Division of Pathobiology, Children's Hospital of Philadelphia, Pennsylvania.
Cell & Molecular Biology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
Mol Cancer Res. 2021 May;19(5):823-833. doi: 10.1158/1541-7786.MCR-20-0721. Epub 2021 Feb 19.
About 10% to 30% of patients with colorectal cancer harbor either loss of or missense mutations in SMAD4, a critical component of the TGFβ signaling pathway. The pathophysiologic function of missense mutations in Smad4 is not fully understood. They usually map to the MH2 domain, specifically to residues that are involved in heterodimeric complex formation with regulatory Smads (such as Smad2/3) and ensuing transcriptional activation. These detrimental effects suggest that SMAD4 missense mutations can be categorized as loss-of-function. However, they tend to cluster in a few hotspots, which is more consistent with them acting by a gain-of-function mechanism. In this study, we investigated the functional role of Smad4 R361 mutants by re-expressing two R361 Smad4 variants in several Smad4-null colorectal cancer cell lines. As predicted, R361 mutations disrupted Smad2/3-Smad4 heteromeric complex formation and abolished canonical TGFβ signaling. In that, they were similar to SMAD4 loss. However, RNA sequencing and subsequent RT-PCR assays revealed that Smad4mut cells acquired a gene signature associated with enhanced Lef1 protein function and increased Wnt signaling. Mechanistically, Smad4 mutant proteins retained binding to Lef1 protein and drove a commensurate increase in downstream Wnt signaling as measured by TOP/FOP luciferase assay and Wnt-dependent cell motility. Consistent with these findings, human colorectal cancers with SMAD4 missense mutations were less likely to acquire activating mutations in the key Wnt pathway gene CTNNB1 (encoding β-catenin) than colorectal cancers with truncating SMAD4 nonsense mutations. IMPLICATIONS: Our studies suggest that in colorectal cancer hotspot mutations in Smad4 confer enhanced Wnt signaling and possibly heightened sensitivity to Wnt pathway inhibitors. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/823/F1.large.jpg.
大约 10% 到 30%的结直肠癌患者存在 SMAD4 的缺失或错义突变，SMAD4 是 TGFβ 信号通路的关键组成部分。错义突变的 Smad4 的病理生理功能尚未完全了解。它们通常位于 MH2 结构域，特别是与调节 Smads（如 Smad2/3）形成异源二聚体复合物并随后进行转录激活的残基。这些有害影响表明 SMAD4 错义突变可以归类为功能丧失。然而，它们往往聚集在少数热点中，这更符合它们通过获得功能机制起作用。在这项研究中，我们通过在几个 Smad4 缺失的结直肠癌细胞系中重新表达两种 R361 Smad4 变体，研究了 Smad4 R361 突变体的功能作用。正如预测的那样，R361 突变破坏了 Smad2/3-Smad4 异源二聚体复合物的形成并消除了经典的 TGFβ 信号。在这方面，它们类似于 SMAD4 缺失。然而，RNA 测序和随后的 RT-PCR 分析表明，Smad4mut 细胞获得了与 Lef1 蛋白功能增强和 Wnt 信号增加相关的基因特征。从机制上讲，Smad4 突变蛋白保留了与 Lef1 蛋白的结合，并通过 TOP/FOP 荧光素酶测定和 Wnt 依赖性细胞迁移来驱动下游 Wnt 信号的相应增加。与这些发现一致，具有 SMAD4 错义突变的人类结直肠癌比具有截断性 SMAD4 无义突变的结直肠癌不太可能获得关键 Wnt 通路基因 CTNNB1（编码 β-连环蛋白）的激活突变。含义：我们的研究表明，在结直肠癌中，Smad4 中的热点突变赋予了增强的 Wnt 信号，并可能增加了对 Wnt 通路抑制剂的敏感性。直观概述：http://mcr.aacrjournals.org/content/molcanres/19/5/823/F1.large.jpg。
