Abstract 4355: Investigating the role of CXCR6 signaling in sorafenib resistance in HCC

Abstract Introduction: Hepatocellular carcinoma (HCC), one of the most common liver cancers and a leading cause of cancer-related deaths globally, is often diagnosed at late stages limited to systemic treatment. Sorafenib, an FDA-approved first in line treatment, efficacy is hindered due to innate or developed resistance in patients. Hyperactivation of CXCR6/CXCL16 signaling has been detected and shown to contribute to a proinflammatory and pro-survival tumor microenvironment driving drug resistance in HCC. Wnt/β-catenin signaling pathway is dysregulated in 50% of HCC and has shown to be involved in sorafenib resistance due to accumulation of its core component, β-catenin. Our hypothesis posits that CXCR6 antagonism will restore potency of sorafenib in HCC cell lines via inhibition of β-catenin-mediated signaling. Experimental Procedure: Basal sorafenib sensitivity assessed via MTT assay, and β-catenin and CXCR6 protein expression via western blot were compared across five HCC cell lines. An orally bioavailable small molecule antagonist, SBI-457, was used to probe CXCR6 signaling. We evaluated sorafenib-, SBI-457- and combination-induced modulation of β-catenin expression via western blot and IF imaging in SKHep1. CXCL16- and SBI-457-induced modulation of pCREB protein expression post forskolin activation in both SKHep1 and CXCR6-overexpressed CHOK1 cells were compared. Secretion of soluble CXCL16 was measured via ELISA in SKHep1 cells. Results: Sorafenib dose-dependently increased β-catenin protein expression via immunofluorescence imaging and whole cell lysates of SKHep1 cells. SBI-457 in combination showed trends towards attenuating sorafenib-induced increases, yet lacked significance. We demonstrate that high CXCR6 protein expression levels match sorafenib growth inhibitory (GI50) values across all five HCC cells tested. 200ng/ml CXCL16 significantly attenuated forskolin-induced increase in pCREB expression and SBI-457 dose-dependently restored in a CXCR6 overexpressed CHOK1 cell line. However, both CXCL16 and SBI-457 were unable to clearly modulate forskolin-induced pCREB in CXCR6 endogenously expressed HCC cell lines. SKHep1 cells significantly cleave increasing levels of soluble CXCL16 as measured up to 24h, of which was blocked by ADAM 10 and 17 inhibition. Conclusion: Sorafenib treatment increased total β-catenin levels in SKHep1 cells. Though we show clear CXCR6-mediated signaling in an overexpressed cell line, it is inconsistent in HCC cells, despite CXCR6 expression. SKHep1 cells secrete significantly increasing CXCL16 and this was attenuated by combined inhibition of ADAM10and17. This data suggests SKHep1 cleave soluble CXCL16 from its transmembrane form and potentially alter traditional forward signaling. Future studies aim to evaluate tmCXCL16 expression levels across HCC cells and evaluate the role this plays in CXCR6-mediated signaling via our small molecule inhibitor to further understand the therapeutic potential of CXCR6 inhibition in HCC. Citation Format: Morgan Reeves, Satyamaheshwar Peddibhotla, Siobhan Malany. Investigating the role of CXCR6 signaling in sorafenib resistance in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4355.