SIRT2 介导的 FOXO3 去乙酰化调控鼻咽癌细胞对拉帕替尼的敏感性。

BACKGROUND: Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. METHODS: Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay. RESULTS: To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1) are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4 mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. CONCLUSION: Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and Lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.

背景：化学耐药性是鼻咽癌（NPC）成功治疗的障碍。拉帕替尼是一种靶向酪氨酸激酶抑制剂治疗药物，也用于治疗 NPC，但通常需要高剂量才能达到效果。为了探讨拉帕替尼耐药的机制，我们对一系列 NPC 细胞系进行了特征描述，以确定 FOXO3 和沉默调节蛋白在调节 NPC 耐药中的作用。

方法：采用磺酰罗丹明 B（SRB）测定、集落形成测定、蛋白质提取、定量和 Western blot、RT-qPCR、免疫共沉淀测定。

结果：为了探索新的治疗策略，我们首先通过 SRB 和集落形成细胞毒性测定对一组 NPC 细胞系的拉帕替尼敏感性进行了特征描述，发现转移性 NPC（C666-1 和 5-8F）细胞对拉帕替尼高度耐药，而低转移性细胞系（6-10B、TW01 和 HK-1）对拉帕替尼敏感。对拉帕替尼敏感的 6-10B 和耐药的 5-8F NPC 细胞的 Western blot 分析表明，磷酸化/失活 FOXO3（P-FOXO3；T32）、其靶标 FOXM1 和其调节剂 SIRT2 的表达与拉帕替尼反应和敏感性呈负相关，表明 SIRT2 介导 FOXO3 去乙酰化以促进拉帕替尼耐药。一致地，使用野生型和 foxo1/3/4 小鼠胚胎成纤维细胞（MEFs）的集落形成细胞毒性测定表明，FOXO1/3/4 缺失显着减弱了拉帕替尼诱导的细胞毒性，证实 FOXO 蛋白对于介导拉帕替尼反应是必需的。使用化学 SIRT 抑制剂（即 sirtinol、Ex527、AGK2 和 AK1）进行的 SRB 细胞活力测定表明，所有 SIRT 抑制剂均可降低 NPC 细胞活力，但只有 SIRT2 特异性抑制剂 AK1 和 AGK2 进一步增强了拉帕替尼的细胞毒性。一致地，集落形成测定表明，SIRT2 抑制剂 AK1 和 AGK2 以及 SIRT2 敲低进一步增强了敏感和耐药 NPC 细胞中的拉帕替尼细胞毒性。免疫共沉淀研究表明，除了拉帕替尼处理外，SIRT2 药物抑制和沉默也导致 FOXO3 乙酰化增加。重要的是，SIRT2 抑制和耗竭进一步增强了 NPC 细胞中拉帕替尼介导的 FOXO3 乙酰化。

结论：总之，我们的结果表明 SIRT2 介导的 FOXO3 去乙酰化参与了拉帕替尼的反应和敏感性，并且 SIRT2 可以通过在敏感和耐药 NPC 细胞中介导 FOXO3 去乙酰化来特异性拮抗拉帕替尼的细胞毒性。本研究还提出 SIRT2 可以作为转移性和拉帕替尼耐药 NPC 的重要生物标志物，并且靶向 SIRT2-FOXO3 轴可能为治疗 NPC 和克服化学耐药性提供新策略。