The tumor suppressor DACT3 sensitizes triple-negative breast cancer to apatinib by inhibiting the Wnt/β-catenin pathway

Abstract

Apatinib, a small-molecule inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2), shows efficient antitumor activity in heavily pretreated metastatic triple-negative breast cancer (TNBC). However, not all patients respond to apatinib, indicating that it is necessary to identify response biomarkers for more precise treatment and investigate the underlying mechanisms of apatinib resistance to develop new treatment strategies for TNBC. In this study, we identified the disheveled binding antagonist of beta-catenin 3 (DACT3) as a biomarker for apatinib sensitivity, as its expression level is significantly higher in apatinib-sensitive patients and positively correlates with longer survival. Furthermore, we explored that the exogenous expression of DACT3 could downregulate the IC₅₀ of apatinib (Vector vs DACT3: 16.04 μM vs 8.81 μM in MDA-MB231 cells, 19.65 μM vs 9.42 μM in YCCB1 cells) by inhibiting the Wnt/β-catenin signaling, a pro-malignancy pathway that leads to apatinib resistance through crosstalk with the VEGF/VEGFR2 pathway. In summary, our results indicate that DACT3 is a potential biomarker for predicting the response to apatinib and a new therapeutic target for improving TNBC sensitivity to apatinib.

Keywords: Apatinib; Breast cancer; DACT3; Drug resistance; Wnt/β-Catenin.

Fig. 1

Expression of DACT3 was lower in BrCa tissues and associated with clinical prognosis and apatinib sensitivity. (A, B) Online analysis of DACT3-expression in BC tissues compared with non-carcinoma counterparts. (C, D) Correlation between the expression of DACT3 and prognostic outcome in BC subclasses. (E) RT-PCR results of DACT3 in BC and normal breast cells. (F) QRT-PCR results of DACT3 in BC tissues relative to non-carcinoma tissues. (G) IHC staining of DACT3 in apatinib-sensitive (left) and apatinib-resistant tumor tissues (right). (H) Average IOD of DACT3 protein level of apatinib-sensitive and apatinib-resistant group. (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 2

Overexpression of DACT3 inhibited the malignant phenotype of breast cancer in vitro. (A, B, C) The expression of DACT3 in MDA-MB231 and YCCB1 cells was detected by qRT-PCR and western blot. (D) The cell viability of DACT3-overexpressing cell lines was measured at 24, 48 and 72 h. (E) The interaction between DACT3 and apatinib was detected by CCK8 assay. (F) The IC50 of apatinib in DACT3-overexpressing group and control group. (G) Colony-forming capacity of Dact3-transfected MDA-MB231 and YCCB1 cells. (H, I) Transwell assay demonstrated the migration and invasion capacity of MDA-MB231 and YCCB1 transfected with DACT3. (J) The motility of transfected-DACT3 MDA-MB231 and YCCB1 cells was detected by wound healing assay. (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3

DACT3 induced cell apoptosis and arrest of cell cycle at G0-G1 phase. DACT3 inhibited BC cell proliferation in vivo and suppressed the stemness of TNBC cells. (A) Apoptosis was detected by flow cytometry. (B, C) The cell cycle was measured by flow cytometry. (D) Tumor images transfected with DACT3 and vector MDA-MB231 cells by subcutaneous injection. (E) Tumor growth curve of DACT3 and vector-transfected MDA-MB231 cells in mice. (F) Weight histograms of two groups of tumors formed by MDA-MB231 cells transfected with DACT3 and vector. (G) The relationship between DACT3 and tumor stem-related markers was analyzed by qRT-PCR. (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4

DACT3 and apatinib both inhibited β-catenin signalling pathway. (A, B, C, D) Immunofluorescence results of interaction between DACT3 and active catenin, total catenin, VEGFR2, p-VEGFR2. (E, F) Expression of Wnt/β-catenin-related proteins in cells transfected with DACT3 and treated with Apatinib.

Fig. 5

Diagram of mechanism of DACT3 synergistically inhibiting Wnt/β-Catenin pathway.