Molecular Mechanisms Of Growth Control And Carcinogenesis

生长控制和致癌的分子机制

• 批准号: 8148624
• 负责人: J Gutkind
• 金额: $ 164.45万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148624
• 关键词: Growth; Molecular; carcinogenesis

50% Effort. Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer. A synthetic biology approach to build, and hence understand, GPCR-regulated signaling networks in cancer: As part of our ongoing efforts addressing normal and aberrant cell growth control by GPCRs, we have begun using a synthetic biology approach consisting in the expression of mutant GPCRs that have lost the ability to respond to their natural ligands, but gained the ability to respond to a pharmacologically inert compound, clozapine-N-oxide (CNO). The use of these receptors activated solely by synthetic ligands (RASSLs) may enable us to build, and hence understand, GPCR-regulated signaling networks in normal and cancer cells. A large number of mitogens activate Gq-coupled GPCRs. Indeed, Gq-RASSL transduces potent mitogenic signals in NIH-3T3 cells, and is transforming if persistently activated, a process that requires the expression of c-Jun and c-Fos AP-1 family members. To investigate how GPCRs regulate AP-1-dependent gene transcription, we performed a genome-wide high-throughput RNAi screen in Drosophila S2 cells expressing Gq-coupled GPCR and an AP-1 reporter system. Both Jun and Fos, members of the AP1 dimer, were hits on our screen. We found that members of the Rho family of GTPases, specifically Rho and Rac, and their downstream effectors such as Pak and multiple regulators of Jun N-terminal kinase (JNK) were integral to AP-1 activation, while a JNK-specific phosphatase puckered, was a negative regulator. While molecules linking GPCRs to the hydrolysis of phosphatidylinositol and PKC activation were dispensable, we found that Trio, a Rho guanine nucleotide exchange factor (GEF) that binds directly to Gq, is essential for AP-1 activation by Gq-coupled GPCRs. Remarkably, Trio was found to be essential for the activation of JNK and p38 MAPKs, c-Jun and c-Fos expression, AP1 activation, and cell proliferation and transformation in mammalian cells. Collectively, the emerging information indicates that GPCRs are linked to the activation of AP-1 through a Rho-GTPase network that is governed by highly specific protein-protein interactions and phosphorylation events rather than by diffusible second messengers. GPCR-regulated signaling circuitries in cancer metastasis: Most tumor cells express numerous GPCRs that are activated by chemokines released to the tumor microenviroment by cancer and stromal cells, thus enhancing the motility and survival of tumor cells in an autocrine and paracrine fashion. In turn, cancer cells may gain, and thus can be selected for, the ability to co-opt the potent pro-migratory activity of chemokines and their GPCRs to metastasize to regional and distant organs. Among all GPCRs implicated in metastasis, we have initiated a focal effort on CXCR4, a GPCR that plays a critical role in many physiological processes involving cell migration, and its contribution to metastatic spread of some of the most prevalent human malignancies is well established. In a recently submitted study, we used multiple experimental strategies, including gene knock down, pharmacological tools, bioluminescence resonance energy transfer (BRET), videomicroscopy, and synthetic biology approaches, to show that in metastatic basal-like breast cancer cells, CXCR4-initiated motility and transendothelial migration strictly requires the activation of Rho through heterotrimeric G proteins of the Gα12/13 family. Furthermore, we provided evidence that interfering with the CXCR4-Rho signaling axis prevents the spontaneous metastasis of breast cancer cells, thus exposing new potential therapeutic targets for mechanism-based metastasis preventive strategies in breast cancer. 