Molecular Mechanisms Of Growth Control And Carcinogenesis

生长控制和致癌的分子机制

• 批准号: 7593367
• 负责人: J Gutkind
• 金额: $ 167.6万
• 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/7593367
• 关键词: Agonist; Behavior; Binding; Blood Vessels; CD100 antigen; CXC Chemokines; CXCL12 gene; CXCR4 gene; Cell Adhesion Molecules; Cell Nucleus; Cell physiology; Cell surface; Cell-Cell Adhesion; Cells; Collaborations; Condition; Data; Development; Dissection; Dominant-Negative Mutation; Endothelial Cells; Epithelial Cells; Excision; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Gene Expression Profile; Gene Transfer; Genes; Genetic; Genome; Growth; Growth Factor Receptors; Guanosine Triphosphate Phosphohydrolases; HIV Infections; Hand; Head and Neck Squamous Cell Carcinoma; Herpesviridae; Homing; Human; Human Herpesvirus 8; Immune response; In Vitro; Integrins; Intercellular Junctions; Kaposi Sarcoma; Knock-out; Lesion; Link; Malignant - descriptor; Malignant Epithelial Cell; Malignant Neoplasms; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Mediating; Membrane; Microarray Analysis; Molecular; Molecular Target; Monomeric GTP-Binding Proteins; Mus; NF-kappa B; Nature; Neoplasm Metastasis; Neoplasms; Neoplasms in Vascular Tissue; Normal Cell; Oncogenes; PH Domain; Pathogenesis; Pathway interactions; Patients; Permeability; Phenotype; Phosphorylation; Physiological Processes; Play; Prevention therapy; Process; Protein Subunits; Protein-Serine-Threonine Kinases; Proteins; Range; Rho-associated kinase; Role; Semaphorins; Signal Pathway; Signal Transduction; Simplexvirus; Sirolimus; Solid Neoplasm; Stem cells; Stress Fibers; Stromal Cell-Derived Factor 1; System; T-Lymphocyte; TSC2 gene; Testing; Thrombin; Thrombin Receptor; Tumor Suppressor Proteins; Vascular Endothelial Growth Factors; Vascularization; analog; angiogenesis; base; cadherin 5; cancer prevention; carcinogenesis; cell growth; cell motility; cell transformation; directional cell; human FRAP1 protein; human TSC2 protein; human disease; in vivo; inhibitor/antagonist; interest; knock-down; mTOR inhibition; migration; mutant; novel; novel therapeutics; paracrine; plexin; prevent; receptor; research clinical testing; research study; response; rho; rho GTP-Binding Proteins; sarcoma; therapeutic target; transcription factor; tumor; tumorigenesis; tumorigenic; vasculogenesis

Certain alterations of proteins involved in mitogenic signaling are known to exert profound effects on cellular behavior, including malignant transformation. Our overall objective is to explore the molecular bases of cancer, approaching this problem through the study of normal and aberrant functioning of molecules that participate in the transduction of proliferative signals. Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer Galpha13-Rho signaling axis is required for SDF-1-induced migration through CXCR4: The CXC chemokine SDF-1 (also known as CXCL12) binds to CXCR4, a G protein coupled receptor (GPCR) that plays a critical role in many physiological processes that involve cell migration, ranging from stem cell homing, angiogenesis, to immune response. CXCR4 is also implicated in various pathological conditions, including metastasis and HIV infection. We