Stress Induction of Glucose Regulated Protein GRP78/BiP

描述（由申请人提供）：最近发现GRP78，传统上被认为是内质网（ER）的主要伴侣和内质网应激信号的调节剂，也可以在癌症等病理生理条件下定位到细胞表面，改变了该蛋白如何在癌症中发挥其促增殖和抗凋亡功能的范式。在过去的研究中，我们发现GRP78单倍不全通过抑制肿瘤增殖、血管生成和增加细胞凋亡来抑制乳腺肿瘤的发生。通过建立一种具有条件双等位基因缺失Grp78和肿瘤抑制基因Pten的新型突变小鼠模型，我们发现Grp78不仅在前列腺肿瘤发生和血液学癌症中是必需的，而且在体内和体外两种类型的癌症中也需要PI3K/AKT的激活。细胞表面GRP78 （sGRP78）作为一种调控细胞信号的共受体正在出现。为了了解GRP78如何调节致癌信号，我们发现内质网应激不仅上调GRP78，而且积极促进GRP78从内质网向细胞表面的重新定位，这一过程受KDEL检索机制的调节。利用GRP78在癌细胞中而非正常器官中的细胞表面定位，我们筛选并鉴定了一种特异性结合GRP78细胞表面，诱导癌细胞凋亡并抑制肿瘤生长的先导单克隆抗体（MAb159）。本研究假设sGRP78通过其调节PI3K/AKT信号通路的能力是肿瘤生长和治疗耐药的主要影响因子，并且靶向sGRP78代表了一种新的有效的抗PI3K治疗方法，可以抑制肿瘤生长和减轻耐药。在Aim 1中，我们通过分析GRP78表面定位所需的功能域和ER检索机制的完整性，结合GRP78动员的活细胞成像，试图了解允许GRP78从内质网逃逸到细胞表面的潜在分子机制。在Aim 2中，我们将通过研究sGRP78与PI3K通路组分的功能和物理相互作用，以及靶向sGRP78对其他致癌通路的影响，来确定sGRP78如何调节PI3K/AKT信号。在Aim 3中，我们将直接测试sGRP78在自发性小鼠癌症模型和使用耐药人类癌细胞系的异种移植模型中的肿瘤发生和治疗耐药性中的作用。作为我们前列腺癌研究的逻辑延伸，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阉割后癌症的发展，进展和复发。从该模型中获得的去势敏感细胞和抗去势细胞也将进行研究。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，这项工作不仅解决了基本机制，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.