Targeting GRP78 for p53 activation as anti-cancer therapy

描述（由申请人提供）：尽管在过去的30年里，制药行业取得了许多重大的科学突破和巨大的研发投资，但癌症仍然是疾病相关死亡率的主要原因，并有待进一步改进治疗。人们一直在集中精力研究抑癌基因p53通路，因为几乎所有的癌症都显示出这一通路的缺陷，其中超过50%的癌症在TP 53基因本身存在突变。我们最近发现Inauhzin（INZ）作为一种新的非遗传毒性p53激活剂。INZ通过靶向SIRT 1激活p53，从而抑制肿瘤生长。在我们正在进行的探索这种化合物的其他可能靶点的研究中，我们确定了一种78 kDa的葡萄糖调节蛋白（GRP 78，也称为BiP）作为INZ靶点候选者之一。GRP 78在传统上被认为是内质网（ER）的主要分子伴侣和未折叠蛋白反应（UPR）的主要调节因子，其促进蛋白质折叠和组装、蛋白质质量控制和调节ER应激（ERS）信号传导。使用小鼠模型和细胞方法的最新进展表明，GRP 78也在ER之外发挥作用。这一发现不仅对于更好地理解GRP 78在癌症发展和预防中的独特和重要作用至关重要，而且还为癌症特异性靶向提供了机会。有趣且令人惊讶的是，我们还发现INZ可以触发GRP 78的核转位，GRP 78与癌细胞核中积累的p53共定位。我们的发现表明，INZ可能通过不同于ERS-UPR途径的机制调节GRP 78。因此，本申请的目的是验证GRP 78作为INZ的另一个真正的靶标，并阐明GRP 78在INZ诱导的p53活化和抗癌活性中的作用机制。我们的中心假设是GRP 78是INZ的直接肿瘤特异性靶点，导致癌症细胞中p53信号通路的ER非依赖性激活，而不是正常细胞，临床使用的ERS/UPR相关调节剂与p53激活剂INZ的组合可能是克服化疗耐药性和消除肿瘤的成功方法。我们将通过以下具体目的来验证这一假设：（1）在体外和细胞中，从生物化学和生物药理学上表征INZ与GRP 78的特异性结合;（2）确定细胞表面GRP 78在INZ摄取和特异性癌症靶向中的作用，确定GRP 78如何通过INZ从ER输入到细胞核以介导p53活化，并评价INZ与临床使用的ERS/UPR相关调节剂的协同作用。如果我们的探索性研究证明GRP 78通过ER非依赖性p53激活作为INZ的癌症特异性靶点，这将不仅为靶向ER应激/UPR相关和p53信号传导途径用于癌症治疗提供概念验证证据，而且还将为GRP 78在癌症发展和治疗中的作用提供新的见解，并对分子领域产生重大影响。特异性抗癌药物的发现，特别是在转化癌症研究领域。

DESCRIPTION (provided by applicant): Despite numerous major scientific breakthroughs and tremendous R&D investments in the pharmaceutical sector over the past 3 decades, cancer still is the leading cause of disease-related mortality and awaits further advances toward improved therapy. Intensive effort has been focusing on the tumor suppressor p53 pathway because nearly all cancers show defects in this pathway, over 50% of which have mutations in the TP53 gene itself. We recently discovered Inauhzin (INZ) as a novel non-genotoxic p53 activator. INZ activates p53 by targeting SIRT1 and consequently suppresses tumor growth. In our ongoing studies to explore other possible targets of this compound, we identified a 78-kDa glucose-regulated protein (GRP78, also called BiP) as one of the top INZ target candidates. GRP78 is traditionally regarded as a major endoplasmic reticulum (ER) chaperone and a master regulator of the unfolded protein response (UPR) facilitating protein folding and assembly, protein quality control and regulating ER stress (ERS) signaling. Recent advances using mouse models and cellular approaches have revealed that GRP78 also functions beyond the ER. This discovery is not only critical for better understanding the unique and essential role of GRP78 in cancer development and prevention, but also offers an opportunity for cancer-specific targeting. Interestingly and surprisingly, we have also found that INZ can trigger the nuclear translocation of GRP78 that co-localized with accumulated p53 in the nucleus of cancer cells. Our finding suggests that INZ may regulate GRP78 through mechanisms distinct from the ERS-UPR pathway. Hence, the objectives of this application are to validate GRP78 as another bona fide target of INZ and to elucidate the mechanism underlying the role of GRP78 in INZ-induced p53 activation and anticancer activities. Our central hypothesis is that GRP78 is the direct tumor-specific target for INZ, leading to ER-independent activation of the p53 signaling pathway in cancer, but not normal, cells, and the combination of clinically used ERS/UPR-associated modulators with the p53 activator INZ may be a successful approach for overcoming chemoresistance and eliminating tumors. We will test this hypothesis by addressing the following Specific Aims: (1) to biochemically and biophysically characterize the specific binding of INZ to GRP78 in vitro and in cells; (2) to determine the role of cell-surface GRP78 in INZ uptake and specific cancer targeting, to determine how GRP78 is imported from ER to the nucleus by INZ to mediate p53 activation, and to evaluate the synergistic effect of INZ with clinically used ERS/UPR-associated modulators. Should our exploratory studies demonstrate GRP78 as a cancer specific target for INZ through an ER-independent p53 activation, this would not only provide proof-of-concept evidence for targeting both of the ER Stress/UPR related and p53 signaling pathways for cancer therapy, but would also offer new insights into the role of GRP78 in cancer development and therapy and have a strong impact on the area of molecule-specific anti- cancer drug discovery, particularly in the field of translational cancer research.