Notch-1 and IGF-1 crosstalk: new therapeutic strategies for NSCLC

描述（由申请人提供）：肺癌是美国癌症相关死亡的主要原因。最常见的肺癌类型是非小细胞肺癌（NSCLC）。在NSCLC的三种主要组织学类型中，以肺腺癌（adenocarmicofthe lung， ACL）最为常见，且其发病率呈稳步上升趋势。ACL在从不吸烟的人群中相对常见（占病例的15%至20%），因此即使在根除吸烟后ACL仍将是一个主要的健康问题。ACL通常在晚期才被诊断出来，因为早期检测方法仍然存在问题。晚期ACL诊断后的中位生存期仅为5个月，化疗仅能略微改善其生存期。因此，迫切需要寻找新的治疗策略来治疗这种致命疾病。我们的初步数据表明，进化上保守的Notch-1受体在缺氧条件下为ACL细胞提供关键的生存信号，这是最能概括ACL微环境的条件。低氧条件是指那些干扰标准化疗并有利于保存肿瘤干细胞龛的条件。因此，靶向有利于癌细胞在缺氧条件下存活的信号通路是治疗ACL的一种特别有前途的治疗策略。为了激活Notch-1， Notch-1需要许多蛋白水解裂解，包括β -分泌酶的参与。由于这种蛋白质也参与阿尔茨海默病中-淀粉样蛋白的积累，因此人们花了很大的努力来合成抑制-淀粉样蛋白的特定小分子。分泌酶(?-分泌酶抑制剂（GSI）。因此，GSI是Notch信号的间接抑制剂，因为在缺乏由？-分泌酶，Notch-1（以及其他Notch受体）不能在细胞核内转位并影响转录。我们使用默克公司（MRK-003）开发的化合物作为GSI。这种化合物被证明在缺氧条件下对ACL细胞的杀伤非常有效。我们的假设是Notch-1向ACL细胞提供生存信号，介导包括PTEN、STAT、IGF-1、IGF-1R、Akt及其下游靶点在内的复杂信号通路网络。我们也假设在体内靶向Notch信号是治疗ACL的有效治疗策略。我们希望根据以下具体目的来验证我们的假设：(1)验证Notch-1与IGF-1信号通路之间是否存在相互作用和正反馈回路，以及这种相互作用在缺氧中的生物学后果。(2)研究缺氧增强Notch-1信号通路的分子机制，以及Notch-1在缺氧条件下特异性保护ACL细胞凋亡的机制。我们将重点关注PTEN和PI3激酶/PDK-1/Akt/mTOR轴的Notch-1调控。(3)在晚期ACL小鼠临床前模型中验证MRK-003单独或联合顺铂、IGF-1R抑制抗体（MK-0646）或Akt特异性抑制剂（MK-2206）的治疗效果。我们将使用顺铂联合MRK-003同时靶向非缺氧和缺氧肿瘤区域。MK-0646和MK-2206也将在单独的实验中使用，因为这些化合物可能与MRK-003具有协同作用，因为我们的初步结果支持目标1和2。我们将利用ACL细胞系来剖析Notch抑制的机制作用，在这些细胞系中我们可以人为下调Notch-1。Aim 1和Aim 2中获得的结果将在小鼠模型和人类临床样本中进行体内验证。本文提出的研究结果将有助于更好地理解导致ACL恶性表型维持的分子过程。此外，该研究将测试靶向Notch信号在使用GSI治疗非小细胞肺癌中的临床有效性。最后，这些实验将阐明Notch-1在体外和体内肺致瘤性中的作用。公共卫生相关性：肺癌是美国癌症相关死亡的最常见原因。本文提出的研究将使人们更好地了解肺癌生物学，并将测试可迅速引入临床的新治疗策略。

DESCRIPTION (provided by applicant): Lung cancer is the leading cause of cancer related deaths in the USA. The most frequent form of lung cancer is non small cell lung cancer (NSCLC). Among the three major histological types of NSCLC, adenocarcinoma of the lung (ACL) is the most prevalent, and its incidence is steadily raising. ACL is relatively common (from 15 to 20% of cases) in people who have never smoked, therefore ACL will remain a major health problem even after cigarette smoking eradication. ACL is normally diagnosed at advanced stages because early detection methods remain problematic. Median survival after advanced ACL diagnosis is only 5 months, and chemotherapy only slightly improves it. It is therefore imperative to find novel therapeutic strategies to treat this deadly disease. Our preliminary data indicate that the evolutionarily conserved Notch-1 receptor provides critical survival signals to ACL cells in hypoxia, a condition that best recapitulates ACL microenvironment. Hypoxic conditions are those that interfere with standard chemotherapy and favor the preservation of niches for cancer stem cells. Therefore, targeting a signaling pathway that favor survival of cancer cells in hypoxia represents a particularly promising therapeutic strategy for the treatment of ACL. To be activated, Notch-1 requires a number of proteolitic cleavages, including the participation of a ?-secretase. Since this protein also participates in the accumulation of ¿-amyloid in Alzheimer's Disease, a great effort has been spent to synthesize specific small molecules that inhibit ?-secretase (?-secretase inhibitors or GSI). Therefore, GSI are indirect inhibitors of Notch signaling because, in the absence of the activating proteolitic cleavage catalyzed by ?-secretase, Notch-1 (as well as other Notch receptors) cannot translocate in the nucleus and affect transcription. We used as GSI a compound developed by Merck (MRK-003). This compound proved very efficient in killing ACL cells specifically under hypoxia. Our hypothesis is that Notch-1 provides survival signals to ACL cells mediating an intricate network of signaling pathways that includes PTEN, STAT, IGF-1, IGF-1R, Akt and its downstream targets. We also hypothesize that targeting Notch signaling in vivo is a valid therapeutic strategy for the treatment of ACL. We want to verify our hypotheses according to the following Specific Aims: (1) Verify whether there is interplay and a positive feedback loop between Notch-1 and the IGF-1 signaling pathway and what are the biological consequences of such interaction in hypoxia. (2) Study the molecular mechanisms through which hypoxia strengthen Notch-1 signaling and how Notch-1 protects ACL cells from apoptosis specifically under hypoxia. We will focus on Notch-1 regulation of PTEN and the PI3 kinase/PDK-1/Akt/mTOR axis. (3) Verify the therapeutic efficacy of MRK-003, alone or in combination with cisplatin, or with an inhibitory antibody for the IGF-1R (MK-0646), or with an Akt specific inhibitor (MK-2206) in a mouse preclinical model of advanced ACL. We will use cisplatin in combination with MRK-003 to simultaneously target non-hypoxic and hypoxic tumor areas. MK-0646 and MK-2206 will be also used in separate experiments because these compounds may have synergistic effects with MRK-003 because of our preliminary results sustaining Aims 1 and 2. We will dissect the contribution of Notch inhibition mechanistically using ACL cell lines in which we can artificially downregulate Notch-1. The results obtained in Aim 1 and 2 will be verified in vivo in the mouse model and in human clinical samples. The outcome of the research proposed here will provide a better understanding of the molecular processes that contribute to the maintenance of the malignant phenotype in ACL. Furthermore, the study will test the clinical usefulness of targeting Notch signaling to treat NSCLC using GSI. Finally, these experiments will elucidate the role of Notch-1 in lung tumorigenicity in vitro and in vivo. PUBLIC HEALTH RELEVANCE: Lung cancer is the most frequent cause of cancer related death in the U.S.A. The studies proposed here will allow a better understanding of lung cancer biology and will test novel therapeutic strategies that could be introduced in the clinic rapidly.