Effects of SDF-1-CXCR4 signaling on microRNA expression and tumorigenesis in estrogen receptor-alpha (ER-α)-positive breast cancer cells

Abstract

The majority of breast cancer cases ultimately become unresponsive to endocrine therapies, and this progression of breast cancer from hormone-responsive to hormone-independent represents an area in need of further research. Additionally, hormone-independent carcinomas are characterized as being more aggressive and metastatic, key features of more advanced disease. Having previously shown the ability of the stromal-cell derived factor-1 (SDF-1)-CXCR4 signaling axis to promote primary tumorigenesis and hormone independence by overexpressing CXCR4 in MCF-7 cells, in this study we further examined the role of SDF-1/CXCR4 in the endogenously CXCR4-positive, estrogen receptor α (ER-α)-positive breast carcinoma cell line, MDA-MB-361. In addition to regulating estrogen-induced and hormone-independent tumor growth, CXCR4 signaling stimulated the epithelial-to-mesenchymal transition, evidenced by decreased CDH1 expression following SDF-1 treatment. Furthermore, inhibition of CXCR4 with the small molecule inhibitor AMD3100 induced CDH1 gene expression and inhibited CDH2 gene expression in MDA-MB-361 cells. Further, exogenous SDF-1 treatment induced ER-α-phosphorylation in both MDA-MB-361 and MCF-7-CXCR4 cells, demonstrating ligand-independent activation of ER-α through CXCR4 crosstalk. qPCR microRNA array analyses of the MDA-MB-361 and MCF-7-CXCR4 cell lines revealed changes in microRNA expression profiles induced by SDF-1, consistent with a more advanced disease phenotype and further supporting our hypothesis that the SDF-1/CXCR4 signaling axis drives ER-α-positive breast cancer cells to a hormone independent and more aggressive phenotype. In this first demonstration of SDF-1-CXCR4-induced microRNAs in breast cancer, we suggest that this signaling axis may promote tumorigenesis via microRNA regulation. These findings represent future potential therapeutic targets for the treatment of hormone-independent and endocrine-resistant breast cancer.

Figure 1. CXCR4 expression drives estrogen-stimulated MDA-MB-361 tumorigenesis

(A) MDA-MB-361 cells were harvested for RNA isolation and basal expression of CXCR4 examined by qPCR compared to MCF-7 and MDA-MB-231 cells. Bars represent mean fold expression ± SEM of triplicate experiments. (B–C) Female, 4–6 week old, ovariectomized Nu/Nu mice (n=5/group) were injected (MFP) with 5×10⁶ MDA-MB-361 cells in 50ul of Matrigel. Animals were implanted with 17β-estradiol (0.72 mg, 60 day time release) pellets subcutaneously in the dorsal neck. (B) Matrigel injections contained isotype IgG or anti-CXCR4 (75 ng/injection) antibodies. (C) Following tumor formation (11 days post cell injection), animals were treated with twice daily i.p. injections of vehicle or AMD3100 (5 mg/kg/animal). Tumors were measured by digital caliper. Data represented as average tumor volume (mm³) ± SEM. *, p<0.05, **, p<0.01.

Figure 2. CXCR4 signaling drives MDA-MB-361 hormone-independent tumorigenesis and stimulates EMT

(A) 4–6 week old Nu/Nu ovariectomized female mice were injected (MFP) with 5×10⁶ MDA-MB-361 cells mixed with matrigel (reduced factor). After tumor formation (day 26 post cell injection), animals were randomized into treatment groups and received once daily i.p. treatment with the CXCR4 inhibitor AMD3100 (5mg/kg/animal) or vehicle control. Tumors were measured by digital caliper. Data represented as average tumor volume (mm³) ± SEM. (B) qPCR analysis of EMT-related genes, CDH1 and CDH2, in MDA-MB-361 cells treated with AMD3100 (5 ug/ml) for 48 hours. Bars represent mean fold change ± SEM of triplicate samples, vehicle normalized to 1. (C) CDH1 protein levels as detected by CDH1 ELISA of MDA-MB-361 cells treated with SDF-1 (100 ng/ml) for 72 hours. Bars represent mean percent positive ± SEM of triplicate samples, vehicle normalized to 100%. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 3. SDF-1 activates ER-α via phosphorylation and alters miR expression consistent with more advanced phenotype

(A–B) Western blot analysis for ER-α phosphorylation at serine 118 and serine 167 in a time course treatment of (A) MDA-MB-361 or (B) MCF-7-CXCR4 cells with SDF-1 (100ng/ml) following 72 hours of serum starvation. Rho GDI or α-tubulin were used as loading controls. Each gel is representative of three independent blots. (C–D) Representative clustergrams of miRNA expression changes as determined by qPCR arrays in (C) MDA-MB-361 or (D) MCF-7-CXCR4 cells following 24 hours of treatment with SDF-1 (100 ng/ml). Red indicates increased expression, while green denotes decreased expression.