Assessment of CXC ligand 12-mediated calcium signalling and its regulators in basal-like breast cancer cells

Abstract

CXC ligand (L)12 is a chemokine implicated in the migration, invasion and metastasis of cancer cells via interaction with its receptors CXC chemokine receptor (CXCR)4 and CXCR7. In the present study, CXCL12-mediated Ca²⁺ signalling was compared with two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which demonstrate distinct metastatic potential. CXCL12 treatment induced Ca²⁺ responses in the more metastatic MDA-MB-231 cells but not in the less metastatic MDA-MB-468 cells. Assessment of mRNA levels of CXCL12 receptors and their potential modulators in both cell lines revealed that CXCR4 and CXCR7 levels were increased in MDA-MB-231 cells compared with MDA-MB-468 cells. Cluster of differentiation (CD)24, the negative regulator of CXCL12 responses, demonstrated increased expression in MDA-MB-468 cells compared with MDA-MB-231 cells, and the two cell lines expressed comparable levels of hypoxia-inducible factor (HIF)2α, a CXCR4 regulator. Induction of epithelial-mesenchymal transition (EMT) by epidermal growth factor exhibited opposite effects on CXCR4 mRNA levels compared with hypoxia-induced EMT. Neither EMT inducer exhibited an effect on CXCR7 expression, however hypoxia increased HIF2α expression levels in MDA-MB-468 cells. Analysis of the gene expression profiles of breast tumours revealed that the highest expression levels of CXCR4 and CXCR7 were in the Claudin-Low molecular subtype, which is markedly associated with EMT features.

Keywords: CXC chemokine receptor type 4; CXC chemokine receptor type 7; breast cancer; calcium signalling; cluster of differentiation 24; hypoxia-inducible factor 2α.

Figure 1.

Effect of CXCL12 on [Ca²⁺]_CYT in MDA-MB-231 and MDA-MB-468 breast cancer cells. (A) Representative [Ca²⁺]_CYT transients following treatment with 100 and 300 ng/ml CXCL12 in MDA-MB-231 cells. (B) Each bar represents the response (peak relative [Ca²⁺]_CYT) to either 100 ng/ml CXCL12 or 300 ng/ml CXCL12 in MDA-MB-231 cells (n=3; mean ± standard deviation). (C) [Ca²⁺]_CYT levels following treatment with 100 and 300 ng/ml CXCL12 in MDA-MB-468 cells. Stimulation with 100 µM ATP was used as a positive control for this assay (representative of three independent experiments). (D) Presents only the CXCL12 response using the same maximum Y-axis scale that was used for MDA-MB-231 cells. Statistical analysis was performed using one-way analysis of variance with Tukey's multiple comparison post-hoc test. **P<0.01. CXCL, CXC ligand; CYT, cytoplasmic; [Ca²⁺], intracellular calcium concentration.

Figure 2.

CXCR4, CXCR7, CD24 and HIF2α mRNA levels in MDA-MB-231 and MDA-MB-468 breast cancer cells normalised to 18S ribosomal RNA (−ΔC_q). Results were taken from three independent experiments and the horizontal line represents the mean (n=3). Statistical analysis was performed using an unpaired t-test. *P<0.05. NS, not significant; CXCR, CXC chemokine receptor; CD, cluster of differentiation; HIF, hypoxia-inducible factor.

Figure 3.

mRNA levels of CXCR4, CXCR7, CD24 and HIF2α mRNA following hypoxia- and EGF-induced EMT in MDA-MB-468 breast cancer cells expressed as fold change. (A) CXCR4, (B) CXCR7, (C) CD24, and (D) HIF2α mRNA levels in MDA-MB-468 cells incubated under normoxic or hypoxic (24 h) conditions or stimulated with EGF (24 h) to induce EMT. Results are presented as the mean ± standard deviation (n=3). Statistical analysis was performed using an unpaired t-test; *P<0.05, **P<0.01, ***P<0.001. NS, not significant; CXCR, CXC chemokine receptor; CD, cluster of differentiation; HIF, hypoxia-inducible factor; EMT, epithelial-mesenchymal transition; EGF, epidermal growth factor.

Figure 4.

The expression of CXCR4 and CXCR7 is enriched in the C-Low molecular subtype of breast tumours compared with the basal molecular subtype. Log2 expression values of (A) CXCR4 and (B) CXCR7 in 6 subtypes of breast tumours, including Basal, C-Low, HER2, LumA, LumB and N-Like. Data were sourced from the METABRIC breast cancer data (29) and analysed using www.cbioportal.org (27,28). Statistical analysis was performed using one-way analysis of variance followed by Tukey's multiple comparisons post-hoc test. *P<0.05, ****P<0.0001. NS, not significant; Basal, Basal-Like; C-Low, Claudin-Low; HER2, human epidermal growth factor receptor 2-enriched; LumA, Luminal A; LumB, Luminal B; N-Like, Normal-like; CXCR, CXC chemokine receptor.