Molecular Dissection of an Arntl2 induced pro-metastatic secretome

 描述(申请人提供)：肺癌是美国男性和女性癌症死亡的主要原因，仅在这个国家每年就有超过155,000名患者死亡。肺癌患者预后不佳的原因有很多，但与大多数实体瘤一样，癌细胞离开原发肿瘤并建立不可手术转移的能力是成功治疗的主要障碍。因此，转移是一个主要的临床挑战，其驱动因素是目前尚不清楚的细胞状态改变。通过对小鼠转移性肺腺癌和人肺腺癌模型的整合素基因表达分析，我们确定转录因子Arntl2是肺腺癌转移能力的关键驱动因素。Arntl2似乎通过控制一个复杂的促转移分泌体的表达来驱动转移的适合性，该分泌体具有极大地提高克隆生长潜力的能力。在Aim1中，我们将从功能上询问Arntl2在人和小鼠肺腺癌细胞系中的功能。我们将进行功能获得和功能丧失的实验，并全面评估Arntl2驱动的细胞表型。移植试验和体内转移部位的初始黏附、增殖和细胞死亡的量化将阐明Arntl2高表达的细胞后果。在AIM2中，我们将研究哪些Arntl2调节的分泌因子协同驱动克隆生长和转移能力。我们将结合细胞培养中重组蛋白的筛选，细胞培养中和体内的功能获得和丧失实验，以及关键的促转移分泌蛋白下游的治疗靶向通路，以更好地了解自分泌转移利基因子的重要性。在AIM3中，我们将使用CRISPR/Cas9介导的基因组编辑和慢病毒介导的cDNA表达的新方法来测试Arntl2和Arntl2调控的基因在促进人肺癌原发小鼠模型转移级联反应中的必要性和充分性。鉴于转移性癌症的巨大临床影响以及目前在了解这种疾病状态的分子基础方面的差距，临床实践和患者结果都将受到任何新疗法的极大影响，这些新疗法可能来自我们拟议的分析所获得的基本知识。通过结合定量方法和强大的体内方法，我们希望揭示控制肿瘤进展和转移扩散的一般原理，并最终揭示跨越癌症进展连续体的新治疗靶点。

 DESCRIPTION (provided by applicant): Lung cancer is the leading cause of cancer deaths in both men and women in the United States, with over 155,000 patients dying each year in this country alone. Several factors contribute to the poor outcome of lung cancer patients, but, as in most solid tumors, the ability of cancer cells to leave the primary tumor and establish inoperable metastases is a major impediment to successful therapy. Metastasis thus represents a major clinical challenge that is driven by as yet poorly understood cell state alterations. By integratin gene expression analyses on murine models of metastatic lung adenocarcinoma and human lung adenocarcinomas we identified the transcription factor Arntl2 as a key driver of lung adenocarcinoma metastatic ability. Arntl2 appears to drive metastatic fitness by controlling the expression of a complex pro-metastatic secretome that has the ability to greatly increase clonal growth potential. In Aim1, we will functionally interrogate Arntl2 function in human and mouse lung adenocarcinoma cell lines. We will perform gain- and loss-of-function experiments and fully assess the cellular phenotypes driven by Arntl2. Transplantation assays and quantification of initial adhesion, proliferation, and cell death within the metastatic site in vivo will elucidate te cellular consequence of high Arntl2 expression. In Aim2, we will investigate which Arntl2-regulated secreted factors cooperate to drive clonal growth and metastatic ability. We will integrate screening of recombinant proteins in cell culture, gain- and loss-of-function experiments in cell culture and in vivo, and therapeutically target pathways downstream of key pro-metastatic secreted proteins to better understand the importance of autocrine metastatic niche factors. In Aim3, we will use novel methods for CRISPR/Cas9-mediated genome editing and lentiviral-mediated cDNA expression to test the requirement and sufficiency of Arntl2 and Arntl2- regulated genes to promote step of the metastatic cascade in autochthonous mouse models of human lung cancer. Given the immense clinical impact of metastatic cancer and the current gap in understanding the molecular underpinnings of this disease state, both clinical practice and patient outcome would be greatly impacted by any new therapies that might result from the fundamental knowledge gained from our proposed analyses. By combining quantitative methods and powerful in vivo methods, we hope to uncover general principles that govern tumor progression and metastatic spread and ultimately reveal novel therapeutic targets across the continuum of cancer progression.