Molecular Mechanisms Of Growth Control And Carcinogenesis

生长控制和致癌的分子机制

• 批准号: 8344119
• 负责人: J Gutkind
• 金额: $ 173.95万
• 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/8344119
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adhesives; Adult; Alleles; Animal Model; Behavior; Binding; Bioluminescence; Breast Cancer Cell; CXCR4 gene; Cell Nucleus; Cell Proliferation; Cells; Cessation of life; Clozapine; Coupled; Development; Dissection; Distant; Drosophila genus; Endothelial Cells; Energy Transfer; Evaluation; Event; FOS gene; Family; Family member; G Protein-Coupled Receptor Genes; G-substrate; Gene Delivery; Gene Dosage; Gene Expression; Genes; Genetic Transcription; Genus Alpharetrovirus; Glycoproteins; Growth; Growth Factor Receptors; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Head and Neck Cancer; Heterotrimeric GTP-Binding Proteins; Human; Human Herpesvirus 8; Hydrolysis; Immune system; Immunosuppression; In Vitro; Individual; Infection; Integrins; JUN gene; Kaposi Sarcoma; Kidney Transplantation; Ligands; Link; Lipids; MAPK14 gene; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Mediating; Metastatic to; Mitogens; Molecular; Molecular Target; Monomeric GTP-Binding Proteins; Mus; N-terminal; NIH 3T3 Cells; Neoplasm Metastasis; Normal Cell; Oncogenic; Organ; Oxides; Pathologic Neovascularization; Pathway interactions; Patients; Pattern; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological Processes; Play; Polyomaviruses Middle T Proteins; Prevention; Prevention strategy; Process; Protein Isoforms; Proteins; RNA Interference; Reporter; Resistance; Retinal; Role; Second Messenger Systems; Semaphorins; Signal Transduction; Sirolimus; Stromal Cells; Structure; System; Therapeutic; Tissues; Transcription Factor AP-1; Vascular Endothelial Growth Factors; Video Microscopy; Virus; Work; angiogenesis; autocrine; base; cancer cell; cancer prevention; cancer therapy; carcinogenesis; cell growth; cell motility; cell transformation; cell type; chemokine; dimer; genome-wide; human FRAP1 protein; human disease; in vivo; in vivo Model; inhibitor/antagonist; knock-down; mTOR Inhibitor; malignant breast neoplasm; member; migration; mutant; neoplastic cell; novel; paracrine; plexin; prevent; protein protein interaction; receptor; research clinical testing; rho; rho GTP-Binding Proteins; second messenger; synthetic biology; therapeutic target; tool; tumor; tumor progression; tumorigenesis

