Cxcl10 is a chemokine that attracts immune cells by binding to the Cxcr3 receptor during a normal immune response. Cxcl10 or Cxcr3 expression has been correlated with aggressive breast cancer. Inhibitor of Growth 4 (ING4) has previously shown to repress CXCL10 expression in breast cancer cells. We investigated the effects of Cxcl10/Cxcr3 in ING4-deficient breast cancer. First, we correlated expression levels of ING4, CXCL10, and CXCR3 with patient survival outcomes using the GDS806 data set. To determine the biological effects Cxcl10/Cxcr3 signaling in ING4-deficient cells, we established ING4 knockout cell lines using CRISPR/Cas9 system in MDAmb231 and MDAmb468 breast cancer cells. We compared proliferation and migration of ING4 wild-type vs. ING4-deleted cells with or without exogenous Cxcl10 using SRB growth assays and Boyden chamber transwell assays, respectively. We used a Cxcr3 inhibitor, AMG-487, or an Egfr inhibitor, erlotinib, to determine a signaling requirement(s) for Cxcl10-induced effects. Immunofluorescence (IF) confocal microscopy was used to evaluate Cxcr3/Egfr interactions. Analysis of the GDS806 gene expression data set showed that patients with tumors containing CXCL10-high/ING4-low had significantly reduced disease-free survival, compared to low CXCL10-low/ING4-high tumors (H.R.= 5.20, 95% CI 2.06-13.09, p = 0.003), suggesting that Cxcl10 may contributed to aggressive progression of ING4 deficient tumors. ING4 deletion with or without Cxcl10 did not affect cell growth. Cxcl10 induced migration of ING4-deleted cells, but not of ING4 wild type cells, suggesting that Cxcl10-induced migration may be responsible for aggressive behavior of ING4-deficient tumors. AMG-487 or erlotinib inhibited Cxcl10 induced cell migration of ING4-deleted cells, indicating that both Cxcr3 and Egfr were required for Cxcl10-induced cell migration. IF results showed that Cxcr3 and Egfr were co-localized in the presence of Cxcl10 at the 10-minute time point in both ING4-wild type and ING4-deleted cells, whereas Cxcr3/Egfr colocalization was present at the 24-hour time point only in ING4-deleted cells. These results demonstrated that Cxcl10-mediated Cxcr3/Egfr co-localization recurred only in the absence of ING4. Cxcr3/Egfr colocalization was inhibited by AMG-487, but not by erlotinib. Taken together, these results indicated that Cxcl10 binding to Cxcr3 induced recurrent colocalization of Egfr only in ING4-deleted cells, which in turn signals downstream components to facilitate cell migration. These results further suggest Cxcl10/Cxcr3/Egfr axis as a novel therapeutic target for ING4-deficient aggressive breast cancer.
Citation Format: Emily Tsutsumi, Jeremiah Stricklin, Emily Arnold, Suwon Kim. ING4 deficiency results in recurrent crosstalk between Cxcl10/Cxcr3 and Egfr in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1978.