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，其特征为平滑肌样LAM细胞的广泛、潜在转移性病变，促进肺囊性破坏，目前尚无治疗方法。我们先前在阐明LAM的分子机制方面的研究导致鉴定肿瘤抑制剂结节性硬化症复合物2（TSC 2）作为LAM中的哺乳动物雷帕霉素靶蛋白（mTOR）/p70 S6激酶（S6 K1）的负调节剂的基本功能。这些临床前发现导致了针对LAM患者的2期雷帕霉素临床试验。在这个项目中，我们建议进一步阐明由TSC 2损失导致LAM的下游组分，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细胞转移扩散到不同的器官，这一过程需要Rho GTP酶和细胞粘附的协调功能，以促进迁移和侵袭。调节LAM细胞迁移的确切机制仍然未知。我们的初步数据表明，TSC 2的损失导致TSC 1依赖性激活RhoA和抑制Rac 1，上调整合素，并增加细胞迁移和侵袭力，这是由TSC 2重新表达或抑制RhoA活性废除。我们的初步数据还表明，RhoA活性有助于增加LAM细胞生长，抑制RhoA活性的辛伐他汀与雷帕霉素联合使用时具有协同、抗增殖和抗肿瘤活性。基于这一证据，我们推测，TSC 2调节肌动蛋白细胞骨架和细胞粘附通过TSC 1介导的调节Rac 1和RhoA GTP酶。我们进一步提出，TSC 2抑制细胞迁移，LAM细胞中TSC 2的缺失促进细胞迁移和侵袭，从而促进LAM肿瘤生长。在目标1中，我们将确定疾病相关的TSC 2突变体的RhoA和Rac 1 GTP酶的活性失调的机制。在目标2中，我们将确定TSC 2和Rho GTP酶是否调节整合素表达、细胞粘附、迁移和侵袭，并鉴定介导这些效应的效应子途径。基于RhoA调节细胞运动和细胞生长的证据，在目标3中，我们将通过使用无胸腺裸鼠中大鼠TSC 2-null细胞的异种移植模型，与单独使用每种药物的作用相比，检测我们的假设，即RhoA与辛伐他汀和雷帕霉素的联合抑制将消除雌激素刺激的TSC 2-null肿瘤的生长。总的来说，这些研究将定义TSC 2和Rho GT3调节LAM细胞粘附、迁移和侵袭的关键细胞和分子机制;此外，我们的研究将提供对可能预防或消除LAM中肿瘤生长的组合治疗靶点的深入了解。项目叙述：淋巴管平滑肌瘤病（LAM）是一种致命的肺部疾病，仅针对女性，在生育年龄期间将其击倒，并可由怀孕引发，进展迅速，通常在十年内导致死亡。这种疾病导致广泛的、异常的平滑肌样细胞增殖，其通过形成囊肿侵入并破坏肺组织，最终阻碍空气流动并导致肺萎陷和衰竭。本研究将阐明肿瘤抑制剂结节性硬化症复合物2（TSC 2）和Rho家族的小GTP酶在LAM中的作用，并将确定新的组合药物干预治疗这种疾病的靶点。

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.