Stress Induction of Glucose Regulated Protein GRP78/BiP

描述（由申请人提供）：最近发现，传统上被认为是主要内质网（ER）伴侣和ER应激信号传导调节剂的GRP 78也可以在病理生理条件如癌症下定位于细胞表面，改变了这种蛋白质如何在癌症中发挥其促增殖和抗凋亡功能的范例。在过去的研究期间，我们确定了GRP 78单倍不足通过抑制肿瘤增殖、血管生成和增加细胞凋亡来抑制乳腺肿瘤的发生。通过建立一种新的突变小鼠模型，该模型具有Grp 78和肿瘤抑制基因Pten的条件性双等位基因缺失，我们发现，不仅GRP 78是前列腺肿瘤发生和血液癌症所必需的，而且在体内和体外两种类型的癌症中PI 3 K/AKT活化也是必需的。细胞表面GRP 78（sGRP 78）作为控制细胞信号传导的共受体出现。在理解GRP 78如何调节致癌信号传导的过程中，我们确定ER应激不仅上调GRP 78，而且还积极促进GRP 78从ER重新定位到细胞表面，并且该过程由KDEL检索机制调节。利用GRP 78在癌细胞中而不是在正常器官中的细胞表面定位，我们筛选并鉴定了特异性结合细胞表面GRP 78的先导单克隆抗体（MAb 159），其诱导癌细胞凋亡并抑制肿瘤生长。在这里，我们假设sGRP 78是通过其调节PI 3 K/AKT信号传导通路的能力来调节肿瘤生长和治疗抗性的主要效应物，并且靶向sGRP 78代表了用于抗PI 3 K治疗的新颖且强大的方法，其将抑制肿瘤生长并减轻药物抗性。在目标1中，我们试图了解潜在的分子机制，使GRP 78从ER逃逸到细胞表面，通过分析GRP 78的功能结构域所需的表面定位和ER检索机制的完整性，再加上活细胞成像GRP 78动员。在目标2中，我们将通过研究sGRP 78与PI 3 K通路组分的功能和物理相互作用以及靶向sGRP 78对其他致癌通路的影响来确定sGRP 78如何调节PI 3 K/AKT信号传导。在目标3中，我们将使用对治疗有抗性的人癌细胞系直接测试sGRP 78在自发性小鼠癌症模型和异种移植模型中的肿瘤发生和治疗抗性中的作用。作为我们对前列腺癌工作的逻辑延伸，我们将利用一种新的Pten无效前列腺癌模型，该模型允许生物发光监测去势后癌症的发展、进展和复发。还将研究源自该模型的去势敏感性和抗性细胞。MAb 159的疗效和安全性将在其他癌症模型中单独或联合治疗中进行测试。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.