took advantage of the potent chemotactic activity of SDF-1 in Jurkat T-cells to examine which G protein subunits contribute to CXCR4-mediated cell migration. Whereas Gi and Gbeta-gamma subunits are involved in Rac activation and cell migration, CXCR4 also stimulated Rho potently, but independently of Gi. Instead, we found that Galpha13 mediates the activation of Rho by CXCR4, and that both Galpha13 and Rho are required for directional cell migration in response to SDF-1. These findings identified the Galpha13-Rho signaling axis as a potential pharmacological target in many human diseases that involve the aberrant function of CXCR4. Rac inhibits thrombin-induced Rho activation: Evidence of a Pak-dependent GTPase crosstalk: The strict spatio-temporal control of Rho GTPases is critical for many cellular functions, including cell motility and growth. The prototypical Rho family GTPases, Rho, Rac, and Cdc42 regulate the activity of each other by a poorly understood mechanism. We found that constitutively active Rac inhibits stress fiber formation and Rho stimulation by thrombin, a GPCR agonist. Because a mutant Rac that does not activate Pak1 failed to inhibit Rho activation, we asked whether Pak1 could regulate GEFs for Rho. We found that Pak1 specifically associates with p115-RhoGEF, and knock down experiments revealed that p115-RhoGEF plays a major role in signaling from thrombin receptors to Rho. We provided evidence that Pak1 binds the DH-PH domain of p115-RhoGEF thereby disrupting receptor-dependent Rho signaling, thus providing a mechanism for cross-talk between these small-GTPases. Molecular basis of developmental and tumor-induced angiogenesis Molecular mechanisms by which Semaphorins and Plexins promote angiogenesis: We have shown that semaphorin 4D (Sema4D) elicits a pro-angiogenic phenotype in endothelial cells by promoting the activation of the PI3K-Akt signaling pathway through its receptor, Plexin-B1. By the use of a receptor chimeric approach, Plexin-B1 mutants, knock down strategies, and dominant negative inhibitors, we found that this response is dependent upon the activation of RhoA and its downstream target, Rho kinase (ROK). Furthermore, we observed that Plexin-B1 utilizes RhoA and ROK to activate an integrin-dependent signaling network that stimulates Pyk2, PI3K, Akt, and ERK, and endothelial motility. On the other hand, we found that Sema4D must be processed and released from its membrane bound form to act in a paracrine manner on endothelial cells. In collaboration with MCU, K. Holmbeck, and T. Bugge (PTRU), we used general and specific inhibitors of MMPs and knockout MEFs to demonstrate that Sema4D is a novel target for membrane type 1-MMP (MT1-MMP). MT1-MMP is not expressed in non-tumorigenic epithelial cells but present in head and neck squamous cell carcinoma (HNSCC) cells. By lentiviral-delivery of shRNAs, we showed that the knock down of MT1-MMP prevents the release of Sema4D into its soluble form from these cells, thereby inhibiting their pro-angiogenic responses in vitro and in vivo. These effects were rescued by re-expression of a catalytically active MT1-MMP, or a truncated form of Sema4D. The proteolytic cleavage of Sema4D may provide a novel