Six2 mediates late-stage metastasis through reversible/irreversible regulation of downstream target genes

项目摘要。绝大多数与乳腺癌相关的死亡不是由原发肿瘤引起的，而是 而是转移。目前的研究主要集中在早期转移的预防上，但自从 肿瘤细胞在诊断时可能已经离开原发肿瘤，抑制这一方面的转移可能 不是抑制转移性负担的最有效方法。因此，重视发展治疗学 抗晚期转移变得至关重要。六种蛋白质是一个转录因子家族，它们不是 不仅对发育至关重要，而且对肿瘤的进展和转移也是至关重要的。SIX2的独特之处在于它特别 影响乳腺癌转移的后期，显示其作为治疗靶点的潜力 减少转移性负担。然而，驱动SIX2介导的转移的分子机制仍然存在 很大程度上是未知的。了解SIX2在这方面的转移中的作用可能有助于深入了解 针对转移负担，特别是针对转移建立的新疗法的开发， 成长和生存。假设：SIX2介导转移的后期阶段，特别是 在次级部位建立肿瘤。SIX2下游调节基因的鉴定 转移可能使更有效的手段来抑制转移进展。目标：通过使用Stage- 特定的体外、体外和体内模型结合全面的生物信息学驱动 候选基因方法和shRNA筛选，我们将探讨SIX2在肿瘤转移后期的作用。 具体目标：1)明确SIX2影响肿瘤转移的晚期(S)。2)确定可逆目标 由SIX2调节，并确定这些下游效应器中的哪些是关键的，并且可能是 治疗靶点，在转移的后期。我们将利用从同一来源分离的细胞系 小鼠乳腺原发肿瘤，代表三阴性乳腺癌，但它们的能力不同 完成转移的后期阶段。66Cl4细胞表达高水平的SIX2，具有高转移性。 4T07细胞表达低水平的SIX2，可以到达肺，但只有当SIX2 介绍。我们已经设计出了带有SIX2基因敲除的66Cl4细胞(66Cl4-Six2KD)，带有SIX2的4T07细胞 过表达(4T07-SIX2)和含诱导型SIX2的4T07细胞(4T07-Six2i)评估转移分期 其中SIX2参与，采用体外、体外和体内转移试验。鉴定可逆的SIX2 目标，我们已经从微阵列数据中生成了候选基因列表，并将结合来自 通过文献、shRNA筛选和全基因组功能相互作用网络分析鉴定出最多 关键的下游效应器，将在我们的体外、体外和体内试验中测试它们的相关性。 最后，我们将对患者肿瘤样本进行免疫组织化学研究，以确定SIX2是否调节 这些靶点在人类乳腺癌的设定中。这项研究将开辟新的途径来探索 抑制晚期转移的治疗，从而更有效地提高患者的存活率。

Project Summary. The vast majority of breast cancer-related deaths are not caused by the primary tumor, but rather metastasis. Current studies mainly focus on the prevention of the early stages of metastasis, but since tumor cells have likely left the primary tumor at the time of diagnosis, inhibiting this aspect of metastasis may not be the most effective way to inhibit metastatic burden. Therefore, an emphasis on developing therapeutics against late-stage metastasis becomes essential. Six proteins are a family of transcription factors that are not only critical for development, but also for tumor progression and metastasis. Six2 is unique in that it specifically affects the later stages of breast cancer metastasis, demonstrating its potential as a therapeutic target to decrease metastatic burden. However, the molecular mechanisms that drive Six2-mediated metastasis remain largely unknown. Understanding the role of Six2 in this aspect of metastasis may provide insight into the development of new therapies targeting metastatic burden, particularly targeting metastatic establishment, outgrowth, and survival. Hypothesis: Six2 mediates the later stages of metastasis, particularly the establishment of tumors at secondary sites. Identification of genes regulated downstream of Six2 that mediate metastasis may enable more effective means to inhibit metastatic progression. Objective: By using stage- specific in vitro, ex vivo and in vivo models in combination with a comprehensive bioinformatics-driven candidate gene approach and shRNA screen, we will examine the role of Six2 in the later stages of metastasis. Specific Aims: 1) Define the later stage(s) of metastasis impacted by Six2. 2) Identify reversible targets regulated by Six2, and determine which of these downstream effectors are critical, and could possibly be therapeutic targets, in the later stages of metastasis. We will utilize cell lines that were isolated from the same mouse mammary primary tumor, representing triple negative breast cancer, but differing in their ability to complete the later-stages of metastasis. The 66cl4 cells express high levels of Six2 and are highly metastatic. The 4T07 cells express low levels of Six2, and can reach the lungs, but can only colonize when Six2 is introduced. We have engineered 66cl4 cells with Six2 knockdown (66cl4-Six2KD), 4T07 cells with Six2 overexpression (4T07-Six2), and 4T07 cells with inducible Six2 (4T07-Six2i) to assess the stage of metastasis in which Six2 is involved, using in vitro, ex vivo, and in vivo metastasis assays. To identify reversible Six2 targets, we have generated candidate gene lists from microarray data and will combine established roles from the literature, shRNA screens and genome-wide functional interaction network analysis to identify the most critical downstream effectors, which will be tested for their relevance in our in vitro, ex vivo, and in vivo assays. Finally, we will perform immunohistochemistry on patient tumor samples to determine whether Six2 regulates these targets in the setting of human breast cancer. This research will open up new avenues to explore for treatments to inhibit the later stages of metastasis, thus more effectively improving patient survival.