Tankyrase Inhibitors Stimulate the Ability of Tankyrases to Bind Axin and Drive Assembly of β-Catenin Degradation-Competent Axin Puncta

Activation of the wnt signaling pathway is a major cause of colon cancer development. Tankyrase inhibitors (TNKSi) have recently been developed to block the wnt pathway by increasing axin levels to promote degradation of the wnt-regulator β-catenin. TNKSi bind to the PARP (poly(ADP)ribose polymerase) catalytic region of tankyrases (TNKS), preventing the PARylation of TNKS and axin that normally control axin levels through ubiquitination and degradation. TNKSi treatment of APC-mutant SW480 colorectal cancer cells can induce axin puncta which act as sites for assembly of β-catenin degradation complexes, however this process is poorly understood. Using this model system, we found that siRNA knockdown of TNKSs 1 and 2 actually blocked the ability of TNKSi drugs to induce axin puncta, revealing that puncta formation requires both the expression and the inactivation of TNKS. Immunoprecipitation assays showed that treatment of cells with TNKSi caused a strong increase in the formation of axin-TNKS complexes, correlating with an increase in insoluble or aggregated forms of TNKS/axin. The efficacy of TNKSi was antagonized by proteasome inhibitors, which stabilized the PARylated form of TNKS1 and reduced TNKSi-mediated assembly of axin-TNKS complexes and puncta. We hypothesise that TNKSi act to stimulate TNKS oligomerization and assembly of the TNKS-axin scaffold that form puncta. These new insights may help in optimising the future application of TNKSi in anticancer drug design.

Wnt信号通路（Wnt signaling pathway）的激活是结直肠癌发生的主要致病诱因。端锚聚合酶抑制剂（Tankyrase inhibitors, TNKSi）作为一类新兴靶向药物，可通过提升轴蛋白（axin）水平、促进Wnt通路调控因子β-连环蛋白（β-catenin）的降解，从而阻断Wnt信号通路。TNKSi可结合端锚聚合酶（Tankyrases, TNKS）的聚(ADP-核糖)聚合酶（poly(ADP-ribose) polymerase, PARP）催化结构域，进而阻断TNKS与轴蛋白的聚ADP核糖基化修饰；而正常生理状态下，这类修饰会通过泛素化与降解过程调控轴蛋白的稳态水平。使用TNKSi处理携带腺瘤性结肠息肉病蛋白（APC）突变的SW480结直肠癌细胞，可诱导轴蛋白斑点（axin puncta）的形成，这类斑点可作为β-连环蛋白降解复合物的组装位点，但目前学界对该过程的认知仍较为匮乏。借助该模型体系，我们发现通过小干扰RNA（siRNA）敲低TNKS1与TNKS2的表达，反而会阻断TNKSi药物诱导轴蛋白斑点形成的能力，这表明轴蛋白斑点的形成既依赖TNKS的表达，也依赖TNKS的失活。免疫沉淀（Immunoprecipitation）实验结果显示，TNKSi处理细胞会显著促进轴蛋白-TNKS复合物的形成，这与TNKS/轴蛋白的不溶性聚集形式增多呈显著相关。蛋白酶体抑制剂（proteasome inhibitors）可拮抗TNKSi的药效：这类抑制剂能够稳定TNKS1经聚ADP核糖基化修饰的形式，并削弱TNKSi介导的轴蛋白-TNKS复合物组装与轴蛋白斑点形成过程。我们提出假说：TNKSi可通过促进TNKS的寡聚化，以及组装形成轴蛋白斑点的TNKS-轴蛋白支架复合物来发挥药理作用。上述全新研究见解或将有助于优化TNKSi在抗癌药物研发中的未来应用。