molecular mechanism by which MT1-MMP controls tumor-induced angiogenesis. The molecular basis of VEGF-induced endothelial cell permeability: VEGF is a pleiotropic factor for endothelial cells that plays a central role in both vasculogenesis and angiogenesis. Deregulated VEGF expression also contributes to the aberrant growth of most solid tumors by promoting their neo-vascularization. In an effort aimed at exploring the mechanism by which VEGF stimulates the proliferation, migration, and survival of endothelial cells, we began investigating how VEGF induces vessel leakiness, its first described function. In a recent study, we identified a novel signaling mechanism, involving the sequential activation of Src, Vav2, Rac1, and Pak1, by which VEGF promotes the rapid removal of a key cell-cell adhesion molecule, VE-cadherin, from the cell surface, leading to the disassembly of endothelial cell junctions and enhanced permeability. These findings opened new therapeutic opportunities for the treatment of many human diseases that involve pathological vessel leakiness. AIDS-associated Kaposi s sarcoma: molecular mechanisms Among the AIDS-associated malignancies, Kaposi s sarcoma (KS) is the most common cancer arising in HIV-infected patients. The Human herpesvirus 8 (HHV-8) or KS associated herpesvirus (KSHV) is the infectious cause of KS. In prior studies, we have developed a high throughput in vivo endothelial specific retroviral gene transfer system, and used it to express candidate KSHV oncogenes in mice. Surprisingly, among the many KSHV genes tested, only one gene, a constitutively active GPCR, vGPCR, was able to promote the development of visible vascular tumors that strikingly resembled human KS lesions. While investigating the nature of the mitogenic and survival pathways utilized by vGPCR to induce tumorigenesis, we observed that vGPCR stimulates the serine/threonine kinase Akt in vivo, and that the activation of Akt and its downstream target, mTOR, represents a hallmark of human KS. vGPCR promotes the activation of mTOR by causing the phosphorylation and inactivation of tuberin (TSC2), a tumor suppressor protein. Interestingly, pharmacologic inhibition of mTOR with rapamycin prevented vGPCR sarcomagenesis, while over-activation of this pathway by haploinsufficiency for TSC2 predisposes mice to the development of KS-like vascular sarcomas. These findings identified the Akt-mTOR pathway as a therapeutic target for KS and provided the rationale for the clinical evaluation of rapamycin and its analogs for the treatment of this AIDS-malignancy. A NF-kB gene expression signature contributes to KSHV vGPCR-induced direct and paracrine neoplasia: Using a full-genome microarray analysis we found that vGPCR promotes a dramatic change in gene expression in endothelial cells either by acting directly in vGPCR-expressing cells, or indirectly through the release of soluble factors. By using gene set enrichment analysis of the microarray data, we found that NF-kB signaling is potently triggered by vGPCR expression and in cells exposed to vGPCR-induced secretions, thus mimicking its paracrine effect. Indeed, we observed that vGPCR activates the NF-kB pathway effectively, and NF-kB activation is a prominent feature in both human and experimental KS. Of interest, constitutive NF-kB signaling is not sufficient to promote endothelial cell transformation. However, using genetic approaches to block NF-kB we found that this transcription factor is strictly required for vGPCR-induced direct and paracrine transformation. Taken together, these results strongly support the role of NF-kB and its regulated genes in KS pathogenesis.

