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Smad2和Smad4是原肠形成所必需的，Smad5是血管生成所必需的，Smad3是建立粘膜免疫反应和骨骼正常发育所必需的。我们最近的工作集中在Smad4，它是转化生长因子-β信号的常见介体。Smad4也被称为DPC4(在胰腺癌基因4中缺失)。已在胰腺癌、结肠癌、胆管细胞癌、胃息肉病和腺癌中检测到Smad4突变。我们的研究表明，Smad4对小鼠的胚胎发育是必不可少的，因为Smad4的缺失会导致E67胚胎死亡，这是因为Smad4的缺失导致胚胎外膜形成受损，上皮细胞增殖减少。Smad4杂合子小鼠由于单倍体功能不全而患上胃息肉和癌症。使用Smad4-Co小鼠，我们已经证明Smad4缺乏可以导致乳腺组织、皮肤、肝脏、前胃和结肠中的肿瘤形成。肝细胞中Smad4的缺失也会导致铁蓄积，这是一种类似人类血色素沉着症的疾病。 在过去的一年里，我们重点研究了Smad4在胆管细胞癌(CC)中的作用。CC是第二常见的原发性肝癌，与预后不良有关。已有研究表明，CCS存在许多抑癌基因和癌基因的改变，但肿瘤发生的关键调控因素仍不清楚。在这里，我们已经建立了一个小鼠模型，它使用肝脏特异性的肿瘤抑制基因Smad4和PTEN的靶向干扰来发展具有高外显性的CC。在没有Smad4和PTEN的情况下，2个月大的突变小鼠的胆管中只会出现增生性病灶，并继续生长，导致4-7个月大的所有动物都形成肿瘤。我们发现CC的形成遵循与显著改变相关的多步骤组织病理学变化，包括磷酸化的AKT、FOXO1、GSK-3β、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，Smad4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的形成。最后，我们对人类CC的分析发现，在大多数p-AKT阳性的CC中，PTEN失活，而大约一半的CC也失去了Smad4的表达。这些发现阐明了Smad4和PTEN之间的关系，扩大了我们对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.