Background: Small cell lung cancer (SCLC) with high c-Myc expression is prone to relapse and metastasis, leading to extremely low survival rate. Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor Abemaciclib plays a key role in the treatment of tumors, but the effects and mechanisms on SCLC remain unclear. This study was to analyze the effect and molecular mechanism of Abemaciclib in inhibiting proliferation, migration and invasion of SCLC with high c-Myc expression, with a view to expanding a new direction for reducing the recurrence and metastasis.
Methods: Proteins interacting with CDK4/6 were predicted using the STRING database. The expressions of CDK4/6 and c-Myc in 31 cases of SCLC cancer tissues and paired adjacent normal tissues were analyzed by immunohistochemistry. The effects of Abemaciclib on the proliferation, invasion and migration of SCLC were detected by CCK-8, colony formation assay, Transwell and migration assay. Western blot was used to detect the expressions of CDK4/6 and related transcription factors. Flow cytometry was used to analyze the effects of Abemaciclib on the cell cycle and checkpoint of SCLC.
Results: The expression of CDK4/6 was associated with c-Myc by STRING protein interaction network. c-Myc can directly modalize achaete-scute complex homolog 1 (ASCL1), neuronal differentiation 1 (NEUROD1) and Yes-associated protein 1 (YAP1). Moreover, CDK4 and c-Myc regulate the expression of programmed cell death ligand 1 (PD-L1). Immunohistochemistry showed that the expressions of CDK4/6 and c-Myc in cancer tissues were higher than those in adjacent tissues(P<0.0001). CCK-8, colony formation assay, Transwell and migration assay verified that Abemaciclib could effectively inhibit the proliferation, invasion and migration of SBC-2 and H446OE(P<0.0001). Western blot analysis further showed that Abemaciclib not only inhibited CDK4 (P<0.05) and CDK6 (P<0.05), but also affected c-Myc (P<0.05), ASCL1 (P<0.05), NEUROD1 (P<0.05) and YAP1 (P<0.05), which are related to SCLC invasion and metastasis. Flow cytometry showed that Abemaciclib not only inhibited the cell cycle progression of SCLC cells (P<0.0001), but also significantly increased PD-L1 expression on SBC-2 (P<0.01) and H446OE (P<0.001).
Conclusions: Abemaciclib significantly inhibits the proliferation, invasion, migration and cell cycle progression of SCLC by inhibiting the expressions of CDK4/6, c-Myc, ASCL1, YAP1 and NEUROD1. Abemaciclib can also increase the expression of PD-L1 in SCLC.
【中文题目：Abemaciclib抑制c-Myc高表达小细胞肺癌 增殖、侵袭和迁移的生物学功能的研究】 【中文摘要：背景与目的 c-Myc高表达小细胞肺癌（small cell lung cancer, SCLC）因易复发转移导致患者生存率极低，细胞周期依赖性激酶4和6（cyclin-dependent kinases 4 and 6, CDK4/6）抑制剂Abemaciclib在多种肿瘤的治疗中均起到关键作用，但其对SCLC的作用及机制仍不明晰。本研究旨在分析Abemaciclib抑制c-Myc高表达的SCLC增殖、侵袭和迁移的作用和分子机制，以期为减少患者的复发和转移拓展新的方向。方法 通过STRING数据库分析了CDK4/6和c-Myc蛋白相互作用网络，免疫组化实验分析了31例SCLC癌组织和配对的相邻正常组织中CDK4/6及c-Myc的表达，CCK-8实验和平板克隆实验检测Abemaciclib对SCLC增殖的影响，Transwell及划痕实验检测Abemaciclib对SCLC的侵袭和迁移能力的影响，Western blot检测Abemaciclib对CDK4/6及相关转录因子的影响，流式细胞术分析了Abemaciclib对SCLC的细胞周期和免疫检查点的调节作用。结果 通过STRING蛋白相互作用网络分析发现CDK4/6的表达均与c-Myc相关，c-Myc可以直接调节ASCL1（achaete-scute complex homolog 1）、NEUROD1（neuronal differentiation 1）和Yes相关蛋白1（Yes-associated protein 1, YAP1）的表达，CDK4和c-Myc均调控程序性死亡配体1（programmed cell death ligand 1, PD-L1）的表达。通过免疫组化实验发现，CDK4/6及c-Myc在SCLC癌组织中的表达高于癌旁组织（P<0.0001）。CCK-8实验、平板克隆实验、Transwell实验及划痕实验验证了Abemaciclib可以有效地抑制SBC-2和H446OE的增殖、侵袭和迁移（P<0.0001）。Western blot进一步分析了Abemaciclib在抑制SBC-2和H446OE细胞内CDK4（P<0.05）和CDK6（P<0.05）的同时，也影响了c-Myc（P<0.05）、ASCL1（P<0.05）、NEUROD1（P<0.05）和YAP1（P<0.05）等与SCLC浸润和转移相关的蛋白。流式细胞术发现Abemaciclib不仅抑制SCLC的细胞周期进程（P<0.0001），还可以提高SBC-2（P<0.01）和H446OE（P<0.001）细胞表面PD-L1的表达。结论 Abemaciclib明显抑制SCLC的增殖、侵袭、迁移和细胞周期进程，其通过抑制CDK4/6下调c-Myc、ASCL1、YAP1和NEUROD1蛋白表达的同时，也提高了PD-L1的表达。 】 【中文关键词：肺肿瘤；细胞周期依赖性激酶4/6；c-Myc】.
Keywords: Cyclin-dependent kinases 4 and 6; Lung neoplasms; c-Myc.