Stress Induction of Glucose Regulated Protein GRP78/BiP

描述(申请人提供)：最近的发现，GRP78，传统上被认为是一个主要的内质网(ER)伴侣和ER应激信号的调节者，也可以在癌症等病理生理条件下定位于细胞表面，改变了该蛋白在癌症中如何发挥其促增殖和抗凋亡功能的范式。在过去的资助期间，我们证实了GRP78单倍体缺陷通过抑制肿瘤增殖、血管生成和增加细胞凋亡来抑制乳腺肿瘤的发生。通过建立具有GRP78和肿瘤抑制基因Pten的条件双等位基因缺失的新型突变小鼠模型，我们发现GRP78不仅在前列腺癌和血液病的发生中是关键的，而且在体内和体外都是PI3K/AKT激活所必需的。细胞表面GRP78(SGRP78)是一种控制细胞信号转导的辅受体。在了解GRP78如何调节致癌信号时，我们建立了内质网应激不仅上调GRP78，还积极促进GRP78从内质网到细胞表面的重新定位，这一过程由KDEL检索机制调节。利用GRP78在癌细胞中的细胞表面定位而不是在正常器官中的定位，我们筛选并鉴定了一种能与细胞表面GRP78特异性结合、诱导肿瘤细胞凋亡并抑制肿瘤生长的先导型单抗MAb159。我们假设sGRP78通过调节PI3K/AKT信号通路是肿瘤生长和治疗耐药的主要效应者，靶向sGRP78代表了一种新的有效的抗PI3K治疗方法，将抑制肿瘤生长和减轻耐药性。在目标1中，我们试图通过分析GRP78表面定位所需的功能结构域和ER检索机制的完整性，以及GRP78动员的Liv细胞成像，来了解允许GRP78从内质网逃逸到细胞表面的潜在分子机制。在目标2中，我们将通过研究sGRP78与PI3K通路的功能和物理相互作用以及靶向sGRP78对其他致癌途径的影响来确定sGRP78如何调控PI3K/AKT信号转导。在目标3中，我们将直接测试sGRP78在自发的小鼠癌症模型和使用耐药的人癌细胞株的异种移植模型中在肿瘤发生和治疗耐药中的作用。作为我们在前列腺癌方面工作的合理扩展，我们将利用一种新的Pten-Null前列腺癌模型，该模型允许生物发光监测癌症的发展、进展和去势后的复发。从这个模型中衍生出来的去势敏感和抵抗细胞也将被研究。MAb159的有效性和安全性将在其他癌症模型中进行测试，无论是单独治疗还是联合治疗。因此，这项工作不仅涉及基本机制，而且具有广泛的临床意义。

DESCRIPTION (provided by applicant): The recent discovery that GRP78, traditionally regarded as a major endoplasmic reticulum (ER) chaperone and regulator of ER stress signaling, can also localize to the cell surface under pathophysiologic conditions such as cancer, changes the paradigm on how this protein may exert its pro-proliferative and anti- apoptotic function in cancer. In the past grant period, we established that GRP78 haploinsufficiency suppresses breast tumorigenesis through inhibition of tumor proliferation, angiogenesis and increased apoptosis. Through creation of a novel mutant mouse model with conditional biallelic deletion of both Grp78 and the tumor suppressor gene Pten, we discovered that not only is GRP78 critically required for prostate tumorigenesis and hematologic cancers, but is also required for PI3K/AKT activation in both types of cancer, both in vivo and in vitro. Cel surface GRP78 (sGRP78) is emerging as a co-receptor controlling cell signaling. In understanding how GRP78 regulates oncogenic signaling, we established that ER stress not only upregulates GRP78 but also actively promotes relocalization of GRP78 from the ER to the cell surface, and this process is regulated by the KDEL retrieval machinery. Taking advantage of cell surface localization of GRP78 in cancer cells but not in normal organs, we screened and identified a lead monoclonal antibody (MAb159) which specifically binds to cell surface GRP78, induces cancer cell apoptosis and suppresses tumor growth. Here we hypothesize that sGRP78 is a major effector of tumor growth and therapeutic resistance through its ability to regulate the PI3K/AKT signaling pathway and that targeting sGRP78 represents a novel and powerful approach for anti-PI3K therapy that will suppress tumor growth and alleviate drug resistance. In Aim 1, we seek to understand the underlying molecular mechanisms that allow GRP78 to escape from the ER to the cell surface, through analysis of the functional domains of GRP78 required for surface localization and the integrity of the ER retrieval machinery, coupled with liv cell imaging of GRP78 mobilization. In Aim 2, we will determine how sGRP78 regulates PI3K/AKT signaling by investigating the functional and physical interactions of sGRP78 with components of the PI3K pathway and the effect of targeting sGRP78 on other oncogenic pathways. In Aim 3, we will directly test the role of sGRP78 in tumorigenesis and therapeutic resistance in spontaneous mouse cancer models and xenograft models using human cancer cell lines resistant to therapy. As a logical extension of our work on prostate cancer, we will utilize a novel Pten-null prostate cancer model which allows bioluminescence monitoring of cancer development, progression and recurrence after castration. Castration sensitive and resistant cells derived from this model will also be studied. The efficacy and safety of MAb159 will be tested in other cancer models, either alone or in combination therapy. Thus, this work not only addresses fundamental mechanisms but also has wide clinical implications.