Defining an LKB1-regulated motility switch in cancer cell invasion and metastasis

描述（由申请人提供）：非小细胞肺癌（NSCLC）是最常见的肺癌类型，由于诊断时发生转移，5年生存率仅为15%。更好地理解转移的机制将通过为癌症治疗提供新的靶点来提高患者的生存率。一种与导致转移潜能增加有关的分子是肝激酶B1（LKB 1）。LKB 1是一种丝氨酸/苏氨酸激酶，通过AMP激酶信号传导参与调节细胞代谢。独立于激酶功能，LKB 1也是正常和癌细胞中已知的细胞极性调节剂。在NSCLC中，LKB 1是第三种最常见的突变基因，其缺失导致动物模型中转移性疾病增加。LKB 1丢失如何影响癌细胞迁移和转移的机制在很大程度上是未知的。我们使用3-D肺癌球体的初步数据显示，LKB 1可能作为不同侵袭机制之间的分子开关的调节剂，因为它的丢失导致间充质向变形虫转变。这种开关可以通过异常极性信号传导为细胞提供迁移优势，并最终为LKB 1耗尽的细胞提供通过肿瘤微环境的运动性的可塑性增加。因此，我们将测试中心假设，即LKB 1丢失破坏正常细胞极性，从而为细胞提供侵入性优势，同时导航微环境。为了验证这一点，我们将确定：1）LKB 1在3-D侵袭中调节间充质和变形虫表型之间的分子开关的机制，以及2）LKB 1缺失是否增强肿瘤细胞的可塑性以促进通过微环境的侵袭。在目标1中，我们将使用活细胞成像来在真实的时间内从3-D球体成像侵袭，以观察和分析侵袭表型，从而允许研究转移级联中的早期事件。具体而言，我们将确定LKB 1的哪个特定区域对于其调节间充质和变形虫侵袭表型之间的转换至关重要。我们还将分析LKB 1的缺失是否会通过异常的Rho GT3活性导致细胞极性调控的丧失。在目标2中，我们将利用我们已经重建的临床相关KrasG 12 DLkb 1fl/fl小鼠模型来研究LKB 1缺失对运动性和转移的影响。这种小鼠模型将允许我们通过离体肺肿瘤切片分析LKB 1缺失是否通过缺陷极性信号传导导致运动表型的可塑性增加，从而在导航微环境时提供侵入性优势。此外，我们将使用这些小鼠来确定间充质和/或变形虫迁移的药理学抑制对LKB 1缺失小鼠转移的影响。这项工作将促进我们对LKB 1生物学及其与通过微环境调节运动的关系的理解。此外，它将提供对指导肺癌侵袭的基本细胞机制的深入了解，最终目标是为LKB 1突变患者开发治疗方法。

DESCRIPTION (provided by applicant): Non-small cell lung carcinoma (NSCLC) is the most prevalent type of lung cancer and has a poor 5-year survival rate of only 15% due to metastatic disease at diagnosis. Better understanding of the mechanisms of metastasis will increase patient survival by providing novel targets for cancer therapeutics. One molecule that has been implicated in leading to an increase in metastatic potential is liver kinase B1 (LKB1). LKB1 is a serine threonine kinase involved in regulating cell metabolism via signaling through AMP kinase. Independently of the kinase function, LKB1 also is a known cell polarity regulator in normal and cancerous cells. In NSCLC, LKB1 is the third most commonly mutated gene, and its loss causes increased metastatic disease in animal models. The mechanism for how LKB1 loss impacts cancer cell migration and metastasis is largely unknown. Our preliminary data using 3-D lung cancer spheroids show that LKB1 may serve as a regulator of a molecular switch between differing invasion mechanisms, as its loss results in a mesenchymal to amoeboid transition. This switch may provide a migratory advantage to cells through aberrant polarity signaling and ultimately provide LKB1-depleted cells increased plasticity in motility through the tumor microenvironment. Therefore, we will test the central hypothesis that LKB1 loss disrupts normal cell polarity and thereby provides cells an invasive advantage while navigating the microenvironment. To test this, we will determine: 1) the mechanism by which LKB1 regulates a molecular switch between mesenchymal and amoeboid phenotypes in 3-D invasion and 2) if LKB1 loss enhances tumor cell plasticity to facilitate invasion through the microenvironment. In Aim 1, we will use live cell imaging to image invasion from a 3-D spheroid in real time to observe and analyze invasive phenotype, allowing for study of early events in the metastatic cascade. Specifically, we will determine which specific region(s) of LKB1 is vital for its regulatin of the switch between mesenchymal and amoeboid invasion phenotypes. We will also analyze if LKB1 loss results in a loss of cell polarity regulation through aberrant Rho GTPase activity. In Aim 2, we will utilize our clinically relevant KrasG12DLkb1fl/fl mouse model, which we have already re-created, to study the impact of LKB1 loss on motility and metastasis. This mouse model which will allow us to analyze via ex vivo lung tumor slices if LKB1-depletion results in increased plasticity of motility phenotype through defective polarity signaling, thus providing an invasive advantage when navigating the microenvironment. Additionally, we will use these mice to determine the impact of pharmacological inhibition of mesenchymal and/or amoeboid migration on metastasis in LKB1-depleted mice. This work will advance our understanding of LKB1 biology and its relationship to regulating motility through the microenvironment. Moreover, it will provide insight into the basic cellular mechanisms guiding lung cancer invasion with the ultimate goal of developing therapeutics for LKB1 mutant patients.