The Role of TSC2 and Rho GTPases in LAM

描述（由申请人提供）：淋巴管平滑肌瘤病（LAM）是一种遗传性疾病，其特征是平滑肌样LAM细胞广泛存在潜在转移性病变，促进肺部囊性破坏，目前尚无治疗方法。我们之前在阐明LAM分子机制的研究中，发现肿瘤抑制因子结节性硬化症复合体2 （TSC2）在LAM中作为雷帕霉素(mTOR)/p70 S6激酶（S6K1）的哺乳动物靶点的负调节因子的基本功能。这些临床前发现导致了针对LAM患者的雷帕霉素2期临床试验。在本项目中，我们建议进一步阐明由TSC2缺失导致LAM的下游成分失调，并探索与雷帕霉素联合治疗LAM的新治疗靶点。目前的证据表明，LAM细胞转移到不同的器官，这一过程需要Rho GTPases和细胞粘附的协调作用来促进迁移和侵袭。调控LAM细胞迁移的确切机制尚不清楚。我们的初步数据表明，TSC2的缺失导致tsc1依赖性的RhoA激活和Rac1的抑制，整合素的上调，以及细胞迁移和侵袭性的增加，这些都被TSC2的重新表达或RhoA活性的抑制所消除。我们的初步数据还表明，RhoA活性有助于增加LAM细胞的生长，而抑制RhoA活性的辛伐他汀与雷帕霉素联用时具有协同、抗增殖和抗肿瘤活性。基于这一证据，我们假设TSC2通过tsc1介导的Rac1和RhoA gtpase的调节来调节肌动蛋白细胞骨架和细胞粘附。我们进一步提出，TSC2抑制细胞迁移，LAM细胞中TSC2的缺失促进细胞迁移和侵袭性，从而促进LAM肿瘤的生长。在Aim 1中，我们将确定疾病相关的TSC2突变体失调RhoA和Rac1 gtpase活性的机制。在Aim 2中，我们将确定TSC2和Rho GTPases是否调节整合素表达、细胞粘附、迁移和侵袭性，并确定介导这些影响的效应通路。基于RhoA调节细胞运动和细胞生长的证据，在Aim 3中，我们将通过在胸腺裸鼠中使用大鼠tsc2缺失细胞的异种图模型，验证我们的假设，即与单独使用每种药物相比，联合抑制RhoA与辛伐他汀和雷帕霉素将消除雌激素刺激的tsc2缺失肿瘤的生长。总的来说，这些研究将确定TSC2和Rho GTPase调节LAM细胞粘附、迁移和侵袭的关键细胞和分子机制；此外，我们的研究将为可能阻止或消除LAM肿瘤生长的联合治疗靶点提供见解。项目描述：淋巴管平滑肌瘤病（LAM）是一种致命的肺部疾病，仅针对女性，在育龄期发作，可由怀孕引发，进展迅速，通常在十年内导致死亡。这种疾病引起广泛的、异常的平滑肌样细胞增殖，通过形成囊肿侵入并破坏肺组织，最终阻碍空气流通，导致肺衰竭和衰竭。本研究将阐明肿瘤抑制性结节性硬化症复合体2 （TSC2）和小gtpase Rho家族在LAM中的作用，并将确定联合药物干预治疗该疾病的新靶点。

DESCRIPTION (provided by applicant): Lymphangioleiomyomatosis (LAM) is a genetic disorder characterized by widespread, potentially metastatic lesions of smooth muscle-like LAM cells promoting cystic destruction of the lung for which there is no therapy. Our previous studies in elucidating the molecular mechanism of LAM led to the identification of essential function of the tumor suppressor tuberous sclerosis complex 2 (TSC2) as a negative regulator of mammalian target of rapamycin (mTOR)/p70 S6 kinase (S6K1) in LAM. These pre-clinical findings led to a Phase 2 rapamycin clinical trial for LAM patients. In this project we propose to further elucidate the downstream components deregulated by the TSC2 loss that contribute to LAM and to explore a new therapeutic target for combination therapy with rapamycin in LAM. Current evidence suggests that LAM cells spread metastatically to distinct organs, a process requiring the coordinated functions of Rho GTPases and cell adhesion to promote migration and invasiveness. The precise mechanism regulating LAM cell migration remains unknown. Our preliminary data demonstrate that loss of TSC2 results in TSC1-dependent activation of RhoA and inhibition of Rac1, the upregulation of integrins, and increased cell migration and invasiveness, which are abolished by TSC2 re-expression or the inhibition of RhoA activity. Our preliminary data also show that RhoA activity contributes to increased LAM cell growth, and simvastatin, which inhibits RhoA activity, has synergistic, anti-proliferative and anti-tumor activities when combined with rapamycin. Based on this evidence, we hypothesize that TSC2 regulates actin cytoskeleton and cell adhesion via TSC1-mediated regulation of Rac1 and RhoA GTPases. We further propose that TSC2 suppresses cell migration and that loss of TSC2 in LAM cells promotes cell migration and invasiveness that contributes to LAM tumor growth. In Aim 1, we will determine the mechanism by which disease-associated TSC2 mutants dysregulate the activity of RhoA and Rac1 GTPases. In Aim 2, we will determine whether TSC2 and Rho GTPases modulate integrin expression, cell adhesion, migration and invasiveness, and identify the effector pathway(s) mediating these effects. Based on evidence that RhoA regulates cell motility and cell growth, in Aim 3, we will test our hypotheses that the combined inhibition of RhoA with simvastatin and rapamycin will abrogate estrogen-stimulated growth of TSC2-null tumors compared to the effects of each agent alone by using a xenographic model of rat TSC2-null cells in athymic nude mouse. Collectively, these studies will define the key cellular and molecular mechanisms by which TSC2 and Rho GTPase regulate LAM cell adhesion, migration, and invasiveness; moreover, our studies will provide insight into the combinational therapeutic targets that may prevent or abrogate tumor growth in LAM. PROJECT NARRATIVE: Lymphangioleiomyomatosis (LAM) is a deadly lung disease that targets only women, striking them down during their childbearing years and can be triggered by pregnancy, progresses rapidly, and often results in death within ten years. The disease causes extensive, abnormal smooth muscle-like cell proliferation, which invades and destroys the tissues of the lung by forming cysts, eventually obstructing the flow of air and leading to lung collapse and failure. This study will elucidate the role of tumor suppressor tuberous sclerosis complex 2 (TSC2) and Rho family of small GTPases in LAM and will identify novel targets for combinational pharmaceutical intervention to treat this disease.