Targeting Cancer through Suppressing Stress Induction of GRP78/BiP

侵袭性癌细胞通常上调GRP 78，GRP 78是一种主要的内质网（ER）伴侣， 未折叠蛋白反应（UPR）的调节剂，以增加蛋白质折叠能力并维持ER 体内平衡，从而促进存活和获得治疗抗性。高通量药物筛选 研究表明，目前，一类被称为强心苷（CG）的化合物，例如地高辛（DIG） 用于治疗心力衰竭，作为GRP 78应激诱导的新型抑制剂，用于多种人类癌症。的 发现CG可以抑制GRP 78的应激诱导，GRP 78对癌症存活、侵袭和转移至关重要。 致癌信号，开辟了一个新的机制，为CGs的抑制作用。在CG中， 夹竹桃苷（OLN）对Na-K ATP酶α3亚基的亲和力比α1亚基高100倍，耐受性高， 与DIG和其他CG相比是最有效的。在这里，我们建议测试OLN抑制GRP 78的功效。 和细胞活力，利用独特的临床前患者来源的乳腺癌和结肠癌模型 这与我们的合作团队开发的体内环境密切相关。我们假设OLN， 通过Na-K ATP酶的α3亚基，急性抑制细胞内和细胞内的应激诱导 通过阻断内质网应激后GRP 78的急性翻译，OLN还可以优选地 抑制在压力下生存的其他关键蛋白质的翻译。因此，ER稳态是 扰动和ER-应激介导的细胞凋亡被触发。这主要是由于 OLN机制在本提案中，我们将：1）确定OLN的疗效，并进行比较，DIG， 抑制患者来源的乳腺循环肿瘤中的GRP 78表达、细胞活力、干性和侵袭性 2）确定OLN和DIG在降低GRP 78表达和活力方面的功效 患者来源的结直肠癌细胞和增强类器官的标准治疗。有前途的治疗 将在体内PDX模型中验证时间表。GRP 78表达水平、α3亚基的状态、 将监测模型系统中血浆和肿瘤中的Na-K ATP酶和OLN/DIG浓度; 和3）研究OLN和DIG抑制GRP 78的应激诱导的机制及其对 UPR和细胞凋亡以及GRP 78在OLN/CG抗肿瘤作用中的重要性。总的来说， 我们的研究将为OLN/DIG在癌症中的疗效提供关键的临床前评估， 患者来源的乳腺癌和结肠癌模型系统，以及模拟 低氧肿瘤微环境用于监测反应。我们的研究还将探索GRP 78的效用 和Na-K ATP酶α3亚基作为OLN/DIG治疗和药物反应患者分层的生物标志物， 并为进一步开发联合或靶向治疗环境中的CG试剂提供概念验证 抑制GRP 78，影响UPR和凋亡。我们的成果将产生深远的影响，因为他们是 也适用于依赖GRP 78生长、侵袭和耐药的其他实体和血液肿瘤。

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.