Targeting Cancer through Suppressing Stress Induction of GRP78/BiP

侵袭性癌细胞通常上调GRP78的表达，GRP78是主要的内质网(ER)伴侣和关键 调节未折叠蛋白反应(UPR)以增强蛋白质折叠能力和维持内质网 动态平衡，从而促进生存和获得治疗抵抗力。一种高通量药物筛查 揭示了一类被称为强心苷(CGs)的化合物，如地高辛(Dig)，在目前的 用于治疗心力衰竭，作为GRP78应激诱导的新型抑制剂，在广泛的人类癌症中。这个 发现CGs可以抑制GRP78的应激诱导，GRP78对癌症的生存、侵袭和 致癌信号，为CGs的抗肿瘤作用开辟了新的机制。在CG中， 齐墩果苷对α3的亲和力是α1亚基的100倍，耐受性高， 与迪格和其他CG相比是最有效的。在这里，我们建议测试OLN抑制GRP78的有效性 和细胞活性，利用独特的临床前患者来源的乳腺癌和结肠癌模型集 这很好地概括了我们合作团队开发的体内环境。我们假设OLN，演戏 通过Na-K三磷酸腺苷酶的α3亚基，强烈抑制细胞内和细胞内的应激诱导 阻断内质网应激后GRP78的急性翻译形成GRP78的表面形态。OLN还可以优先 抑制对压力下的生存至关重要的其他蛋白质的翻译。因此，内质网的动态平衡 扰动和内质网应激介导的细胞凋亡被触发。这些都是抗肿瘤药物的主要成分。 OLN的发病机制。在本方案中，我们将：1)建立OLN的有效性，并进行比较，挖掘， 抑制患者来源的乳腺循环肿瘤中GRP78的表达、细胞活性、干性和侵袭性 2)建立OLN和Dig降低GRP78表达和活性的效果 患者来源的结直肠癌细胞和加强有机类药物的标准治疗。前景看好的治疗方法 时间表将在体内的PDX模型中得到验证。GRP78的表达水平，α3亚单位的状态 将监测模型系统中Na-K-ATPase和OLN/DIG在血浆和肿瘤中的浓度； 3)研究OLN和DIG抑制GRP78应激诱导的机制及其对GRP78的影响 UPR和细胞凋亡以及GRP78在OLN/CG抗癌作用中的重要性。总而言之， 我们的研究将利用新技术对OLN/DIG在癌症中的疗效提供关键的临床前评估 患者派生的乳腺癌和结肠癌模型系统，以及模拟 用于监测肿瘤缺氧微环境的反应。我们的研究还将探索GRP78的用途 和α-K-ATPase 3亚单位作为对OLN/DIG治疗和药物反应进行分层的生物标志物， 并为在联合或靶向治疗环境中进一步开发CG试剂提供概念验证 抑制GRP78，影响UPR和细胞凋亡。我们的结果将产生深远的影响 也适用于其他依赖GRP78生长、侵袭和耐药的实体癌和血癌。

Aggressive cancer cells often upregulate GRP78, a major endoplasmic reticulum (ER) chaperone and key regulator of the unfolded protein response (UPR) to augment protein folding capacity and maintain ER homeostasis, thereby promoting survival and acquiring therapeutic resistance. A high-throughput drug screen revealed that a class of compounds referred to as cardiac glycosides (CGs) such as digoxin (DIG), in current use to treat heart failure, as novel inhibitors of GRP78 stress induction in a wide range of human cancer. The discovery that CGs can suppress stress induction of GRP78, which is pivotal for cancer survival, invasion and oncogenic signaling, opens up a new mechanism for the antineoplastic action of the CGs. Among the CGs, oleandrin (OLN) with 100-fold higher affinity for the α3 versus the α1 subunit of Na-K ATPase and high tolerability, is most potent compared to DIG and other CGs. Here we propose to test the efficacy of OLN to suppress GRP78 and cell viability, taking advantage of unique sets of preclinical patient-derived breast and colon cancer models that closely recapitulate the in vivo milieu developed by our collaborative team. We hypothesize that OLN, acting through the α3 subunit of the Na-K ATPase, acutely suppresses stress induction of both intracellular and the cell surface form of GRP78 through blocking its acute translation following ER stress. OLN may also preferentially suppress translation of other proteins critical for survival under stress. As a consequence, ER homeostasis is perturbed and ER-stress mediated apoptosis is triggered. These contribute majorly to the antineoplastic mechanism of OLN. In this proposal, we will: 1) establish the efficacy of OLN and for comparison, DIG, in suppressing GRP78 expression, cell viability, stemness and invasion in patient-derived breast circulating tumor cells both in vitro and in vivo; 2) establish the efficacy of OLN and DIG in lowering GRP78 expression and viability of patient-derived colorectal cancer cells and enhancing standard therapy in organoids. Promising treatment schedules will be validated in PDX models in vivo. The GRP78 expression levels, the status of the α3 subunit of Na-K ATPase and the OLN/DIG concentration in the plasma and tumor in the model systems will be monitored; and 3) examine the mechanisms whereby OLN and DIG suppress stress induction of GRP78 and its impact on the UPR and apoptosis as well as the importance of GRP78 in the anti-cancer effect of OLN/CG. In summary, our studies will provide critical preclinical evaluation of the efficacy of OLN/DIG in cancer leveraging novel patient-derived breast and colon cancer model systems, and a high content imaging platform that mimics the hypoxic tumor microenvironment for monitoring the response. Our studies will also explore the utility of GRP78 and α3 subunit of Na-K ATPase as biomarkers for stratifying patients for OLN/DIG therapy and drug response, and provide proof-of-concept for further development of CG agents in combination or targeted therapy settings in suppressing GRP78, impacting the UPR and apoptosis. Our results will have far-reaching impact as they are also applicable to other solid and blood cancers dependent on GRP78 for growth, invasion and resistance.