30% Effort. Molecular basis of developmental and tumor-induced angiogenesis. Molecular mechanisms by which Semaphorins and Plexins control angiogenesis: Semaphorin 3E (Sema3E) and its receptor Plexin-D1 control the patterning of the developing vasculature. However, little is known whether Sema3E-Plexin-D1 signals in adult and pathological angiogenesis. We have recently observed that Sema3E behaves as the most potent natural anti-angiogenic molecule in a number of in vivo models of developmental and tumor-induced angiogenesis. In particular, we observed that Sema3E provokes the rapid retraction of endothelial tip cells in the post-natal developing retinal vasculature, diminish the pro-angiogenic activity of VEGF in vitro and in vivo, and prevents the pro-angiogenic effect of head and neck cancer cells when grown in angioreactors in vivo. At the cellular level, Sema3E induces dramatic morphological changes in endothelial cells associated with the loss of integrin-mediated focal adhesive structures. Plexins are known to function as GTPase-activating proteins (GAPs) for R-Ras, which is required for repulsive responses to semaphorins in axon guidance. However, activation of Plexin-D1 by Sema3E induces cytoskeletal collapse without inactivating R-Ras. Furthermore, we found that Sema3E activates potently the small GTPase Arf6. Depletion of Arf6 by siRNA or expression of dominant negative mutants of Arf6 rescues Sema3E-induced focal adhesion disassembly. Emerging data suggest that the Sema3E-Plexin-D1 system negatively regulates angiogenesis by modulating integrin signal transduction via Arf6, thus revealing a novel anti-angiogenic molecular mechanism. 20% effort. AIDS-associated Kaposis sarcoma: molecular mechanisms. Development of alternative therapeutic approaches for KS management and prevention: Early work from our group led to the identification of the Akt/mTOR pathway as a critical signaling axis for KSHV-induced cancer progression, which contributed to the evaluation of rapamycin, an inhibitor of mTOR, for the treatment of KSHV-infected individuals who develop KS upon renal transplantation. As there is a possibility that the immunosuppressive effects of rapamycin may limit its therapeutic benefit, we have begun investigating the possibility of interfering with the Akt/mTOR pathway in an endothelial-specific manner without disturbing the critical function of mTOR in the immune system. We have recently found that vGPCR relies specifically on PI3Kγ to initiate the activation of the Akt/mTOR pathway. PI3Kγ, unlike the other PI3K isoforms α, β and δ, does not play a critical role in the immune system. RNA interference approaches suggest that PI3Kγ is strictly required for vGPCR-induced tumorigenesis, and PI3Kγ-specific inhibitors are as efficient as rapamycin in halting the development of vGPCR-induced tumors. We have now challenged these studies using genetically defined animal models. In particular, we used mice expressing Tva, a glycoprotein that acts as a receptor for avian retroviruses (ALV), in endothelial cells, thereby enabling the somatic expression of genes, including vGPCR, in vivo by