Abstract 1522: Cxcr3 chemokine receptor crosstalk with EGFR recurs independently of the Cxcl10 chemokine to signal for cell migration in the absence of the tumor suppressor ING4
Authors: Tsutsumi Emily , Kim Suwon 
Published: June 2022

Abstract
Cxcr3 is a G-protein coupled receptor expressed on various cell types including cancer cells. Cxcr3 is activated via binding of the chemokine Cxcl10, which mediates immune cell migration during normal immune response. Pharmacological inhibition of Cxcr3 was shown to mitigate metastasis in a murine breast cancer model, suggesting it is critical for cancer metastasis, but the mechanism is not well understood. We have previously shown that exogenous Cxcl10 induced migration of ING4-deleted breast cancer cells, but not of ING4-intact cells. Cxcl10 signaling required the growth factor receptor Egfr, suggesting a crosstalk between Cxcr3 and Egfr. Confocal immunofluorescent (IF) colocalization assays showed that Cxcr3 and Egfr rapidly and transiently colocalized upon Cxcl10 treatment, which recurred only in ING4-deleted cells, suggesting that the recurrent receptor association may drive Cxcl10-induced cell migration. Here we characterized the temporal dynamics of the Cxcl10-induced Cxcr3/EGFR receptor associations and migration using IF and transwell migration assays, respectively. We also used inhibitors of Cxcr3 (AMG-487), Egfr (erlotinib and SAH-EJ1), and Gβγ (gallein) in order to delineate the signaling mechanism. The IF results demonstrated that, the first Cxcr3/EGFR association occurred rapidly within 10 minutes of Cxcl10 treatment, which disappeared by 1 hour post Cxcl10 treatment. The Cxcr3/Egfr complex formation recurred again transiently at 8 hours post Cxcl10 treatment. This transient receptor complex formation repeatedly occurred at 8-hour intervals up to 24 hours, suggesting a cyclic complex formation. 1 hour treatment of cells with Cxcl10 induced the cyclic formation of the Cxcr3/Egfr complexes, indicating that continuous presence of Cxcl10 was not required for recurrent Cxcr3/EGFR association. Likewise, 1 hour Cxcl10 treatment of cells induced cell migration, indicating that the initial binding of Cxcl10 to Cxcr3 was the critical event in the subsequent recurrent receptor colocalization and cell migration. Inhibition of the early Cxcr3/Egfr complex formation by AMG-487 or SAH-EJ1 blocked subsequent receptor association and cell migration, corroborating the idea that the initial receptor complex formation is the critical event. Erlotinib did not block the receptor colocalization, but inhibited cell migration when added for the initial 1 hour time duration of Cxcl10 treatment, but not when added after 4 hours. These results indicated that the early Cxcr3/Egfr complex signaled via the Egfr kinase. In contrast, gallein inhibited cell migration irrespective of when it was added, indicating that Gβγ was a required signaling component downstream of Cxcr3/EGFR. These findings present the Cxcl10/Cxcr3/Egfr/Gβγ signaling axis as the potential therapeutic targets for ING4-deficient aggressive breast cancer.
Citation Format: Emily Tsutsumi, Suwon Kim. Cxcr3 chemokine receptor crosstalk with EGFR recurs independently of the Cxcl10 chemokine to signal for cell migration in the absence of the tumor suppressor ING4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1522.