通过突变和竞争性抑制破坏TWIST1-RELA结合，以验证TWIST1 WR结构域作为治疗靶点。

Disruption of TWIST1-RELA binding by mutation and competitive inhibition to validate the TWIST1 WR domain as a therapeutic target.

作者信息

Roberts Cai M, Shahin Sophia A, Loeza Joana, Dellinger Thanh H, Williams John C, Glackin Carlotta A

机构信息

City of Hope, 1500 E Duarte Rd, Duarte, CA, 91010, USA.

Present address: Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06520, USA.

出版信息

BMC Cancer. 2017 Mar 10;17(1):184. doi: 10.1186/s12885-017-3169-9.

DOI:10.1186/s12885-017-3169-9

PMID:28283022

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5345230/

Abstract

BACKGROUND

Most cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance. Across many cancer types, patients with advanced stage disease would benefit from a novel therapy preventing or reversing these changes. To this end, we have investigated the unique WR domain of the transcription factor TWIST1, which has been shown to play a role in driving metastasis and drug resistance.

METHODS

In this study, we identified evolutionarily well-conserved residues within the TWIST1 WR domain and used alanine substitution to determine their role in WR domain-mediated protein binding. Co-immunoprecipitation was used to assay binding affinity between TWIST1 and the NFκB subunit p65 (RELA). Biological activity of this complex was assayed using a dual luciferase assay system in which firefly luciferase was driven by the interleukin-8 (IL-8) promoter, which is upregulated by the TWIST1-RELA complex. Finally, in order to inhibit the TWIST1-RELA interaction, we created a fusion protein comprising GFP and the WR domain. Cell fractionation and proteasome inhibition experiments were utilized to elucidate the mechanism of action of the GFP-WR fusion.

RESULTS

We found that the central residues of the WR domain (W190, R191, E193) were important for TWIST1 binding to RELA, and for increased activation of the IL-8 promoter. We also found that the C-terminal 245 residues of RELA are important for TWIST1 binding and IL-8 promoter activation. Finally, we found the GFP-WR fusion protein antagonized TWIST1-RELA binding and downstream signaling. Co-expression of GFP-WR with TWIST1 and RELA led to proteasomal degradation of TWIST1, which could be inhibited by MG132 treatment.

CONCLUSIONS

These data provide evidence that mutation or inhibition of the WR domain reduces TWIST1 activity, and may represent a potential therapeutic modality.

摘要

背景

大多数癌症死亡是由已转移至其起源组织之外的肿瘤细胞或已产生耐药性的肿瘤细胞导致的。在许多癌症类型中，晚期疾病患者将从预防或逆转这些变化的新型疗法中获益。为此，我们研究了转录因子TWIST1独特的WR结构域，该结构域已被证明在驱动转移和耐药性方面发挥作用。

方法

在本研究中，我们鉴定了TWIST1 WR结构域内进化上高度保守的残基，并使用丙氨酸替代来确定它们在WR结构域介导的蛋白质结合中的作用。采用免疫共沉淀法检测TWIST1与NFκB亚基p65（RELA）之间的结合亲和力。使用双荧光素酶检测系统检测该复合物的生物学活性，其中萤火虫荧光素酶由白细胞介素-8（IL-8）启动子驱动，该启动子由TWIST1-RELA复合物上调。最后，为了抑制TWIST1-RELA相互作用，我们创建了一种包含绿色荧光蛋白（GFP）和WR结构域的融合蛋白。利用细胞分级分离和蛋白酶体抑制实验来阐明GFP-WR融合蛋白的作用机制。

结果

我们发现WR结构域的中心残基（W190、R191、E193）对于TWIST1与RELA的结合以及IL-8启动子的激活增强很重要。我们还发现RELA的C末端245个残基对于TWIST1结合和IL-8启动子激活很重要。最后，我们发现GFP-WR融合蛋白拮抗TWIST1-RELA结合和下游信号传导。GFP-WR与TWIST1和RELA共表达导致TWIST1的蛋白酶体降解，这可通过MG132处理来抑制。