Molecular Mechanisms Of Growth Control And Carcinogenesis

生长控制和致癌的分子机制

• 批准号: 7967054
• 负责人: J Gutkind
• 金额: $ 168.55万
• 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/7967054
• 关键词: Active Sites; Acute; Affect; Apoptosis; Architecture; Behavior; Binding; Blood Vessels; Calcium; Catalysis; Cell Adhesion Molecules; Cell Nucleus; Cell Proliferation; Cell physiology; Cell-Cell Adhesion; Cells; Chronic; Coagulants; Coagulation Process; Complex; Coupled; Development; Disease; Dissection; Drosophila genus; Endothelial Cells; Event; Extracellular Matrix; Extravasation; Family; Fibrin; Focal Adhesions; G Protein-Coupled Receptor Genes; GTP-Binding Proteins; Genetic Transcription; Growth; Growth Factor Receptors; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Herpesviridae; Homologous Gene; Human; Human Herpesvirus 8; Human Pathology; Hydrolysis; IL8 gene; IL8RB gene; Inflammation; Intercellular Junctions; Kaposi Sarcoma; Knowledge; Lesion; Link; MAPK1 gene; MAPK14 gene; MAPK7 gene; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Mitogen-Activated Protein Kinases; Modeling; Molecular; Molecular Target; Monomeric GTP-Binding Proteins; Mus; Muscarinics; N-terminal; Neoplasm Metastasis; Neoplastic Cell Transformation; Normal Cell; Nuclear; PIK3CG gene; Pathway interactions; Patients; Peptides; Permeability; Phenotype; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Plasma Proteins; Play; Prevention therapy; Process; Protein Isoforms; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Quantum Mechanics; RNA Interference; Reaction; Reporter; Retinal; Role; Second Messenger Systems; Serine Protease; Signal Pathway; Signal Transduction; Specificity; Stimulus; Structure; System; Thrombin; Transcription Factor AP-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vascular Permeabilities; Vascularization; Wound Healing; angiogenesis; autocrine; base; cadherin 5; cancer cell; cancer prevention; cancer therapy; carcinogenesis; cell growth; chemokine; chemokine receptor; computer studies; dimer; extracellular; genome-wide; in vivo; inhibitor/antagonist; interest; macrophage; member; migration; molecular mechanics; mouse model; neutrophil; novel; novel strategies; prevent; protein protein interaction; receptor; rho; rho GTP-Binding Proteins; sarcoma; second messenger; transcription factor; tumor; tumor initiation

40% Effort. Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer. Mitogen-activated protein kinase (MAPK) signaling pathways play an essential role in the transduction of environmental stimuli to the nucleus, therefore regulating a variety of cellular processes, including cell proliferation, differentiation, programmed cell death, and neoplastic transformation. The aberrant activation of the MAPK extracellular activated protein kinase (ERK) is a frequent event among highly prevalent human cancers. Thus, the components of the ERK kinase cascade represent an attractive target for cancer therapy. The MAPK mechanism of catalysis is still poorly understood due to the lack of complex structures of MAPKs with their bound targets in the active site. Recently, we performed the first computational study of the phosphoryl transfer reaction catalyzed by ERK2 using a Quantum Mechanics/Molecular Mechanics (QM/MM) approach. We modeled the interaction of ERK2 with a target peptide and analyzed the specificity towards S/T-P motifs. Our results provided a detailed description of the molecular events involved in the phosphorylation catalyzed by ERK2. This dynamic model of how MAPKs function may contribute to the understanding of how these kinases phosphorylate their growth promoting downstream targets. Key molecules linking GPCRs to the activation of AP1: GPCRs stimulate cell proliferation by regulating the