Cripto Antagonism of Activin and TGF-Beta Signalling

激活素和 TGF-β 信号传导的 Cripto 拮抗作用

• 批准号: 6769849
• 负责人: WYLIE W. VALE
• 金额: $ 30.54万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769849
• 关键词: MCF7 cell; activins; biological signal transduction; breast neoplasms; cell proliferation; growth factor receptors; hormone inhibitor; laboratory rabbit; molecular oncology; neutralizing antibody; oncoproteins; phosphorylation; protein binding; protein structure function; transcription factor; transforming growth factors

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.

描述（由申请人提供）：TGF-β和激活素是生长和分化因子TGF-β超家族的功能相关成员。TGF-β和激活素激活Smad 2和Smad 3，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。对激活素和/或TGF-β的抗增殖反应的破坏与失调的细胞增殖、肿瘤发生和向恶性表型的进展相关。Cripto是一种发育癌蛋白，在多种人类肿瘤中过表达，包括约80%的人类乳腺肿瘤。Cripto过表达转化乳腺上皮细胞并激活促生长Erk/MAPK和PI 3 K信号通路。Cripto和相关的EGF-CFC蛋白在胚胎发育过程中也起着重要的信号作用，Cripto是中胚层诱导和心脏发生所必需的。已经显示Cripto作为辅助受体，通过TGF-β超家族成员nodal、Vgl和GDFl经由激活素II/I型受体促进受体组装和信号传导。这使我们测试Cripto是否可以调节激活素和可能的其他TGF-β配体组装功能性受体复合物的能力，并且我们表明Cripto在II型激活素受体存在下结合激活素，与ALK 4竞争结合激活素并拮抗激活素信号传导。我们现在有证据表明，Cripto在TBRII存在下也结合TGF-β，与ALK 5竞争TGF-β结合并拮抗TGF-β信号传导;因此，我们建议Cripto通常能够阻断抗增殖Smad 2/3信号。在本提案的目标I下，我们将鉴定Cripto上介导Cripto与激活素/TGF-β结合以及激活素/TGF-β信号传导拮抗作用的分子决定簇。我们还建议开发包括抗Cripto中和抗体的试剂，旨在防止Cripto拮抗激活素和TGF-β信号传导的能力。目的II提出表征Cripto在体外响应激活素/TGF-β阻断I型受体募集至配体-II型受体复合物和阻断乳腺癌细胞中I型受体磷酸化的能力，以及Cripto在体外阻断乳腺癌细胞经激活素/TGF-β处理后Smad 2/3磷酸化的能力。在目标III下，我们将表征Cripto对三种乳腺上皮衍生细胞系（MCF-7、MCF-10A和CID-9细胞）中激活素/TGF-β诱导的生长抑制的影响。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。