The Role of TSC2 and Rho GTPases in LAM

描述(申请人提供)：淋巴管肌瘤病(LAM)是一种遗传性疾病，其特征是广泛存在的、可能转移的平滑肌样LAM细胞病变导致肺囊性破坏，目前尚无治疗方法。我们前期对LAM分子机制的研究发现，肿瘤抑制因子结节性硬化症复合体2(TSC2)在LAM中作为哺乳动物雷帕霉素靶标(MTOR)/p70 S6激酶(S6K1)的负调控因子，具有重要的功能。这些临床前发现导致了雷帕霉素在LAM患者中的第二阶段临床试验。在这个项目中，我们建议进一步阐明TSC2缺失导致LAM的下游成分，并探索与雷帕霉素联合治疗LAM的新靶点。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 GTP酶和细胞黏附的协调功能来促进迁移和侵袭。调节LAM细胞迁移的确切机制尚不清楚。我们的初步数据表明，TSC2的缺失导致了TSC1依赖的RhoA的激活和rac1的抑制，整合素的上调，以及细胞迁移和侵袭力的增加，这些都可以被TSC2的重新表达或RhoA活性的抑制所抵消。我们的初步数据还表明，RhoA活性有助于促进LAM细胞的生长，抑制RhoA活性的辛伐他汀与雷帕霉素联合使用具有协同、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 GTP酶调节肌动蛋白细胞骨架和细胞黏附。我们进一步认为，TSC2抑制了细胞的迁移，而TSC2在LAM细胞中的缺失促进了细胞的迁移和侵袭，从而促进了LAM肿瘤的生长。在目标1中，我们将确定与疾病相关的TSC2突变体失调RhoA和rac1 GTP酶活性的机制。在目标2中，我们将确定TSC2和Rho GTP酶是否调节整合素的表达、细胞的黏附、迁移和侵袭，并确定介导这些作用的效应途径(S)。基于RhoA调节细胞运动和细胞生长的证据，在目标3中，我们将使用裸鼠TSC2缺失细胞的异种移植模型，验证我们的假设，即RhoA与辛伐他汀和雷帕霉素联合抑制将与单独使用两种药物相比，消除雌激素刺激的TSC2缺失肿瘤的生长。总之，这些研究将确定TSC2和Rho GTPase调控LAM细胞黏附、迁移和侵袭性的关键细胞和分子机制；此外，我们的研究将为预防或消除LAM肿瘤生长的联合治疗靶点提供洞察力。项目简介：淋巴管肌瘤病(LAM)是一种致命的肺部疾病，仅针对女性，在育龄期间发病，可由怀孕引发，进展迅速，通常会导致十年内死亡。这种疾病会导致广泛的、异常的平滑肌样细胞增殖，通过形成包囊来入侵和破坏肺组织，最终阻碍空气流动，导致肺衰竭和衰竭。本研究将阐明肿瘤抑制因子结节性硬化症复合体2(TSC2)和Rho家族小GTP酶在LAM中的作用，并将为联合药物干预治疗LAM寻找新的靶点。

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.