Mechanism and role of DLC-1 tumor suppressor loss in lung cancer

描述（由申请人提供）：DLC-1在肺癌和其他癌症中表达缺失，DLC-1的异位再表达会损害DLC-1缺陷肿瘤细胞系的转化和致瘤性生长。因此，DLC-1表现出肿瘤抑制因子的特性。DLC-1编码GTPase激活蛋白（GAP）和Ras同源（Rho）小GTPase的负调节因子。Rho GTPases的组成性激活导致肿瘤细胞的生长转化，促进肿瘤细胞的侵袭、转移和血管生成。因此，我们假设DLC-1功能的丧失可能导致Rho GTPase持续激活并促进NSCLC的癌变。然而，除了RhoGAP催化结构域外，DLC-1还含有START脂质结合和SAM蛋白-蛋白相互作用结构域。我们最近的研究结果表明，DLC-1通过RhoGAP依赖性和非依赖性机制抑制NSCLC的生长。除了RhoA，我们还确定RhoGAP结构域调节生物学上不同的RhoB和RhoC亚型，以及Cdc42，为我们建立被DLC-1灭活的Rho GTPases的完整库（Aim 1）提供了基础。我们还发现SAM结构域是调节DLC-1 RhoGAP活性的自抑制结构域。该结构域如何调节DLC-1活性，以及MEK-ERK-RSK蛋白激酶和PI3K-AKT脂质激酶信号通路介导的DLC-1磷酸化是否调节DLC-1活性将被确定（Aim 3）。DLC-1与局灶粘连（FAs）相关，这种关联对DLC-1肿瘤抑制至关重要，但令人惊讶的是，对体内Rho GTPase失活却没有作用。最近已经确定DLC-1与FAs的关联是通过与紧张素蛋白结合介导的。我们还确定SAM和START结构域也可能调节DLC-1亚细胞定位，而不是与局灶性粘附相关。我们发现DLC-1的异位表达在体外使迁移细胞前沿的RhoA失活，抑制肿瘤细胞的侵袭。这些观察结果为我们确定空间限制性dlc -1介导的Rho GTPase失活对抑制肿瘤生长的重要性的研究提供了理论基础（目的3）。这些研究将确定SAM、START和fa靶向的DLC-1是否优先失活对肿瘤抑制重要的特定亚细胞区室中的Rho gtpase。最后，为了补充我们显示DLC-1抑制肿瘤生长的异位再表达研究，我们提出了干扰RNA和显性阴性DLC-1研究，以确定DLC-1表达缺失的生物学后果，并评估肺肿瘤组织微阵列，以确定DLC-1蛋白表达缺失是否与非小细胞肺癌的特定遗传特性和临床结果相关（目的4）。公共卫生相关性：肺癌仍然是美国男性（31%）和女性（28%）中最常见的致命癌症，非小细胞肺癌占所有肺癌病例的80%，是癌症死亡的主要原因（http://www.cancer.org/）。最近的统计数据发现，肺癌死亡率的增长速度比过去慢得多。不幸的是，这种适度的改善主要归功于吸烟的减少，而不是治疗的改善。尽管分子靶向治疗最近取得了进展，但晚期肺癌的治疗结果仍然令人失望。最近快速批准的EGFR抑制剂（吉非替尼和厄洛替尼）用于常规化疗失败的晚期非小细胞肺癌，已被证明仅对约10%的非小细胞肺癌有效，其影响不大，延长了2个月的生存期（厄洛替尼）。此外，吉非替尼未能显示出生存益处，这促使FDA撤销了对新肺癌患者的治疗批准。因此，虽然有一部分EGFR突变的患者是有反应的，但普遍的共识是，EGFR抑制剂对非小细胞肺癌的治疗效果令人失望。因此，在非小细胞肺癌的治疗中，显然需要新的靶向治疗策略。一类可能的靶标是Ras同源的Rho小GTPases。越来越多的证据表明，Rho GTPase在肿瘤发生中的异常功能，特别是在乳腺癌、胰腺癌、头颈癌和黑色素瘤中。然而，迄今为止，关于异常Rho gtpase在非小细胞肺癌生长中的作用的研究非常有限。我们建议研究在大多数非小细胞肺癌中Rho GTPase功能失调的一个关键机制，即DLC-1肿瘤抑制因子的缺失。我们最近的证据支持我们的假设，即DLC-1的缺失导致非小细胞肺癌中Rho gtpase的过度激活。因此，我们相信，我们对DLC-1缺失可能解除Rho gtpase调控并促进NSCLC生长的机制的阐明，可能为肺癌治疗的靶向治疗确定新的方向。

DESCRIPTION (provided by applicant): DLC-1 expression is lost in lung and other cancers and ectopic re-expression of DLC-1 impairs the transformed and tumorigenic growth of DLC-1-deficient tumor cell lines. Thus, DLC-1 exhibits properties of a tumor suppressor. DLC-1 encodes a GTPase activating protein (GAP) and negative regulator of Ras homologous (Rho) small GTPases. Constitutive activation of Rho GTPases causes growth transformation and promotes tumor cell invasion, metastasis and angiogenesis. Therefore, we hypothesize that the loss of DLC-1 function may result in persistent Rho GTPase activation and promotion of NSCLC oncogenesis. However, in addition to a RhoGAP catalytic domain, DLC-1 also contains START lipid-binding and SAM protein-protein interaction domains. Our recent results determined that DLC-1 suppresses NSCLC growth by both RhoGAP- dependent and -independent mechanisms. In addition to RhoA, we also determined that the RhoGAP domain regulates the biologically distinct RhoB and RhoC isoforms, as well as Cdc42, providing the basis for our studies to establish the full repertoire of Rho GTPases inactivated by DLC-1 (Aim 1). We also identified the SAM domain as an autoinhibitory domain that regulates DLC-1 RhoGAP activity. How this domain may regulate DLC-1 activity, and whether MEK-ERK-RSK protein kinase and PI3K-AKT lipid kinase signaling pathway-mediated phosphorylation of DLC-1 may regulate DLC-1 activity will be determined (Aim 3). DLC-1 is associated with focal adhesions (FAs) and this association is critical for DLC-1 tumor suppression but