activity of transcription factors, including AP1, whose aberrant activation has been implicated in tumor initiation and progression. To investigate how GPCRs regulate AP1-dependent gene transcription, we performed a genome-wide high-throughput RNAi screen in Drosophila S2 cells expressing muscarinic 1 receptors, a typical GPCR, and an AP-1 reporter system. Both Jun and Fos, members of the AP1 dimer, were hits on our screen. Interestingly, 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 AP1 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 M1. We have now extended these studies in mammalian cells, and obtained evidence that Gq stimulates the activity of multiple MAPK cascades, including JNK, p38, and ERK5 through Rho GTPases and their downstream targets. 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. 40% Effort. Molecular basis of developmental and tumor-induced angiogenesis. PRK and ROCK are required for thrombin-induced endothelial cell permeability downstream from G12/13 and G11/q and RhoA: Increase in vascular permeability occurs under many physiological conditions such as wound repair, inflammation, and thrombotic reactions, and is central in diverse human pathologies, including tumor-induced angiogenesis. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. We used pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We showed that thrombin signals through PAR-1 and its coupled G proteins G12/13 and G11/q to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK and PRK, two Rho-dependent serine/threonine kinases that are required for endothelial permeability and the remodeling of cell-extracellular matrix and cell-cell adhesions. The signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK, and the remodeling of focal adhesion components through PRK. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage. A role for a CXCR2/PI3Kgamma signaling axis in acute and chronic vascular permeability: Aberrant angiogenesis and vessel leakiness occur in numerous pathological conditions, such as in acute and chronic inflammation, tumor-induced angiogenesis and metastasis, and is central in the progression of many ocular diseases. Of interest, IL-8 (CXCL-8) exerts multiple functions in angiogenesis by acting directly on endothelial cell growth, permeability and migration and by serving as a potent chemo-attractant factor for macrophages and neutrophils. In search for the underlying mechanism by which IL-8 promotes vascular leakage, we found that IL-8 stimulates the redistribution of VE-cadherin, a key endothelial cell-cell adhesion molecule, by a signaling pathway involving CXCR2, the small GTPase Rac1, and its downstream target, PAK, resulting in the phosphorylation-dependent internalization of VE-cadherin. This process required the activation of a particular PI(3)K catalytic isoform, PI3Kgamma. Indeed, we obtained evidence that IL-8 induces a rapid and severe vascular leakage that can be efficiently prevented by the pharmacological inhibition of CXCR2 and PI3Kgamma in vivo. These results prompted us to investigate the potential role of IL-8 signaling in a mouse model for retinal vascular hyper-permeability. We showed that interfering with the CXCR2/PI3Kgamma signaling axis may represent a novel strategy to prevent and treat retinal hyper-permeability, found in numerous ocular diseases and inflammation conditions. These findings may also raise the possibility of targeting IL-8 signaling in the tumor microenvironment, as many angiogenic mechanisms are similarly deregulated in ocular diseases and tumor vascularization. 