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

项目总结 作为内质网中普遍存在的HSP90类似物，grp94在蛋白质质量中起着重要的作用。 通过参与未折叠蛋白反应和内质网相关的内质网调节分泌途径 蛋白质降解途径。它在癌症中过度表达，水平越高，预后越差。 但表达水平和病理之间的潜在关系尚不清楚。从历史上看，人类的生物学 Grp94已经在宿主对癌症的免疫反应的背景下进行了研究，但没有在 癌症生物学本身。在过去的十年里，我们的实验室一直专注于确定内在性质 通过遗传方法获得grp94基因。使用cre-loxP系统，我们产生了条件grp94基因敲除(KO) 老鼠模型。对各种细胞类型特异性grp94缺失小鼠的功能分析导致发现 Grp94是一种专有的伴侣蛋白，控制着细胞生长、迁移和分化的几个关键途径， 包括整合素、Toll样受体、Wnt共受体LRP6和细胞表面转化生长因子β对接 分子GARP。因此，grp94已成为癌症治疗的一个具有重要战略意义的靶点。这个项目 将重点放在乳腺癌生物学的GRP94-GARP-转化生长因子β轴上。我们最重要的假设是 GRP94是通过GARP调节细胞表面转化生长因子β表达的关键分子伴侣 它通过整合素的激活过程。强劲的初步数据支持这一假设。这两项都是我们在 体外和体内证据表明，grp94控制细胞表面GARP和整合素的表达。加普是 潜伏的转化生长因子β与细胞表面对接所需，而整合素有助于潜在的转化生长因子β激活。这个 新的初步数据证实了GRP94-GARP-转化生长因子β轴与肿瘤的相关性 包括对多种原发人类癌症的肿瘤/组织微阵列进行广泛的免疫组织化学研究， 证明GARP蛋白在癌症中的异常表达与预后不良的相关性。 同时观察GARP过表达在促进乳腺癌和免疫耐受中的作用。 因此，我们准备研究grp94-garp-转化生长因子β轴的作用机制和生物学影响。 癌症生物学。我们的第一个目标是将GARP建立为一种新的grp94客户蛋白，确定grp94的作用 在调节表面转化生长因子β的生物发生和激活方面，揭示了grp94的详细机制 监管GARP的成熟和贩运。我们的第二个目标将确定grp94的生物学意义- 利用几种新的小鼠模型在新生肿瘤中表达GARP，这些模型允许精确控制 GARP基因表达。使用一组独特的GARP单抗，Aim 3将确定 Grp94-GARP在乳腺癌中表达的临床意义及GARP能否作为一种新的基因 治疗靶点。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的调节电路铺平了道路- GARP-转化生长因子β通路对人类癌症的基本认识、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.