Functions of SMAD4 in development and cancers

我们对突变小鼠的初步分析揭示了SMAD基因在多种生物学过程中的不同功能。我们发现SMAD 2和SMAD 4是原肠胚形成所需的，SMAD 5是血管生成所需的，SMAD 3是建立粘膜免疫应答和骨骼正常发育所需的。我们最近的努力集中在SMAD 4上，它是TGF-β信号的常见介导剂。SMAD 4也被称为DPC 4（在胰腺癌基因座4中缺失）。SMAD 4突变已在胰腺癌、结肠癌、胆管细胞癌、胃息肉病和腺癌中检测到。我们的研究表明SMAD 4对于小鼠的胚胎发育是必不可少的，因为SMAD 4的缺失导致E67时由于胚外膜形成受损和上胚层增殖减少而致死。SMAD 4-杂合小鼠由于单倍不足而发生胃息肉病和癌症。使用SMAD 4-Co小鼠，我们已经证明SMAD 4缺乏可导致乳腺组织、皮肤、肝脏、前胃和结肠中的肿瘤形成。肝细胞中SMAD 4的缺失也会导致铁积累，这是一种类似于人类血色病的疾病。 在过去的一年中，我们集中研究SMAD 4在胆管细胞癌（CC）中的作用。CC是第二常见的原发性肝癌，并且与预后不良相关。它已被证明，CC港的一些肿瘤抑制基因和癌基因的改变，但肿瘤发生的关键调控仍然未知。在这里，我们已经产生了一个小鼠模型，使用肝脏特异性靶向破坏肿瘤抑制因子SMAD 4和PTEN，以高转移率发展CC。在缺乏SMAD 4和PTEN的情况下，增生灶仅出现在2月龄的突变小鼠的胆管中，并继续生长，导致4-7月龄的所有动物中的肿瘤形成。我们发现CC的形成遵循与显著改变相关的组织病理学变化的多步骤进展，包括磷酸化AKT、FOXO 1、GSK-3 β、mTOR和ERK水平的增加以及细胞周期蛋白D1的核水平的增加。我们进一步证明SMAD 4和PTEN通过一种新的反馈机制相互调节，以维持表达平衡并协同抑制CC形成。最后，我们对人CC的分析在大多数p-AKT阳性CC中检测到PTEN失活，而大约一半也失去了SMAD 4表达。这些发现阐明了SMAD 4和PTEN之间的关系，并扩展了我们对CC形成的理解。

Our preliminary analysis on the mutant mice has revealed distinct functions of SMAD genes in multiple biological processes. We showed that SMAD2 and SMAD4 are needed for gastrulation, SMAD5 for angiogenesis, and SMAD3 for establishment of the mucosal immune response and proper development of the skeleton. Our recent effort is focused on SMAD4, which is a common mediator of TGF-beta signals. SMAD4 is also called DPC4 (deleted in pancreastic cancer locus 4). Mutatons of SMAD4 has been detected in pancreatic cancer, colon cancer, cholangiocellular carcinoma, gastric polyposis, and adenocarcinomas. Our study indicates that SMAD4 is essential for embryonic development in mice, as loss of SMAD4 results in lethality at E67 due to impaired extraembryonic membrane formation and decreased epiblast proliferation. SMAD4-heterozygous mice developed gastric polyposis and cancer due to haploinsufficiency. Using SMAD4-Co mice, we have demonstrated that SMAD4 deficiency could cause tumor formation in mammary tissue, skin, liver, forestomach, and colon. Deletion of SMAD4 in hepatic cells also results in iron accumulation, a disease mimicking human hemochromatosis. In the past year, we focused on studying SMAD4 in cholangiocellular carcinoma (CC). CC is the second most common primary liver cancer, and is associated with a poor prognosis. It has been shown that CCs harbor alterations of a number of tumor-suppressor genes and oncogenes, yet key regulators for tumorigenesis remain unknown. Here we have generated a mouse model that develops CC with high penetrance using liver-specific targeted disruption of tumor suppressors SMAD4 and PTEN. In the absence of SMAD4 and PTEN, hyperplastic foci emerge exclusively from bile ducts of mutant mice at 2 months of age and continue to grow, leading to tumor formation in all animals at 4-7 months of age. We show that CC formation follows a multistep progression of histopathological changes that are associated with significant alterations, including increased levels of phosphorylated AKT, FOXO1, GSK-3beta, mTOR, and ERK and increased nuclear levels of cyclin D1. We further demonstrate that SMAD4 and PTEN regulate each other through a novel feedback mechanism to maintain an expression balance and synergistically repress CC formation. Finally, our analysis of human CC detected PTEN inactivation in a majority of p-AKT-positive CCs, while about half also lost SMAD4 expression. These findings elucidate the relationship between SMAD4 and PTEN and extend our understanding of CC formation.