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，由于诊断时的转移性疾病，其5年生存率仅为15%。更好地了解转移机制将为癌症治疗提供新的靶点，从而提高患者的生存率。肝激酶B1 （LKB1）是导致转移潜能增加的一个分子。LKB1是一种丝氨酸苏氨酸激酶，通过AMP激酶的信号传导参与调节细胞代谢。除了激酶的功能外，LKB1在正常细胞和癌细胞中也是一种已知的细胞极性调节剂。在非小细胞肺癌中，LKB1是第三个最常见的突变基因，在动物模型中，它的缺失导致转移性疾病增加。LKB1丢失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表明，LKB1可能作为不同侵袭机制之间分子转换的调节剂，因为它的丢失导致间质向变形虫转变。这种开关可能通过异常极性信号传导为细胞提供迁移优势，并最终通过肿瘤微环境使lkb1缺失的细胞在运动中增加可塑性。因此，我们将验证LKB1缺失破坏正常细胞极性的中心假设，从而为细胞在微环境中导航时提供侵袭性优势。为了验证这一点，我们将确定：1)LKB1在3-D侵袭过程中调节间充质和变形虫表型之间分子转换的机制；2)LKB1缺失是否会增强肿瘤细胞的可塑性，从而促进微环境的侵袭。在Aim 1中，我们将使用活细胞成像从三维球体实时成像侵袭，以观察和分析侵袭表型，从而研究转移级联中的早期事件。具体来说，我们将确定LKB1的哪个特定区域对其调节间充质和变形虫入侵表型之间的转换至关重要。我们还将分析LKB1缺失是否会通过Rho GTPase活性异常导致细胞极性调节丧失。在Aim 2中，我们将利用我们已经重建的临床相关KrasG12DLkb1fl/fl小鼠模型，研究LKB1丢失对运动和转移的影响。该小鼠模型将允许我们通过离体肺肿瘤切片分析lkb1耗竭是否通过极性信号缺陷导致运动表型可塑性增加，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药物抑制间充质和/或变形虫迁移对lkb1缺失小鼠转移的影响。这项工作将促进我们对LKB1生物学及其通过微环境调节运动的关系的理解。此外，它将为指导肺癌侵袭的基本细胞机制提供深入的见解，最终目标是开发针对LKB1突变患者的治疗方法。

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.