The demographic and clinicopathological characteristics of HRNBC patients
Overall, 144 HRNBC patients were studied, and the demographic and clinicopathological characteristics are shown in the Table 1. All biopsies were collected from women aged from 24 to 86 years at the time of surgery, with an average age of 50.4 years. The follow-up period ranged from 27 to 130 months, with an average of 75 months. A total of 113 (78.5%) and 31 (21.5%) patients had tumors with TNM stages I–II and III, respectively. Thirty (20.8%) and 114 (79.2%) patients had T classifications of T1 and T2-T4, respectively. In total, 57 (39.6%) patients were devoid of lymph node invasion. Forty-eight (33.3%) and 96 (66.7%) patients had well + moderate cell differentiation and poor cell differentiation of IDC, respectively. Fifty (34.7%) patients had HER-2 positivity. 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 its relationships with clinicopathological parameters
IHC analysis was performed to investigate Btbd7 expression in HRNBC and benign breast lesion tissues. Positive immunohistochemical staining of Btbd7 was mainly observed in the cytoplasm of the cells. Benign breast lesions and NAT cells exhibited negative or weaker staining when compared with that in HRNBC cells (Figure 1 A). The total positive rate of Btbd7 expression in HRNBC tumor tissue was 66.7% (96/144), which was higher than that in NAT (52.1% 75/144, P<0.001) and benign breast lesion tissues (20%, 6/30) (Figure 1 B, P<0.001). Increased Btbd7 expression in HRNBC was significantly associated with larger tumor volume and poorer TNM stage (Figure 1 B, P<0.05). However, no significant association was observed between the expression of Btbd7 and other clinicopathological factors in HRNBC.
Survival analysis
The data from TCGA showed that in HRNBC patients and in those in HER-2 overexpression breast cancer subgroup, the higher BTBD7 mRNA expression tended to be associated with shorter DFS (P<0.001 Figure 1C-D). However, the DFS in patients with TNBC was insufficient to adequately analyze prognosis. As shown by the Kaplan-Meier survival curves in Figure 1E-F, the median estimated DFS (93.5 ± 5.0 vs. 72.7 ± 5.8 P=0.003) and OS (98.4±3.8 vs 92.2 ± 5.2 P=0.028) times in the patients with low Btbd7 expression were longer than those in patients with high Btbd7 expression.
In order to analyze the function of Btbd7 in TNBC and HER-2+ patients, we performed a subgroup analysis of these two molecular subtypes.
In the TNBC subgroup analysis, the patients with high Btbd7 expression had significantly shorter DFS (P=0.049) and OS (P=0.048) than those with low Btbd7 expression (Figure 1 G-H). As shown in Supplementary figure 2 A-J, among the patients aged <50 years and with TNM stages 1–2 and well + moderately differentiated IDC, a higher Btbd7 expression level was associated with shorter DFS time. As shown in Supplementary figure 2 K-T, among the patients aged >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 significantly shorter DFS (P=0.026) than those with low Btbd7 expression (P=0.026, Figure 1 I). However, no significant differences were observed in the median estimated survival time between HER-2+ patients with low and high Btbd7 expression (P=0.338 Figure 1 J). As shown in Supplementary figure 3 A-J, among the patients aged >50 years, tumor diameter >2cm, lymph node metastasis, and poorly differentiated IDC, a higher Btbd7 expression level was associated with shorter DFS time. However, as shown in Supplementary figure 3 K-T, a higher Btbd7 expression level was not associated with shorter OS time among subgroups.
Tumor recurrence and metastasis were the main causes of death in breast cancer patients. In our study, we found that high Btbd7 expression was significantly associated with a higher lung metastasis rate (28.1% 27/96) in comparison to patients with low Btbd7 expression (4.2% 2/48, P=0.001), while no association could be detected between Btbd7 expression and bone, liver, brain metastasis, and chest wall recurrence (P>0.05) (Table 2).
Correlation between Btbd7 and Slug expression in HRNBC patients
As Slug is recognized to be an important regulatory factor in EMT, in order to verify the level of influence of Btbd7 on EMT, correlation analyses on Btbd7 and Slug were performed. IHC (Figure 2A) and immunofluorescent (Figure 2F) staining in the HRNBC tumor tissue showed that Slug protein was observed mainly in the cytoplasm and the cell nucleus, and co-expression of Btbd7 and Slug could be observed in the tumor tissue.