cell-type specific retroviral gene delivery. When these mice were crossed with mice lacking PI3Kγ, we observed that even littermates expressing a single allele of PI3Kγ were partially resistant to vGPCR-induced tumorigenesis and death, and PI3Kγ-deficient mice are completely protected. However, PI3Kγ gene dosage had no effect on the lethality caused by infection with ALV-viruses expressing polyomavirus middle T antigen, which uses PI3Kα for its oncogenic activity. These studies may provide a rationale for the clinical evaluation of therapies exploiting the tissue-specific distribution of a critical component of the PI3K-mTOR pathway to treat KS in AIDS patients.

50％的努力。将生长因子受体与细胞核联系起来的途径的分子解剖：它们在正常细胞生长和癌症中的作用。 一种合成生物学方法来构建癌症中GPCR调节的信号网络：作为我们正在进行的努力解决GPCR正常和异常细胞生长控制的努力的一部分，我们已经开始使用一种合成生物学方法，该方法包括突变GPCR的表达，这些方法表达了突变的GPCR，这些方法却失去了对其自然依赖的能力响应的能力，但该方法既响应了自然的态度，又涉及了该方法。 （CNO）。这些受体仅通过合成配体（RASSL）激活的受体可能使我们能够构建正常和癌细胞中GPCR调节的信号网络。大量有丝分裂原激活GQ耦合的GPCR。实际上，GQ-RASSL在NIH-3T3细胞中传递了有效的有丝分裂信号，并且如果持续激活，则会转化，这一过程需要C-JUN和C-FOS AP-1家族成员的表达。为了研究GPCR如何调节AP-1依赖性基因转录，我们在表达GQ耦合GPCR和AP-1报告基因系统的果蝇S2细胞中进行了全基因组高通量RNAi筛选。 AP1二聚体的成员Jun和Fos都在我们的屏幕上受到打击。我们发现，GTPases的Rho家族的成员，特别是RHO和RAC，以及它们的下游效应子，例如PAK和Jun N末端激酶（JNK）的多个调节剂（JNK）是AP-1激活不可或缺的，而JNK特异性磷酸酶则是负调节剂。虽然将GPCR连接到磷脂酰肌醇和PKC激活的水解的分子是可分配的，但我们发现Trio是直接与GQ结合的Rho Guanine核苷酸交换因子（GEF），对于通过GQ耦合GPCRS激活AP-1是必不可少的。值得注意的是，发现三人对于JNK和p38 MAPK，C-JUN和C-FOS表达，AP1激活以及哺乳动物细胞中细胞增殖和转化至关重要。总的来说，新兴信息表明GPCR与AP-1通过Rho-GTPase网络的激活相关，该网络受高度特定的蛋白质 - 蛋白质相互作用和磷酸化事件的控制，而不是通过可扩散的第二使者。 癌症转移中GPCR调节的信号传导回路：大多数肿瘤细胞表达了许多GPCR，这些GPCR被癌症和基质细胞释放到肿瘤微视线的趋化因子激活，从而增强了自身分泌和旁乳核的运动性和肿瘤细胞的活力和存活。反过来，癌细胞可能会增加，因此可以选择趋化因子及其GPCR的有效迁移活性的能力转移到区域和远处器官。在与转移有关的所有GPCR中，我们已经开始了对CXCR4的焦点，这是一种在许多涉及细胞迁移的生理过程中起着关键作用的GPCR，并且它对某些最普遍的人类恶性肿瘤的转移蔓延的贡献得到了很好的确定。 In a recently submitted study, we used multiple experimental strategies, including gene knock down, pharmacological tools, bioluminescence resonance energy transfer (BRET), videomicroscopy, and synthetic biology approaches, to show that in metastatic basal-like breast cancer cells, CXCR4-initiated motility and transendothelial migration strictly requires the activation of Rho through heterotrimeric G proteins of the Gα12/13 家庭。此外，我们提供的证据表明，干扰CXCR4-RHO信号轴可防止乳腺癌细胞的自发转移，从而暴露了基于机制的基于机制的转移预防策略的新潜在治疗靶标。 30％的努力。发育和肿瘤诱导的血管生成的分子基础。 信号蛋白和丛蛋白控制血管生成的分子机制：Semaphorin 3E（SEMA3E）及其受体Plexin-D1控制发育中的脉管系统的模式。然而，鲜为人知的是成人和病理血管生成中的Sema3e-plexin-d1信号。我们最近观察到，SemA3E在许多具有发育和肿瘤诱导的血管生成的体内模型中是最有效的天然抗血管生成分子。特别是，我们观察到SEMA3E会引起产后发育后的视网膜脉管系统中内皮尖端细胞的快速缩回，从而减少了VEGF在体外和体内的促血管生成活性，并阻止了血管比在体内生长的头部和颈部癌细胞的促血管生成效应。在细胞水平上，SEMA3E诱导内皮细胞中与整合素介导的局灶性粘合剂结构丧失相关的内皮细胞的急剧变化。丛蛋白被称为R-RAS的GTPase激活蛋白（GAP），这对于在Axon引导中对Semaphorins的排斥反应是必需的。但是，Sema3e激活Plexin-D1会诱导细胞骨架塌陷而不会失活R-RAS。此外，我们发现SEMA3E有效地激活了小的GTPase ARF6。 siRNA的ARF6耗竭或ARF6的显性负突变体的表达挽救了SEMA3E诱导的局灶性粘附拆卸。新兴数据表明，SEMA3E-PLEXIN-D1系统通过通过ARF6调节整联蛋白信号转导来负调节血管生成，从而揭示了一种新型的抗血管生成分子机制。 20％的努力。与艾滋病相关的kaposis肉瘤：分子机制。 开发用于KS管理和预防的替代治疗方法：我们小组的早期工作导致AKT/MTOR途径是KSHV诱导的癌症进展的关键信号轴，这有助于评估Rapamycin（MTOR的抑制剂，MTOR的抑制剂），以对KSHV感染的个体进行KSHV感染的肾脏转换。由于雷帕霉素的免疫抑制作用可能会限制其治疗益处，因此我们开始研究以内皮特异性方式干扰Akt/MTOR途径的可能性，而不会干扰免疫系统中MTOR的关键功能。我们最近发现，VGPCR特别依赖于PI3Kγ来启动Akt/MTOR途径的激活。与其他PI3K同工型α，β和δ不同，PI3Kγ在免疫系统中不起作用。 RNA干扰方法表明，PI3Kγ严格需要VGPCR诱导的肿瘤发生，而PI3Kγ特异性抑制剂在停止VGPCR诱导的肿瘤的发展方面与雷帕霉素一样有效。现在，我们使用遗传定义的动物模型对这些研究提出了挑战。特别是，我们使用了表达TVA的小鼠，TVA是一种糖蛋白，在内皮细胞中充当禽逆转录病毒（ALV）的受体，从而实现了包括VGPCR的体细胞表达，包括VGPCR，通过细胞类型特异性逆转录病毒基因递送。当这些小鼠与缺乏PI3Kγ的小鼠交叉时，我们观察到，即使表达单个PI3Kγ等位基因的同窝窝也对VGPCR诱导的肿瘤发生和死亡有部分抗性，并且PI3Kγ缺陷型小鼠也得到了完全保护。然而，PI3Kγ基因剂量对表达多瘤病毒中间T抗原的ALV病毒感染引起的致死性没有影响，后者使用PI3Kα进行致癌活性。这些研究可能为利用PI3K-MTOR途径的关键成分的组织特异性分布的疗法评估提供了理由，以治疗AIDS患者的KS。