FAK blockade enhances antitumor effect of BTK inhibitor in esophageal squamous cell carcinoma via EGFR-ERK-Akt pathway inhibition

Background: Esophageal squamous cell carcinoma (ESCC) is a fatal disease with poor prognosis and lack of effective targeted therapy. Recently, some researches have shown that BTK can be a novel therapeutic target for esophageal cancer, while the mechanism remains unknown. In addition, FAK is one of the important factors to promote the metastasis of esophageal cancer. In this study, we sought to explore the synergistic antitumor effect and underline mechanism of combining BTK inhibitor Ibrutinib with a novel FAK inhibitor APG-2449 for esophageal squamous cell carcinoma. Methods: Cell viability and growth inhibition were determined by CCK-8 assay and colony formation assay. Migration ability was detected by Transwell assay. EMT markers and ki67 were measured by RT-qPCR. Cell cycle analysis and apoptosis were confirmed by flow cytometry. Western Blot analysis and siRNA interference for mechanism exploration. ESCC xenograft models were established to evaluated the synergistic antitumor effect in vivo. Results: We found that ESCC cell lines harbor EGFR or c-myc-amplified exhibited more sensitive to Ibrutinib inhibition. Moreover, Ibrutinib combined with APG-2449 showed synergistic inhibition of cell proliferation in ESCC cell lines. By analyzing data from the Gene Expression Profiling Interactive Analysis database, we found that EGFR expression positively correlated with FAK expression in ESCC samples. The combination of Ibrutinib and APG-2449 exerted obviously synergistic apoptosis induction and colony formation inhibition. Furthermore, we found that Ibrutinib combined with APG-2449 could induce more cancer cells arrested in G1/S phase. In addition, the co-treatment of Ibrutinib and APG-2449 could significantly suppress ESCC cells migration by upregulating E-cadherin. Ibrutinib alone could down-regulate the protein expression of p-EGFR and p-ERK. Moreover, combination treatment significantly reduced the expression of p-Akt and p-c-myc, and upregulated P21 and PUMA expression. In ESCC xenotransplantation models, single therapy with either Ibrutinib or APG-2449 was equivalent in delaying tumor growth, while the combination therapy demonstrated more significantly tumor growth suppression. Conclusions: Taken together, our data strongly suggest that the combination of FAK inhibitor APG-2449 and Ibrutinib provide an effective and precise therapeutic strategy for treating esophageal squamous cell carcinoma. Citation Format: Qiu-Yun Luo, Su-Na Zhou, Wen-Tao Pan, Meng-xian Pan, Lu-ping Yuan, Lin Zhang, Xiang-Lei Yan, Yu-xin Zhang, Jian Sun, Miaozhen Qiu, Da-Jun Yang. FAK blockade enhances antitumor effect of BTK inhibitor in esophageal squamous cell carcinoma via EGFR-ERK-Akt pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 309.