Fig 2: Reduction of CTNND1 upregulates the CXCR4/CXCL12 axis to facilitate TNBC cells homing to the bone. (A) Expression of bone metastasis-related genes was detected by qRT-PCR in human TNBC cell lines (MDA-MB-231, MBA-MB-468) with and without CTNND1 knockdown. The result was showed as a heatmap. (B) Cytokines secreted by MDA-MB-231 with CTNND1 knockdown were detected by human Cytokine Array Kit. (C) Immunoblotting of CXCR4 in TNBC cells with CTNND1 knockdown. (D) Immunofluorescence of CXCR4 in TNBC cells with CTNND1 knockdown. Scale bar = 50 µm. (E) Images and quantification of chemotaxis of TNBC cells with CTNND1 knockdown, fibroblast (upper panel), osteoblast (middle panel), CXCL12 (lower panel) in bottom chamber, respectively. Scale bar = 200 µm. *** p < 0.005. Error bars indicate Standard Error of Mean (S.E.M.).

Fig 3: Downregulation of CTNND1 enhances EMT in TNBC cells. (A) CTNND1 expression level in human normal breast epithelial cells (MCF-10A) and various breast cancer cells (MDA-MB-231-1833, MDA-MB-231, MDA-MB-468, BT549, Hs578T, MDA-MB-453, HCC1937, MCF-7, T47D, BT474, SKBR3) by qRT-PCR. (B) Immunoblotting of the protein encoded by CTNND1 in human breast normal cell and breast cancer cells as the same as cell lines detected by qRT-PCR. (C) Immunoblotting of verifying knocking down CTNND1 stably in human TNBC cell lines (MDA-MB-231, MDA-MB-468, BT549). (D) Images and quantification of transwell migrated and invaded cells after CTNND1 knockdown. Scale bar represents 200 μm. (E) Immunoblotting of EMT markers in cells with CTNND1 knockdown or not. (F) Immunofluorescence of EMT markers in cells with CTNND1 knockdown or not. Scale bar represents 50 μm. All experiments were repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.005. Error bars indicate Standard Error of Mean (S.E.M.).

Fig 4: Knockdown of CTNND1 upregulates CXCR4 via activation of PI3K/AKT/HIF-1a pathway. (A) KEGG pathway analysis of RNA sequencing between cells with sh-NC and sh-CTNND1 in MDA-MB-231 and MDA-MB-468 cells. (B) Immunoblotting of verifying activation of PI3K/AKT/HIF-1a pathway. (C) Expression of CXCR4 by qRT-PCR (left) and immunoblotting (right) in cells with CTNND1 knockdown or BKM120. (D) Images and quantification of chemotaxis of cells with CTNND1 knockdown or BKM120 in the upper chamber and CXCL12 in the bottom chamber. Scale bar: 200 µm. (E) IHC staining of AKT, p-AKT and CXCR4 in the bone of mice withEO771CTNND1KD. Scale bar: 200 µm, T: tumor, B: bone, M: bone marrow. (F) Chromatin immunoprecipitation (ChIP) assay of the enrichment of HIF-1a at the CXCR4 promoter relative to IgG in MDA-MB-231 cells transduced with sh-CTNND1. (G) The enrichment of HIF-1a at the CXCR4 promoter relative to Input in MDA-MB-231 cells transduced with sh-CTNND1 by CHIP-qPCR. (H) Luciferase reporter assay for the CXCR4 promoter construct transfected in MDA-MB-231 cells. * p < 0.05, ** p < 0.01, *** p < 0.005. Error bars indicate Standard Error of Mean (S.E.M.).

Fig 5: (A) Kaplan–Meier analysis of the correlation between CTNND1 expression and overall survival (OS), distant metastasis-free survival (DMFS) in other types of breast cancer (hormone receptor-positive breast cancer and HER2-positive breast cancer). (B) The infiltration of immune cells in the bone of mice with EO771 cells that had downregulated expressing of CTNND1 (EO771CTNND1KD)). All experiments were repeated at least 3 times. Error bars indicate Standard Error of Mean (S.E.M.).