecting HRNBC prognosis including DFS (Table 3) and OS (Table 4) of the patients were analysed by Cox regression model. Concerning DFS, the univariate cox regression analysis showed that good prognostic factors were with no lymphatic metastasis (P = 0.038), lower TNM stage (P = 0.007), Well or moderate IDC differentiation (P=0.026), lower Btbd7 (P = 0.003) and lower Slug expression level (P = 0.001). In multivariate cox regression analysis, however, age (P = 0.014), Btbd7 (P = 0.013) and Slug (P=0.011) were independent prognostic factors. It was found that following the univariate cox regression analysis of the overall survival, only a younger age (P = 0.006), lower Btbd7 expression levels (P = 0.035) and lower SLUG expression levels (P = 0.006) were good prognostic factors. In the multivariate cox regression analysis, we found that the age (P = 0.001) the Btbd7 (P=0.036) and Slug (P=0.021) were the independent prognostic factors.
BTBD7 was involved in regulating extracellular components.
To explore the function roles of BTBD7 in breast cancer, we screened out 171 DEGs according to the expression level of BTBD7. DAVID was used to analyse the DEGs and enriched several GO terms, such as extracellular space and extracellular region (Fig 5A). To enlarge this observation, KOBAS 3.0 was performed. Similar GO term ‘extracellular region’ (Figure 5B) and several KEGG pathways such as Ras signalling pathway, MAPK signalling pathway, Estrogen signalling pathway and Breast cancer (Figure 5C) were obtained. These results revealed BTBD7 was associated with extracellular components and indicated it is closely related to tumor invasion and metastasis.