FAK Inhibition Reduces Tumor Infiltration of Tregs via Restricting CCL5 Release and Boosts the Combination Therapy for BRAF V600E Mutant Colorectal Carcinoma

Abstract

Background: BRAFV600E mutant colorectal carcinoma (CRC) is a refractory disease with poor prognosis and has little response to regular chemotherapy and targeted therapy. Recruitment of Tregs in tumor tissue promoted by BRAFV600E is responsible for the weakened antitumor immunity. FAK, a therapeutic target, has an impact on the release of Treg chemokines. Methods: Transwell assay, flow cytometry, western blot, Elisa and RT-qPCR analysis were conducted for mechanism exploration. Fluorescent multiplex immunohistochemistry analysis was performed to confirm the connection of BRAFV600E, FAK and Tregs. CRC xenograft models were established to detect the Treg recruitment and evaluate the synergistic antitumor effect in vivo. Results: CRC cells harboring BRAFV600E mutation was of higher expression of FAK, and hence more sensitive to the tumor inhibitory effect of FAK blockade. FAK inhibitor APG-2449 inhibited the proliferation via inducing G1 phase arrest and suppressed the migration of CRC cells, and APG-2449 reduced the tumor infiltrated Tregs through FAK-IL33-CCL5 axis. Overexpressed FAK in BRAFV600E mutant CRC led to larger amount of tumor infiltrated Tregs. BRAFV600E mutant CRC can benefit from FAK inhibition through downregulating Tregs in tumor site. Moreover, a synergistic antitumor effect of APG-2449 with Vemurafenib and/or Cetuximab in BRAFV600E mutant CRC was observed. Conclusions: BRAFV600E mutation in CRC is associated with higher expression level of FAK, and more Tregs infiltrated in tumors. FAK inhibitor APG-2449 has an impact on antitumor immunity of CRC by decreasing infiltrated Treg numbers, and boosts the targeted combination therapy of BRAF inhibitor and/or EGFR inhibitor for BRAFV600E mutant CRC. Funding Statement: This work was supported by National Natural Science Foundation of China (Grant number. 81602066 and 81772587); Guangdong Esophageal Cancer Institute Science and Technology Program (grant number M201809); CSCO-HengRui Oncology Research Fund (grant number Y-HR2018-184); the third outstanding young talents training plan and Medical Scientist program of Sun Yat-sen University cancer center; Science and Technology Planning Project of Guangzhou, China (Grant number. 201510161726583); National Science and Technology Major Project, China (Grant number. 2016ZX09101004); Natural Science Foundation of Guangdong Province, China (grant number 2018A030310260) and Medical Scientific Research Foundation of Guangdong Province, China (grant no. 2018102516469945). Declaration of Interests: The authors stated: No potential conflicts of interest were disclosed. Ethics Approval Statement: The animal experiments were approved by Animal Use and Care Committee of Sun Yat-sen University.