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. 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 in pathological angiogenesis. We have recently observed that Sema3E behaves as a very 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. In a recent study, we have shown that Sema3E potently activates the small GTPase Arf6, and that the Sema3E-Plexin-D1 system negatively regulates angiogenesis by modulating integrin signal transduction via Arf6. We have now explored how Plexin-D1 activates Arf6, and shown that this process involves the direct association of Sema3E-activated Plexin-D1 with a lipid kinase, PIP5K, and the consequent recruitment and activation of the Arf6 GEF known as GEP100. These studies provide the molecular basis for anti-angiogenic signaling by Sema3E, which may have broad implications in many human diseases, including cancer. 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。连接生长因子受体与细胞核途径的分子解剖：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，用于构建并理解癌症中 GPCR 调节的信号网络：作为我们持续努力解决 GPCR 控制正常和异常细胞生长问题的一部分，我们已开始使用包含突变 GPCR 表达的合成生物学方法失去了对其天然配体作出反应的能力，但获得了对药理学惰性化合物氯氮平-N-氧化物（CNO）作出反应的能力。使用这些仅由合成配体 (RASSL) 激活的受体可以使我们能够在正常细胞和癌细胞中构建并从而了解 GPCR 调节的信号网络。大量有丝分裂原激活 Gq 偶联的 GPCR。事实上，Gq-RASSL 在 NIH-3T3 细胞中转导有效的有丝分裂信号，如果持续激活，就会发生转化，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 偶联 GPCR 和 AP-1 报告系统的果蝇 S2 细胞中进行了全基因组高通量 RNAi 筛选。 Jun 和 Fos 都是 AP1 二聚体的成员，都在我们的屏幕上受到了关注。我们发现 GTPases 的 Rho 家族成员，特别是 Rho 和 Rac，及其下游效应子（例如 Pak 和 Jun N 末端激酶 (JNK) 的多个调节因子）是 AP-1 激活的组成部分，而 JNK 特异性磷酸酶则起皱。 ，是负调节因子。虽然将 GPCR 与磷脂酰肌醇水解和 PKC 激活联系起来的分子是可有可无的，但我们发现 Trio（一种直接与 Gq 结合的 Rho 鸟嘌呤核苷酸交换因子 (GEF)）对于 Gq 偶联 GPCR 激活 AP-1 至关重要。值得注意的是，Trio 被发现对于哺乳动物细胞中 JNK 和 p38 MAPK 的激活、c-Jun 和 c-Fos 表达、AP1 激活以及细胞增殖和转化至关重要。总的来说，新出现的信息表明，GPCR 通过 Rho-GTPase 网络与 AP-1 的激活相关，该网络由高度特异性的蛋白质-蛋白质相互作用和磷酸化事件控制，而不是由可扩散的第二信使控制。 癌症转移中 GPCR 调节的信号通路：大多数肿瘤细胞表达大量 GPCR，这些 GPCR 被癌症和基质细胞释放到肿瘤微环境的趋化因子激活，从而以自分泌和旁分泌的方式增强肿瘤细胞的运动和存活。反过来，癌细胞可能获得并因此能够被选择利用趋化因子及其 GPCR 的有效促迁移活性转移到局部和远处器官的能力。在所有与转移有关的 GPCR 中，我们对 CXCR4 发起了重点研究，CXCR4 是一种在涉及细胞迁移的许多生理过程中发挥关键作用的 GPCR，并且其对一些最常见的人类恶性肿瘤转移扩散的贡献已得到充分证实。在最近提交的一项研究中，我们使用了多种实验策略，包括基因敲除、药理学工具、生物发光共振能量转移（BRET）、视频显微镜和合成生物学方法，以表明在转移性基底细胞样乳腺癌细胞中，CXCR4启动运动和跨内皮迁移严格要求通过 Gα12/13 家族的异三聚体 G 蛋白激活 Rho。此外，我们提供的证据表明，干扰 CXCR4-Rho 信号轴可以防止乳腺癌细胞的自发转移，从而为基于机制的乳腺癌转移预防策略提供新的潜在治疗靶点。 30% 的努力。发育和肿瘤诱导的血管生成的分子基础。 Semaphorin 3E (Sema3E) 及其受体 Plexin-D1 控制发育中的脉管系统的模式。然而，Sema3E-Plexin-D1 是否在成人和病理性血管生成中发出信号尚不清楚。我们最近观察到，Sema3E 在许多发育和肿瘤诱导的血管生成的体内模型中表现出非常有效的天然抗血管生成分子的作用。特别是，我们观察到 Sema3E 会引起出生后发育中的视网膜脉管系统中内皮尖端细胞的快速回缩，在体外和体内降低 VEGF 的促血管生成活性，并防止头颈癌的促血管生成作用细胞在体内血管反应器中生长时。在细胞水平上，Sema3E 诱导内皮细胞发生与整合素介导的局灶性粘附结构丧失相关的显着形态变化。在最近的一项研究中，我们发现 Sema3E 有效激活小 GTP 酶 Arf6，并且 Sema3E-Plexin-D1 系统通过 Arf6 调节整合素信号转导来负调节血管生成。我们现在已经探索了 Plexin-D1 如何激活 Arf6，并表明该过程涉及 Sema3E 激活的 Plexin-D1 与脂质激酶 PIP5K 的直接关联，以及随后被称为 GEP100 的 Arf6 GEF 的招募和激活。这些研究为 Sema3E 的抗血管生成信号传导提供了分子基础，这可能对包括癌症在内的许多人类疾病具有广泛的影响。 20%的努力。艾滋病相关的卡波西肉瘤：分子机制。 开发 KS 管理和预防的替代治疗方法：我们小组的早期工作发现 Akt/mTOR 通路是 KSHV 诱导的癌症进展的关键信号轴，这有助于评估 mTOR 抑制剂雷帕霉素，用于治疗肾移植后出现 KS 的 KSHV 感染者。由于雷帕霉素的免疫抑制作用可能会限制其治疗效果，因此我们已开始研究以内皮特异性方式干扰 Akt/mTOR 通路而不干扰 mTOR 在免疫系统中的关键功能的可能性。我们最近发现 vGPCR 特异性依赖 PI3Kγ 来启动 Akt/mTOR 通路的激活。与其他 PI3K 亚型 α、β 和 δ 不同，PI3Kγ 在免疫系统中并不发挥关键作用。 RNA干扰方法表明PI3Kγ是vGPCR诱导的肿瘤发生所必需的，并且PI3Kγ特异性抑制剂在阻止vGPCR诱导的肿瘤的发展方面与雷帕霉素一样有效。我们现在使用基因定义的动物模型对这些研究提出了挑战。特别是，我们使用在内皮细胞中表达Tva（一种作为禽逆转录病毒（ALV）受体的糖蛋白）的小鼠，从而通过细胞类型特异性逆转录病毒基因传递在体内实现基因（包括vGPCR）的体细胞表达。当这些小鼠与缺乏 PI3Kγ 的小鼠杂交时，我们观察到即使是表达 PI3Kγ 单一等位基因的同窝小鼠也能部分抵抗 vGPCR 诱导的肿瘤发生和死亡，而 PI3Kγ 缺陷小鼠则受到完全保护。然而，PI3Kγ基因剂量对表达多瘤病毒中T抗原的ALV病毒感染引起的致死率没有影响，该病毒利用PI3Kα发挥其致癌活性。这些研究可能为利用 PI3K-mTOR 通路关键成分的组织特异性分布来治疗 AIDS 患者 KS 的疗法的临床评估提供依据。