Department of General Surgery, Beijing Jishuitan Hospital, The 4th Medical College of Peking University, Beijing, China.
Eur Rev Med Pharmacol Sci. 2018 Dec;22(23):8273-8280. doi: 10.26355/eurrev_201812_16524.
MLN4924 is a second-generation small molecule inhibitor with anti-cancer activity that inhibits neddylation activation enzyme (NAE), subsequently blocking the neddylation-dependent activation of Cullin-RING E3 ligases (CRLs). Mof4 family associated protein 1 (MRFAP1) is a highly conserved, short half-life protein and one of the most up-regulated proteins in response to MLN4924 treatment. MRFAP1 has been identified as a novel cell cycle-related protein and a regulatory component monitoring and preventing genomic instability. However, whether MRFAP1 plays a role in MLN4924-mediated cancer cell death remains elusive.
The expression of MRFAP1 in gastric cancer clinic samples was detected by Real-time PCR and Western blot. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system was used to knockout MRFAP1 gene in both AGS and SGC-7901 cells. The proliferation of GC cells was measured by CCK8 assay. The cell cycle distribution of GC cells was determined by fluorescence-activated cell sorting (FACS) assay. Co-immunoprecipitation assay was used to determine the interaction between MRFAP1 and P27.
MRFAP1 was downregulated in clinic gastric cancer samples at post-translational level. Overexpression of MRFAP1 decreased gastric cancer cells proliferation. CRISPR-mediated knockout of MRFAP1 increased the cytotoxicity of MLN4924 by augmenting MLN4924-induced G2/M arrest and apoptosis against gastric cancer cells. At the molecular level, we found that MLN4924 induced the interaction between P27 and MRFAP1, the latter associated with P27, which was further stabilized in response to MLN4924 treatment.
We showed a protective role of MRFAP1 in gastric cancer cells with MLN4924 treatment and suggested the potential possibility to combine MLN4924 with MRFAP1 inhibition to treat gastric cancer.
MLN4924 是一种具有抗癌活性的第二代小分子抑制剂，它能抑制 neddylation 激活酶（NAE），从而阻断 Cullin-RING E3 连接酶（CRLs）的 neddylation 依赖性激活。Mof4 家族相关蛋白 1（MRFAP1）是一种高度保守、半衰期短的蛋白质，是对 MLN4924 治疗反应最上调的蛋白质之一。MRFAP1 已被确定为一种新型细胞周期相关蛋白和一种调节元件，用于监测和防止基因组不稳定性。然而，MRFAP1 是否在 MLN4924 介导的癌细胞死亡中发挥作用仍不清楚。
通过实时 PCR 和 Western blot 检测胃癌临床样本中 MRFAP1 的表达。利用 CRISPR（成簇规律间隔短回文重复序列）/Cas9 系统敲除 AGS 和 SGC-7901 细胞中的 MRFAP1 基因。通过 CCK8 测定法测定 GC 细胞的增殖。通过荧光激活细胞分选（FACS）测定法测定 GC 细胞的细胞周期分布。通过免疫共沉淀测定法确定 MRFAP1 和 P27 之间的相互作用。
MRFAP1 在胃癌临床样本中在翻译后水平下调。MRFAP1 的过表达降低了胃癌细胞的增殖。CRISPR 介导的 MRFAP1 敲除通过增强 MLN4924 诱导的 G2/M 阻滞和凋亡来增加 MLN4924 对胃癌细胞的细胞毒性。在分子水平上，我们发现 MLN4924 诱导了 P27 和 MRFAP1 之间的相互作用，后者与 P27 结合，并在 MLN4924 处理后进一步稳定。
我们表明 MRFAP1 在 MLN4924 治疗的胃癌细胞中具有保护作用，并提出了将 MLN4924 与 MRFAP1 抑制相结合治疗胃癌的潜在可能性。
