Project 1: Definition of grp94-GARP-TGFbeta Axis in Cancer Biology and Clinical Significance

项目1：grp94-GARP-TGFbeta轴在癌症生物学中的定义及其临床意义

• 批准号: 8934513
• 负责人: Zihai Li
• 金额: $ 22.38万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8934513
• 关键词: Address; Binding; Biochemistry; Biogenesis; Biological; Biological Markers; Biology; Breast Cancer cell line; Breast Carcinoma; Cancer Biology; Cell surface; Client; Clip; Collection; Colon; Colon Carcinoma; Data; Degradation Pathway; Development; Diagnostic; Disease; Docking; ERBB2 gene; Elements; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Foundations; Future; Genetic; Heat-Shock Proteins 90; Human; Immune; Immune Tolerance; Immune response; Immunohistochemistry; In Vitro; Inflammation; Insulin-Like Growth Factor II; Integrin Binding; Integrins; Investigation; Knock-in Mouse; Knockout Mice; Laboratories; Lead; Link; Lung; Malignant Neoplasms; Malignant neoplasm of liver; Malignant neoplasm of lung; Mammary Neoplasms; Mammary gland; Membrane; Modeling; Molecular; Molecular Chaperones; Monoclonal Antibodies; Multiple Myeloma; Mus; Neoplasm Metastasis; Null Lymphocytes; Oncogenic; Pathology; Pathway interactions; Play; Primary carcinoma of the liver cells; Process; Promoter Regions; Property; Prostate; Proteins; Quality Control; Reagent; Regulation; Research Personnel; Role; Surface; System; Tetanus Helper Peptide; Therapeutic; Tissue Microarray; Toll-like receptors; Transforming Growth Factor beta; Transforming Growth Factors; Tumor Tissue; cancer therapy; cell growth; cell type; clinically significant; genetic approach; human TLR7 protein; in vivo; inhibitor/antagonist; lipoprotein receptor-related protein 6; malignant breast neoplasm; malignant mouth neoplasm; malignant stomach neoplasm; migration; mouse model; new therapeutic target; novel; outcome forecast; overexpression; paralogous gene; protein degradation; response; targeted cancer therapy; therapeutic target; trafficking; tumorigenesis

PROJECT SUMMARY As a ubiquitous HSP90 paralog in the endoplasmic reticulum, grp94 plays important roles in protein quality control in the secretory pathway by participating in both the unfolded protein response and the ER-associated protein degradation pathway. It is over-expressed in cancers, with higher levels conferring poorer prognosis, but the underlying relationship between expression levels and pathology is unclear. Historically, the biology of grp94 has been studied in the context of the host's immune response to cancer, but not in the context of cancer biology per se. For the past decade, our laboratory has focused on determining the intrinsic properties of grp94 via genetic approaches. Using the cre-loxP system, we generated a conditional grp94 knockout (KO) mouse model. Functional analyses of various cell type-specific grp94 null mice have led to the discovery that grp94 is an obligate chaperone controlling several key pathways for cell growth, migration and differentiation, including integrins, Toll-like receptors (TLRs), Wnt co-receptor LRP6, and the cell surface TGFβ-docking molecule GARP. Thus, grp94 has emerged as a strategically important target for cancer therapy. This project will focus on the breast cancer biology of the grp94-GARP-TGFβ axis. Our overarching hypothesis is that grp94 is a key molecular chaperone in regulating the expression of cell surface TGFβ through GARP as well as its activation process via integrins. Strong preliminary data support this hypothesis. We have obtained both in vitro and in vivo evidence that grp94 controls cell surface expression of GARP as well as integrins. GARP is required for docking latent TGFβ to cell surface, whereas integrins contribute to latent TGFβ activation. The cancer relevance of grp94-GARP-TGFβ axis in cancer has been established by new preliminary data including extensive immunohistochemistry of tumor/tissue microarrays of multiple primary human cancers, demonstrating the correlation between aberrant expression of GARP protein in cancers and worse survival. The impact of GARP overexpression in promoting breast cancer and immune tolerance also was observed. Thus, we are poised to study both the mechanism and the biological impact of the grp94-GARP-TGFβ axis in cancer biology. Our first aim will establish GARP as a novel grp94 client protein, determine the roles of grp94 in regulating surface TGFβ biogenesis and activation, and uncover the detailed mechanism by which grp94 regulates GARP maturation and trafficking. Our second aim will pinpoint the biological significance of grp94- GARP expression in de novo oncogenesis using several novel mouse models that allow precise control of GARP expression genetically. Using a panel of unique GARP monoclonal antibodies, Aim 3 will determine the clinical significance of grp94-GARP expression in breast cancer and address if GARP can serve as a novel therapeutic target. This project will advance the field of grp94 biology in cancer by providing timely answers to many outstanding questions and by paving the way to eventually unlock the regulatory circuit of the grp94- GARP-TGFβ pathway for fundamental understanding, prognosis and treatment of human cancers.

项目概要 作为内质网中普遍存在的 HSP90 旁系同源物，grp94 在蛋白质质量中发挥着重要作用 通过参与未折叠蛋白反应和 ER 相关的分泌途径来控制 蛋白质降解途径。它在癌症中过度表达，水平越高预后越差， 但从历史上看，表达水平与病理学之间的潜在关系尚不清楚。 grp94 已在宿主对癌症的免疫反应的背景下进行了研究，但并未在 在过去的十年里，我们的实验室一直致力于确定癌症生物学的内在特性。 使用 cre-loxP 系统，我们生成了 grp94 的条件敲除 (KO)。 对各种细胞类型特异性 grp94 缺失小鼠的功能分析发现： grp94 是专性伴侣，控制细胞生长、迁移和分化的几个关键途径， 包括整合素、Toll 样受体 (TLR)、Wnt 辅助受体 LRP6 和细胞表面 TGFβ 对接 因此，grp94 已成为该项目的战略重要靶点。 我们将重点关注 grp94-GARP-TGFβ 轴的乳腺癌生物学。 grp94 是通过 GARP 调节细胞表面 TGFβ 表达的关键分子伴侣 它通过整合素的激活过程。我们已经获得了这一假设的有力初步数据。 体外和体内证据表明 grp94 控制 GARP 以及整合素的细胞表面表达。 需要将潜在的 TGFβ 对接至细胞表面，而整合素则有助于潜在的 TGFβ 激活。 新的初步数据已确定 grp94-GARP-TGFβ 轴与癌症的癌症相关性 包括多种原发性人类癌症的肿瘤/组织微阵列的广泛免疫组织化学， 证明癌症中 GARP 蛋白的异常表达与较差的生存率之间的相关性。 还观察到 GARP 过度表达对促进乳腺癌和免疫耐受的影响。 因此，我们准备研究 grp94-GARP-TGFβ 轴的机制和生物学影响 我们的首要目标是将 GARP 作为一种新型 grp94 客户蛋白，确定 grp94 的作用。 调控表面TGFβ生物合成和激活，并揭示grp94的详细机制 我们的第二个目标是确定 grp94- 的生物学意义。 使用几种新的小鼠模型在从头肿瘤发生中表达 GARP，这些模型可以精确控制 目标 3 将使用一组独特的 GARP 单克隆抗体来确定 GARP 表达。 grp94-GARP 在乳腺癌中表达的临床意义以及 GARP 是否可以作为一种新的治疗方法 该项目将通过提供及时的答案来推进 grp94 癌症生物学领域的发展。 许多悬而未决的问题，并为最终解锁 grp94- 的监管电路铺平道路 GARP-TGFβ 通路对人类癌症的基本了解、预后和治疗。