The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou 510632, China; MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
J Adv Res. 2023 Sep;51:135-147. doi: 10.1016/j.jare.2022.11.006. Epub 2022 Nov 14.
Acquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation.
We identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study.
DIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co‑immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays.
We found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently.
This study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib.
在转移性结直肠癌（CRC）中，BRAF 抑制剂 vemurafenib 的获得性耐药经常发生，这导致治疗失败，是一个棘手的问题。作为 vemurafenib 治疗的适应性反应，一系列细胞旁路反应是 ERK 信号适应性反馈再激活的原因，这需要进一步研究。
我们鉴定出 ARF1（ADP-核糖基化因子 1）是一种新型的 vemurafenib 耐药和癌症转移的调节剂，本研究对其分子机制和潜在抑制剂进行了研究。
基于 DIA 的定量蛋白质组学和 RNA-seq 分别对vemurafenib 耐药的 RKO 细胞（RKO-VR）和高侵袭性 RKO 细胞（RKO-I8）进行了系统分析。通过共免疫沉淀实验检测 ARF1 和 IQGAP1（IQ 结构域 GTP 酶激活蛋白 1）的相互作用。建立基于 ELISA 的 FDA 批准药物库药物筛选系统，筛选针对 ARF1-IQGAP1 相互作用的化合物。通过 Transwell、Western blot、Annexin V-FITC/PI 染色和体内实验转移实验确定 ARF1 和 LY2835219 的生物学功能。
我们发现 ARF1 与 IQGAP1 强烈相互作用，在 VR 和 I8 CRC 细胞中激活 ERK 信号。缺失 IQGAP1 或 ARF1 失活（ARF-T48S）恢复了 ARF1 诱导的侵袭能力。由于 ARF1-IQGAP1 相互作用对于 ERK 激活是必不可少的，我们筛选出 LY2835219 作为 ARF1-IQGAP1 相互作用的新型抑制剂，该抑制剂在体外和体内均能抑制 ERK 信号转导，并抑制 CRC 转移和 vemurafenib 耐药，且无明显副作用。此外，LY2835219 与 vemurafenib 联合治疗对 ARF1 介导的癌症转移的抑制作用明显优于单独使用。
本研究揭示了 ARF1-IQGAP1 相互作用介导的 ERK 信号再激活对于 vemurafenib 耐药和癌症转移是至关重要的，并且 LY2835219 作为单一药物和与 vemurafenib 联合使用，都是 CRC 的一种很有前途的治疗药物。
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