The data from TCGA revealed that in HRNBC patients, the patients with high SLUG mRNA expression tended to have shorter DFS than those with low expression (P=0.014, Figure 2B), but there was no significant difference in OS between these groups (Figure 2C). Our IHC results also indicated that high Slug protein expression in the HRNBC patient tissue was associated with shorter DFS (P=0.001) and OS (P=0.003) time (Figure 2D-E), which indicated that Slug was also a prognostic indicator in HRNBC patients. 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 those with low Btbd7 (Supplementary Table 1). 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 was weakly positively correlated with Slug expression (Figure 2F).
Considering the prognostic significance of Btbd7 and Slug, we generated receiver operating characteristic (ROC) curves to assess the predictive value of the 3- and 5-year DFS rate. As showed in figure 2G-H, the area the curve (AUC) in both Btbd7 and Slug expression positive was 0.745 for 3 years, and 0.735 for 5 years, which was higher than that in pT, pN, and Btbd7 or Slug expression positive alone (P<0.05), which indicated that combination of Slug and Btbd7 proteins expression levels had the better prognostic value in evaluating HRNBC DFS rate.
Univariate and multivariate analyses
Furthermore, the factors potentially affecting HRNBC prognosis, including DFS (Table 3) and OS (Table 4), were analyzed using 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, the 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 OS, only 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 age (P=0.001) the Btbd7 (P=0.036), and Slug (P=0.021) were independent prognostic factors.
BTBD7 promotes MDA-MB-231 cell proliferation and metastasis via EMT pathway
As the clinical data and IHC analyses demonstrated that high Btbd7 and Slug were poor prognostic factors in HRNBC patients, and the EMT pathway may be the key element for understanding the molecular mechanism of Btbd7 and Slug in breast cancer, it is important to identify the localization and function of these proteins within the cells.
MDA-MB-231 and SKBR-3—the cell lines with negative sex hormone receptor—had higher Btbd7 expression, than immortalized mammary cells did (MCF-10A) (P<0.01; Figure 3B). Immunofluorescence staining results (Figure 3A) demonstrated that MDA-MB-231 cells showed co-expression of Slug and Btbd7 protein, which was consistent with the results obtained from HRNBC tissues.
Subsequently, we knocked down the expression of Btbd7 using short interfering RNA in MDA-MB-231 cells. As shown in Figure 3C-D using qRT-PCR and western blot analysis, the Btbd7 expression was successfully knocked down. While Btbd7 expression was silenced in MDA-MB-231 cells, the epithelial marker E-cadherin was up-regulated (P<0.05), and the mesenchymal markers α-smooth muscle actin protein (α-SMA) and Slug (P<0.05) were down-regulated, indicating that the EMT process was inhibited (Figure 5D).
To corroborate the function of Btbd7 in MDA-MB-231 cells, the CCK-8 and cell invasion assays were used to analyze cell proliferation and invasion ability. The CCK-8 assay showed that relative cell growth was significantly decreased in BTBD7-siRNA groups at 48 and 72 h compared with those in the control group (Figure 3E). Cell invasive assay showed lower invasive properties in BTBD7-siRNA groups than in the control group (Figure 3F).
BTBD7 is associated with generic transcription of the breast cancer.
To explore the functional roles of BTBD7 in breast cancer, we screened out 332 DEGs (Supplementary Table 2) according to the expression level of BTBD7. The top three up-regulated genes were TRIP11, DICER1, and ATG2B, and the top 3 down-regulated genes were LSM4, PAM16, and SNRPA. DAVID was used to analyze the DEGs and enriched 30 GO terms (Figure 4A, Supplementary Table 3), such as DNA-templated transcription, regulation of transcription, and integral component of the membrane. To enlarge this observation, KOBAS 3.0 was performed, and 25 KEGG terms (Figure 4B Supplementary Table 4), such as signal transduction and generic transcription pathways, were obtained. These results revealed that BTBD7 was associated with generic transcription, which may be the mechanism by which BTBD7 promotes tumor invasion and metastasis.