surprisingly, not for Rho GTPase inactivation in vivo. It has been determined recently that DLC-1 association with FAs is mediated by binding to tensin proteins. We also determined that the SAM and START domains may also regulate DLC-1 subcellular localization distinct from association with focal adhesions. We found that ectopic expression of DLC-1 inactivated RhoA at the leading edge of migrating cells and inhibited tumor cell invasion in vitro. These observations provide the rationale for our studies to determine the importance of spatially-restricted DLC-1-mediated Rho GTPase inactivation for inhibition of tumor growth (Aim 3). These studies will determine if SAM, START and FA-targeted DLC-1 preferentially inactivates Rho GTPases in specific subcellular compartments important for tumor suppression. Finally, to complement our ectopic re- expression studies that show DLC-1 inhibition of tumor growth, we propose interfering RNA and dominant negative DLC-1 studies to determine the biological consequences of DLC-1 loss of expression, and to evaluate a lung tumor tissue microarray to determine if loss of DLC-1 protein expression is associated with specific genetic properties and clinical outcomes of NSCLCs (Aim 4). PUBLIC HEALTH RELEVANCE: Lung cancer remains the most common fatal cancer in men (31%) and women (28%) in the US, and NSCLC accounts for 80% of all lung cancer cases and is the leading cause of cancer mortality (http://www.cancer.org/). The most recent statistics found that lung cancer death rates were increasing at a much slower rate than in the past. Unfortunately, this modest improvement is attributed primarily to decreased smoking, rather than improved therapy. Despite recent advances in molecularly targeted therapies, treatment outcomes for advanced lung cancer remain disappointing. The recent fast-track approval of EGFR inhibitors (gefitinib and erlotinib) for advanced NSCLC that have failed conventional chemotherapy have proven effective against only ~10% of NSCLCs and their impact has been modest, increasing survival by two months (erlotinib). Furthermore, the failure of gefitinib to show a survival benefit has prompted the FDA to reverse its approval for the treatment new lung cancer patients. Thus, while a subset of patients with EGFR mutations is responsive the general consensus is that EGFR inhibitors have been a disappointment for NSCLC treatment. Therefore, new target-based treatment strategies are clearly needed in NSCLC therapy. One possible class of targets is the Ras homologous Rho small GTPases. There is considerable and growing evidence for aberrant Rho GTPase function in oncogenesis, in particular in breast, pancreatic, and head and neck carcinomas, and melanomas. However, to date, there has been surprisingly limited study of the role of aberrant Rho GTPases in NSCLC growth. We propose studies to address one key mechanism by which Rho GTPase function may be deregulated in a majority of NSCLCs, the loss of the DLC-1 tumor suppressor. Our recent evidence supports our hypothesis that loss of DLC-1 causes hyperactivation of Rho GTPases in NSCLC. Hence, we believe that our elucidation of the mechanism by which DLC-1 loss may deregulate Rho GTPases and promote NSCLC growth may define novel directions for targeted therapies for lung cancer treatment.