CXCL10 Chemokine Induces Migration of ING4-deficient Breast Cancer Cells by Signaling Through CXCR3 and Receptor Tyrosine Kinases
CXCL10 is a chemoattractant for immune cells expressing its cognate receptor CXCR3 during inflammation. High expression of CXCL10 is associated with advanced disease and poor patient prognosis in breast cancer, suggesting CXCL10/CXCR3 signaling may contribute to aggressive breast cancer. We previously showed that CXCL10 is repressed by Inhibitor of Growth 4 (ING4). As low ING4 expression was correlated with advanced tumors, we hypothesized that CXCL10 contributes to breast cancer progression in the context of ING4 deficiencies. To test the hypothesis, the ING4 gene was deleted in breast cancer cell lines using a CRISPR/Cas9 system. ING4-deleted cells were tested for an aggressive tumor phenotype, cell migration, in the presence or absence of CXCL10 using Boyden chamber assays. As previous studies have shown that chemokine receptors cross-activate receptor tyrosine kinases (RTKs), we investigated CXCR3/RTK requirement in cell migration assays by using a CXCR3 inhibitor, AMG-487, and two RTK inhibitors, Linsitinib and Erlotinib, that inhibits the insulin growth factor-like receptor (IGFR) in T47D cells and epithelial growth factor receptor (EGFR) in triple negative breast cancer cells, respectively. The results showed that CXCL10 induced migration in ING4-deleted cells by 3-fold compared to the vector control (T47D v2 vs. v2h1, p=0.003). Additionally, CXCL10 induced migration of T47D cells expressing a dominant negative form of ING4, DC-1, by 2.5-fold (T47D pMP vs. DC-1, p=0.002). These results indicated that CXCL10-induced migration was specific to ING4-deficient breast cancer cells. AMG-487 inhibited CXCL10-induced migration of ING4-deleted cells (T47D, p=0.005; MDA-mb-231, p=0.002, MDA-mb-468, p<0.0001), indicating that CXCR3 is required for CXCL10 induced migration of breast cancer cells. CXCL10-induced migration of ING4-deleted cells was inhibited by Linsitinib to basal level (T47D, p=0.003). Taken together, these results demonstrated that CXCL10-induced migration required CXCR3 and IGFR in T47D cells. In TNBC cells, CXCL10-induced migration of ING4-deleted cells was inhibited by Erlotinib (MDA-mb-231, p=0.002; MDA-mb-468, p<0.0001). Taken together, these results demonstrated that CXCL10/CXCR3 signals through RTKs to induce migration in ING4-deficient breast cancer cells. This study suggests the CXCL10/CXCR3/RTK axis as a potential therapeutic target to treat aggressive ING4-deficient breast cancer.