Downregulation of annexin A3 inhibits tumor metastasis and decreases drug resistance in breast cancer

Abstract

Annexin A3 (ANXA3) is dysregulated and plays an important role in various cancers. However, the role of ANXA3 in breast cancer is still unclear. Here, we observed that the expression level of ANXA3 was significantly upregulated in breast cancer tissues. ANXA3 knockdown inhibited cell invasion but promoted cell proliferation in both in vitro and in vivo assays. Furthermore, we found that ANXA3 knockdown inhibited the NFκB pathway via upregulating IκBα, resulting in mesenchymal-epithelial transition (MET) and a heterogeneity change of breast cancer stem cells (BCSCs). In addition, we demonstrated that ANXA3 knockdown increased the sensitivity of breast cancer cells to doxorubicin by increasing the drug uptake. The combination of ANXA3 knockdown and doxorubicin treatment simultaneously inhibited tumor growth and metastasis in vivo. This study described the role and mechanisms of ANXA3 in regulating BCSCs and breast cancer growth and metastasis, indicating that downregulating ANXA3 together with chemotherapy might be a novel therapeutic strategy for treating breast cancer.

Fig. 1. ANXA3 is upregulated in breast cancer tissues and correlated with poor overall survival

a The mRNA expression level of ANXA3 was examined in clinical breast tumor tissues and paratumor tissues by qRT-PCR. The two connected dots represent the ANXA3 levels in the tumor and paratumor from the same patient (n = 16). **p < 0.01. b The ANXA3 protein level in clinical breast tumor tissues and paratumor tissues are shown by immunohistochemistry (Brown: ANXA3). c Kaplan–Meier survival curves of breast cancer patients (log-rank test, ***p < 0.001), data from the Oncomine database (n = 471). d The ANXA3 protein level was measured in several breast cancer cell lines by western blotting

Fig. 2. ANXA3 knockdown significantly inhibits in vitro cell invasion but promotes cell proliferation, as well as induces an epithelial phenotype

a Human ANXA3 or mouse Anxa3 was knocked down in human breast cancer cell lines MDA-MB-231 and MDA-MB-468 or mouse mammary tumor cell line 4T1 via infection with shCTRL, shANXA3, or shAnxa3 lentivirus. ANXA3 or Anxa3 expression was detected by western blotting. b Cell invasion ability was measured by the Matrigel invasion as described in the methods. c Quantitative analysis of the total invaded cells in b. **p < 0.01; ***p < 0.001. d Cell proliferation activity was measured using an MTT assay as described in the methods. **p < 0.01; ***p < 0.001. e The protein expression levels of E-cadherin, γ-catenin, Vimentin, and N-cadherin were detected by western blotting

Fig. 3. ANXA3 knockdown inhibits in vivo tumor metastasis but promotes tumor growth

a For each group, 50,000 MDA-MB-231 cells were implanted into the mammary glands of 6- to 8-week-old female nude mice and tumor size was monitored weekly. **p < 0.01; ***p < 0.001. b The tumor images (left) and tumor weight (right) from a are shown. The tumor growth from a was halted when the largest tumor reached 15 mm in diameter for humane reasons. **p < 0.01; ***p < 0.001. c IHC staining of ANXA3 and Ki67 in the tumors from b. d Quantification of Ki67-positive cells (%) as shown in c. **p < 0.01; ***p < 0.001. e HE staining in slices of lungs harvested from mice in a. Arrow shows the metastatic nodules. f Quantitative analysis of the metastatic nodules in e. *p < 0.05

Fig. 4. ANXA3 differentially regulates two states of breast cancer stem cells

a The mRNA levels of ANXA3 were examined in FACS-sorted BCSCs from the MDA-MB-231 cell line by RT-PCR. *p < 0.05. b Flow cytometry analyses of BCSCs by the ALDEFLUOR assay (upper) and the CD24/CD44 assay (lower) in shCTRL- and shANXA3-infected MDA-MB-231 cells. c Quantification of ALDEFLUOR-positive or CD24⁻/CD44⁺ cells in shCTRL- and shANXA3-infected MDA-MB-231 and MDA-MB-468 cells. *p < 0.05; **p < 0.01; ***p < 0.001. d Quantification of ALDEFLUOR-positive or CD24⁻/CD44⁺ cells in digested tumor cells from Fig. 3b. *p < 0.05; **p < 0.01

Fig. 5. ANXA3-knockdown induces MET and affects breast cancer cell invasion and proliferation via inhibiting the NFκB pathway

a Heatmap of gene expression in shCTRL- and shANXA3-infected MDA-MB-231 cells; each group has been repeated twice. b Pathway enrichment analysis of transcriptome profiling results from a. c Diagram showing the fold-changes of both the transcriptome sequencing and RT-PCR experiments of several typical genes to confirm and validate the sequencing data. d The protein levels of NFKBIA (IκBα) and phospho-p65 (p-p65) were detected by western blotting. e The protein levels of ANXA3, IκBα, phospho-p65, E-cadherin, and Vimentin were detected by western blotting. f Cell invasion ability was measured by the Matrigel invasion assay, as described in the methods. g Quantitative analysis of the total number of invaded cells in f. **p < 0.01. h Cell proliferation activity was measured by an MTT assay as described in the methods. *p < 0.05; ***p < 0.001. i Flow cytometry analyses of BCSCs by the ALDEFLUOR assay (upper) and the CD24/CD44 assay (lower) in shCTRL-, shANXA3-, shIκBα-, and shANXA3 + shIκBα-infected MDA-MB-231 cells. *p < 0.05; ***p < 0.001

Fig. 6. ANXA3 knockdown enhances the efficacy of doxorubicin on tumor growth and metastasis in vivo by increasing the uptake of doxorubicin in breast cancer cells

a Flow cytometry analysis of MDA-MB-231 and 4T1 cells after incubation with doxorubicin for 4 h. The dose of doxorubicin was 5 μg/mL. b 4T1 cells (20,000) were implanted into the fourth mammary glands of 6- to 8- week-old female BALB/c mice, and the mice were divided into two groups for treatment: saline (control) and doxorubicin (Dox), and tumor size was monitored weekly. *p < 0.05; **p < 0.01; ***p < 0.001. c Mice were sacrificed at the end of treatment when the largest tumor reached to 15 mm in diameter for humane reasons, and the tumor image is shown. d HE staining in slices of lungs harvested from mice in b. Arrow show metastatic nodules. e Quantitative analysis of the metastatic nodules in d. *p < 0.05