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

项目摘要 grp 94作为一种普遍存在于内质网中的HSP 90蛋白，对蛋白质的质量起着重要的作用 通过参与未折叠蛋白质反应和ER相关蛋白质反应来控制分泌途径。 蛋白质降解途径它在癌症中过度表达，水平越高，预后越差， 但表达水平和病理学之间的潜在关系尚不清楚。从历史上看， 已经在宿主对癌症的免疫应答的背景下研究了grp 94，但没有在 癌症生物学本身在过去的十年里，我们的实验室一直致力于确定 GRP 94基因的表达。使用cre-loxP系统，我们产生了条件性grp 94敲除（KO） 小鼠模型对各种细胞类型特异性grp 94缺失小鼠的功能分析发现， GRP 94是控制细胞生长、迁移和分化的几个关键途径的专性伴侣蛋白， 包括整合素、Toll样受体（TLR）、Wnt共受体LRP 6和细胞表面TGFβ-对接， GARP分子。因此，grp 94已经成为癌症治疗的重要战略目标。这个项目 将专注于grp 94-GARP-TGFβ轴的乳腺癌生物学。我们的首要假设是 grp 94是通过GARP调节细胞表面TGFβ表达的关键分子伴侣， 它的激活过程通过整合素。强有力的初步数据支持这一假设。我们已经获得了这两个在 体外和体内证据表明grp 94控制GARP以及整联蛋白的细胞表面表达。GARP是 整合素是将潜伏性TGFβ与细胞表面对接所需的，而整合素有助于潜伏性TGFβ活化。的 通过新的初步数据已经确定了grp 94-GARP-TGFβ轴在癌症中的癌症相关性 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， 证实了癌症中GARP蛋白的异常表达与较差的存活率之间的相关性。 还观察了GARP过表达在促进乳腺癌和免疫耐受中的影响。 因此，我们准备研究grp 94-GARP-TGFβ轴的机制和生物学影响， 癌症生物学我们的第一个目标是将GARP作为一种新的grp 94客户蛋白，确定grp 94的作用， 在调节表面TGFβ生物合成和活化方面，并揭示了grp 94 调节GARP成熟和贩运。我们的第二个目标是确定grp 94的生物学意义- 使用几种新的小鼠模型在从头肿瘤发生中的GARP表达， GARP基因表达。使用一组独特的GARP单克隆抗体，Aim 3将确定 乳腺癌中grp 94-GARP表达的临床意义及GARP是否可作为一种新的乳腺癌治疗方法的探讨 治疗靶点该项目将通过提供及时的答案来推进grp 94在癌症中的生物学领域， 许多悬而未决的问题，并通过铺平道路，最终解开监管电路的grp 94- GARP-TGFβ通路对人类癌症的基本理解，预后和治疗。

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.