已知参与有丝分裂信号传导的蛋白质的某些改变会对细胞行为产生深远的影响，包括恶性转化。我们的总体目标是探索癌症的分子碱基，通过研究参与扩散信号转导的分子的正常和异常功能来解决这个问题。 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 SDF-1通过CXCR4诱导的迁移需要Galpha13-RHO信号轴：CXC趋化因子SDF-1（也称为CXCL12）与CXCR4结合，G蛋白偶联受体（GPCR）在许多物理学中涉及aNGRANE compration compration compration compration compration compration cont range ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging ranging s ranging ranging ranging。 CXCR4还与各种病理条件有关，包括转移和HIV感染。我们利用了SDF-1在Jurkat T细胞中的有效趋化活性，以检查哪种G蛋白亚基有助于CXCR4介导的细胞迁移。 gi和gbeta-gamma亚基参与RAC激活和细胞迁移，而CXCR4也有效地刺激了Rho，但独立于GI。取而代之的是，我们发现galpha13介导了CXCR4的Rho激活，并且Galpha13和Rho都是响应SDF-1的方向细胞迁移所必需的。这些发现将Galpha13-RHO信号轴确定为许多涉及CXCR4异常功能的人类疾病的潜在药理靶标。 RAC抑制凝血酶诱导的RHO激活：PAK依赖性GTPase串扰的证据：Rho GTPases的严格时空控制对许多细胞功能，包括细胞运动和生长至关重要。原型的Rho家族GTPases，Rho，RAC和CDC42通过较知的机制来调节彼此的活性。我们发现，组成性活跃的RAC抑制了GPCR激动剂凝血酶的胁迫纤维形成和RHO刺激。由于未激活PAK1的突变体RAC无法抑制RHO激活，因此我们询问PAK1是否可以调节GEF的RHO。我们发现PAK1专门与P115-Rhogef相关，并击倒实验表明，P115-Rhogef在从凝血酶受体到RHO的信号传导中起主要作用。我们提供的证据表明PAK1结合了P115-RhoGEF的DH-PH结构域，从而破坏了受体依赖性的RHO信号传导，从而为这些小型GTPase之间提供了一种机制。 发育和肿瘤诱导的血管生成的分子基础 词素和丛蛋白促进血管生成的分子机制：我们已经表明，词素4D（SEMA4D）通过促进PI3K-AKT信号通过其受体Plexin-B1的激活而激活内皮细胞中的促血管生成表型。通过使用受体嵌合方法，plexin-b1突变体，敲低策略和显性负抑制剂，我们发现这种反应取决于RhoA及其下游靶标Rho激酶（ROK）的激活。此外，我们观察到Plexin-B1利用RhoA和ROK激活整合素依赖性信号网络，该信号网络刺激PYK2，PI3K，AKT和ERK和内皮运动。另一方面，我们发现必须从其膜结合形式进行处理并释放SEMA4D，以在内皮细胞上以旁分泌方式作用。与MCU，K。Holmbeck和T. Bugge（PTRU）合作，我们使用了MMP和基因敲除MEF的一般和特定抑制剂，以证明SEMA4D是膜1-MMP（MT1-MMP）的新目标。 MT1-MMP在非肿瘤上皮细胞中不表达，而是在头颈部鳞状细胞癌（HNSCC）细胞中。通过shrnas的慢病毒分娩，我们表明MT1-MMP的敲低阻止了Sema4d从这些细胞中释放到其可溶性形式中，从而抑制了它们的体外和体内促血管生成反应。通过重新表达催化活性的MT1-MMP或SEMA4D截断形式来挽救这些效果。 SEMA4D的蛋白水解裂解可能提供了一种新型的分子机制，MT1-MMP控制肿瘤诱导的血管生成。 VEGF诱导的内皮细胞通透性的分子基础：VEGF是内皮细胞的多效因子，在血管生成和血管生成中起着核心作用。放松管制的VEGF表达也通过促进其新的血管形成有助于大多数实体瘤的异常生长。为了探索VEGF刺激内皮细胞的增殖，迁移和存活的机制，我们开始研究VEGF如何诱导血管泄漏，这是其首次描述的功能。 在最近的一项研究中，我们确定了一种新颖的信号传导机制，涉及SRC，VAV2，RAC1和PAK1的顺序激活，通过该激活，VEGF促进了从细胞表面快速去除钥匙细胞 - 细胞粘附分子VE-cadherin，从细胞表面中，导致内皮细胞连接固定和增强的渗透性。这些发现为治疗许多涉及病理血管泄漏的人类疾病打开了新的治疗机会。 与艾滋病相关的卡波西肉瘤：分子机制 在与AIDS相关的恶性肿瘤中，Kaposi的肉瘤（KS）是HIV感染的患者最常见的癌症。人类疱疹病毒8（HHV-8）或KS相关的疱疹病毒（KSHV）是KS的感染原因。在先前的研究中，我们已经开发了体内特异性逆转录病毒基因转移系统的高通量，并将其用来表达小鼠中的候选KSHV Oncogenes。令人惊讶的是，在测试的许多KSHV基因中，只有一个基因，一种组成型活性的GPCR VGPCR，能够促进与人类KS病变非常相似的可见血管肿瘤的发展。在研究VGPCR使用的有丝分裂和生存途径的性质时，我们观察到VGPCR在体内刺激丝氨酸/苏氨酸激酶Akt，而Akt的激活AKT及其下游靶标MTOR代表了人类KS的Hallmark。 VGPCR通过引起肿瘤抑制蛋白Tuberin（TSC2）的磷酸化和失活（TSC2）来促进MTOR的激活。有趣的是，用雷帕霉素对MTOR的药理抑制阻止了VGPCR肌瘤作用，而通过单倍不足的TSC2使小鼠对KS样血管肉瘤的发育过度激活这种途径。这些发现将AKT-MTOR途径确定为KS的治疗靶标，并为雷帕霉素的临床评估及其类似物提供了治疗这种艾滋病 - 恶性的基本原理。 NF-KB基因表达签名有助于KSHV VGPCR诱导的直接和旁分泌肿瘤：使用完整基因组微阵列分析，我们发现VGPCR通过直接在表达VGPCR的细胞中作用或通过释放的释放来促进内皮细胞中基因表达的急剧变化，或者可以促进内皮细胞中的急剧变化。通过使用微阵列数据的基因集富集分析，我们发现NF-KB信号传导是由VGPCR表达和暴露于VGPCR诱导的分泌的细胞中触发的，从而模仿了其旁分泌作用。确实，我们观察到VGPCR有效地激活NF-KB途径，而NF-KB激活是人类和实验性KS的突出特征。感兴趣的是，本构NF-KB信号不足以促进内皮细胞转化。但是，使用遗传方法来阻断NF-KB，我们发现该转录因子严格对于VGPCR诱导的直接和旁分泌转化需要。综上所述，这些结果强烈支持NF-KB及其调节基因在KS发病机理中的作用。