Cripto Antagonism of Activin and TGF-Beta Signalling

描述（由申请人提供）：TGF-B和激活素是tgf - β生长和分化因子超家族中功能相关的成员。tgf - β和激活素激活Smad2和Smad3，它们都通过有效抑制多种细胞类型（包括上皮细胞）的增殖来调节组织稳态。激活素和/或tgf - β抗增殖反应的破坏与细胞增殖失调、肿瘤发生和恶性表型的进展有关。Cripto是一种发育性癌蛋白，在多种人类肿瘤中过表达，包括大约80%的人类乳腺肿瘤。Cripto过表达转化乳腺上皮细胞，激活促生长的Erk/MAPK和PI3K信号通路。Cripto和相关的EGF-CFC蛋白在胚胎发育过程中也发挥着重要的信号作用，并且在中胚层诱导和心脏发生中也需要Cripto。研究表明，Cripto作为一种辅助受体，通过tgf - β超家族成员节点、Vgl和GDF1激活II/I型受体，促进受体组装和信号传导。这让我们测试了Cripto是否可以调节激活素和其他可能的TGF-Beta配体组装功能受体复合物的能力，我们发现在II型激活素受体存在的情况下，Cripto与激活素结合，与ALK4竞争与激活素的结合，并拮抗激活素信号传导。我们现在有证据表明，在TBRII存在的情况下，Cripto也能与tgf - β结合，与ALK5竞争tgf - β结合，并拮抗tgf - β信号传导；因此，我们提出，Cripto可能通常能够阻断抗增殖的Smad2/3信号。根据本提案的目的1，我们将确定介导Cripto与激活素/ tgf - β结合和激活素/ tgf - β信号拮抗的Cripto分子决定因子。我们还建议开发包括抗Cripto中和抗体在内的试剂，旨在阻止Cripto拮抗激活素和tgf - β信号传导的能力。Aim II提出在体外实验中，在激活素/ tgf - β的作用下，在乳腺癌细胞中，Cripto阻断I型受体向配体- II型受体复合物募集和阻断I型受体磷酸化的能力，以及在体外实验中，在激活素/ tgf - β治疗乳腺癌细胞后，Cripto阻断Smad2/3磷酸化的能力。在Aim III下，我们将在三种乳腺上皮源性细胞系中描述Cripto对激活素/ tgf - β诱导的生长抑制的影响：MCF-7；MCF-10A;和CID-9细胞。总之，这些研究将导致对Cripto作用机制的更好理解，并将有助于阐明肿瘤疾病管理的治疗靶点。

DESCRIPTION (provided by applicant): TGF-B and activin are functionally related members of the TGF-Beta superfamily of growth and differentiation factors. TGF-Beta and activin activate Smad2 and Smad3 and they both regulate tissue homeostasis by potently inhibiting proliferation of multiple cell types, including epithelial cells. Disruption of the antiproliferative response to activin and/or TGF-Beta is associated with dysregulated cellular proliferation, oncogenesis and progression toward the malignant phenotype. Cripto is a developmental oncoprotein that is overexpressed in multiple human tumors including approximately 80% of human breast tumors. Cripto overexpression transforms mammary epithelial ceils and activates the growth promoting Erk/MAPK and PI3K signaling pathways. Cripto and related EGF-CFC proteins also play essential signaling roles during embryonic development and Cripto is required for mesoderm induction and cardiogenesis. It has been shown that Cripto acts as a coreceptor to facilitate receptor assembly and signaling via activin type II/I receptors by the TGF-Beta superfamily members nodal, Vgl and GDF1. This led us to test whether Cripto may regulate the ability of activin and possibly other TGF-Beta ligands to assemble functional receptor complexes and we showed that Cripto binds activin in the presence of type II activin receptors, competes with ALK4 for binding to activin and antagonizes activin signaling. We now have evidence showing that Cripto also binds TGF-Beta in the presence of TBRII, competes with ALK5 for TGF-Beta binding and antagonizes TGF-Beta signaling; we propose, therefore, that Cripto may be generally capable of blocking antiproliferative Smad2/3 signals. Under Aim I of this proposal we will identify the molecular determinants on Cripto that mediate Cripto binding to activin/TFG-Beta and antagonism of activin/TGF-Beta signaling. We also propose to develop reagents including anti-Cripto neutralizing antibodies designed to prevent the ability of Cripto to antagonize activin and TGF-Beta signaling. Aim II proposes to characterize the ability of Cripto to block type I receptor recruitment to ligand-type II receptor complexes and block type I receptor phosphorylation in breast cancer cells in vitro in response to activin/TGF-Beta and the ability of Cripto to block phosphorylation of Smad2/3 following activin/TGF-beta treatment of breast cancer cells in vitro. Under Aim III we will characterize the effects of Cripto on activin/TGF-Beta-induced growth inhibition in three mammary epithelial-derived cell lines: MCF-7; MCF-10A; and CID-9 cells. Together, these studies will lead to an improved understanding of the mechanisms of Cripto action and will help illuminate therapeutic targets for the management of neoplastic disease.