20% Effort. AIDS-associated Kaposi s sarcoma: molecular mechanisms. Kaposis sarcoma (KS) is the most common cancer arising in HIV-infected patients. HHV-8 or KS associated herpesvirus (KSHV) is the infectious cause of KS. In prior studies, we have shown that a KSHV-encoded constitutively active GPCR, vGPCR, can initiate the formation of KS lesions in mice, and that vGPCR promotes the expression of VEGF-A under the normoxic conditions that characterize KS. We took advantage of the observation that the chemokine IL-8, which acts on CXCR2 that is the closest human homolog of vGPCR, can rescue the proangiogenic phenotype in HIF-1 deficient cancer cells, probably by promoting VEGF expression and secretion by a HIF-1-independent mechanism, to investigate how GPCRs regulate VEGF-A expression in endothelial cells. Indeed, we showed here that IL-8 induces the expression of VEGF message and protein, thereby promoting the autocrine activation of its receptor, VEGFR2, in endothelial cells. Surprisingly, this effect was found to be independent on HIF-1, but instead involves the activation of NFkappaB by the CXCR2 chemokine receptor, which utilizes the CBM (Carma3/Bcl10/Malt1) complex to stimulate IKKalpha/beta, thus causing the nuclear activation of NFkappaB. These findings have important implications regarding tumor and inflammation-induced angiogenesis, and may explain how vGPCR stimulates the release of VEGF-A in KS.

40% 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。 丝裂原激活蛋白激酶 (MAPK) 信号通路在环境刺激向细胞核的转导中发挥着重要作用，因此调节多种细胞过程，包括细胞增殖、分化、程序性细胞死亡和肿瘤转化。 MAPK 细胞外激活蛋白激酶 (ERK) 的异常激活是高度流行的人类癌症中的常见事件。因此，ERK 激酶级联的成分代表了癌症治疗的一个有吸引力的靶标。由于缺乏具有活性位点结合靶点的 MAPK 的复杂结构，人们对 MAPK 的催化机制仍知之甚少。最近，我们使用量子力学/分子力学（QM/MM）方法对 ERK2 催化的磷酰基转移反应进行了首次计算研究。我们模拟了 ERK2 与目标肽的相互作用，并分析了对 S/T-P 基序的特异性。我们的结果提供了 ERK2 催化磷酸化所涉及的分子事件的详细描述。这种 MAPK 如何发挥作用的动态模型可能有助于理解这些激酶如何磷酸化其促进生长的下游靶标。 将 GPCR 与 AP1 激活联系起来的关键分子：GPCR 通过调节转录因子（包括 AP1）的活性来刺激细胞增殖，AP1 的异常激活与肿瘤的发生和进展有关。为了研究 GPCR 如何调节 AP1 依赖性基因转录，我们在表达毒蕈碱 1 受体、典型 G​​PCR 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。有趣的是，我们发现 GTPase 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 AP1 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 M1 激活 AP-1 至关重要。我们现在将这些研究扩展到哺乳动物细胞中，并获得证据表明 Gq 通过 Rho GTPases 及其下游靶点刺激多个 MAPK 级联的活性，包括 JNK、p38 和 ERK5。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。 40% 的努力。发育和肿瘤诱导的血管生成的分子基础。 PRK 和 ROCK 是 G12/13、G11/q 和 RhoA 下游凝血酶诱导的内皮细胞通透性所必需的：血管通透性增加发生在许多生理条件下，例如伤口修复、炎症和血栓反应，并且在多种人类中至关重要病理学，包括肿瘤诱导的血管生成。凝血酶是一种促凝丝氨酸蛋白酶，它会导致内皮屏障完整性局部丧失，从而使血浆蛋白快速外渗并局部形成含纤维蛋白的凝块。我们使用药理学抑制剂、敲除方法以及关于通透性因素如何影响内皮连接的新兴知识来详细研究凝血酶诱导的内皮通透性的机制。我们发现凝血酶通过 PAR-1 及其偶联的 G 蛋白 G12/13 和 G11/q 发出信号来诱导 RhoA 激活和细胞内钙升高，并且这些事件是相互关联的。反过来，这会刺激 ROCK 和 PRK，这两种 Rho 依赖性丝氨酸/苏氨酸激酶是内皮通透性以及细胞-细胞外基质和细胞-细胞粘附重塑所必需的。凝血酶引发的信号在 RhoA 水平上分叉，通过 ROCK 促进细胞骨架结构的变化，并通过 PRK 促进粘着斑成分的重塑。最终，两条途径汇聚导致细胞-细胞连接破坏并引起血管渗漏。 CXCR2/PI3Kgamma 信号轴在急性和慢性血管通透性中的作用：异常血管生成和血管渗漏发生在许多病理状况中，例如急性和慢性炎症、肿瘤诱导的血管生成和转移，并且是许多疾病进展的核心。眼部疾病。有趣的是，IL-8 (CXCL-8) 通过直接作用于内皮细胞生长、通透性和迁移以及作为巨噬细胞和中性粒细胞的有效化学吸引因子，在血管生成中发挥多种功能。在寻找 IL-8 促进血管渗漏的潜在机制时，我们发现 IL-8 通过涉及 CXCR2（小 GTPase Rac1）的信号通路刺激 VE-钙粘蛋白（一种关键的内皮细胞-细胞粘附分子）的重新分布，及其下游靶标 PAK，导致 VE-钙粘蛋白的磷酸化依赖性内化。该过程需要激活特定的 PI(3)K 催化异构体 PI3Kgamma。事实上，我们获得的证据表明 IL-8 会诱导快速而严重的血管渗漏，而这种血管渗漏可以通过体内 CXCR2 和 PI3Kgamma 的药理学抑制有效地预防。这些结果促使我们研究 IL-8 信号在小鼠视网膜血管通透性过高模型中的潜在作用。我们发现，干扰 CXCR2/PI3Kgamma 信号轴可能代表一种预防和治疗视网膜高通透性的新策略，这种通透性存在于许多眼部疾病和炎症状况中。这些发现还可能提高在肿瘤微环境中靶向 IL-8 信号传导的可能性，因为许多血管生成机制在眼部疾病和肿瘤血管化中类似地失调。 20% 的努力。艾滋病相关卡波西肉瘤：分子机制。 卡波西肉瘤 (KS) 是 HIV 感染者中最常见的癌症。 HHV-8 或 KS 相关疱疹病毒 (KSHV) 是 KS 的感染原因。在之前的研究中，我们已经表明，KSHV 编码的组成型活性 GPCR（vGPCR）可以启动小鼠 KS 病变的形成，并且 vGPCR 在具有 KS 特征的常氧条件下促进 VEGF-A 的表达。我们利用这一观察结果，即作用于 CXCR2（vGPCR 最接近的人类同源物）的趋化因子 IL-8 可以通过 HIF-1 促进 VEGF 表达和分泌来挽救 HIF-1 缺陷癌细胞中的促血管生成表型。 1独立机制，研究GPCR如何调节内皮细胞中VEGF-A的表达。事实上，我们在这里表明，IL-8 诱导 VEGF 信息和蛋白的表达，从而促进内皮细胞中其受体 VEGFR2 的自分泌激活。令人惊讶的是，这种效应被发现与 HIF-1 无关，而是涉及 CXCR2 趋化因子受体激活 NFkappaB，该受体利用 CBM (Carma3/Bcl10/Malt1) 复合物刺激 IKKα/β，从而引起核激活NFκB。这些发现对于肿瘤和炎症诱导的血管生成具有重要意义，并可能解释 vGPCR 如何刺激 KS 中 VEGF-A 的释放。