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影响，包括恶性转化。我们的总体目标是探索癌症的分子基础，通过研究参与增殖信号转导的分子的正常和异常功能来解决这个问题。 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 Galpha13-Rho 信号轴是 SDF-1 通过 CXCR4 诱导的迁移所必需的：CXC 趋化因子 SDF-1（也称为 CXCL12）与 CXCR4 结合，CXCR4 是一种 G 蛋白偶联受体 (GPCR)，在许多生理过程中发挥着关键作用涉及细胞迁移，范围从干细胞归巢、血管生成到免疫反应。 CXCR4 还与多种病理状况有关，包括转移和 HIV 感染。我们利用 Jurkat T 细胞中 SDF-1 的强大趋化活性来检查哪些 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 也能有效刺激 Rho，但与 Gi 无关。相反，我们发现 Galpha13 介导 CXCR4 对 Rho 的激活，并且 Galpha13 和 Rho 都是响应 SDF-1 的定向细胞迁移所必需的。这些发现确定 Galpha13-Rho 信号轴是许多涉及 CXCR4 功能异常的人类疾病的潜在药理学靶点。 Rac 抑制凝血酶诱导的 Rho 激活：Pak 依赖性 GTP 酶串扰的证据：Rho GTP 酶的严格时空控制对于许多细胞功能（包括细胞运动和生长）至关重要。典型的 Rho 家族 GTPases、Rho、Rac 和 Cdc42 通过一种鲜为人知的机制相互调节活性。我们发现，组成型活性 Rac 抑制应力纤维形成和凝血酶（一种 GPCR 激动剂）对 Rho 的刺激。由于不激活 Pak1 的突变 Rac 无法抑制 Rho 激活，因此我们询问 Pak1 是否可以调节 Rho 的 GEF。我们发现 Pak1 与 p115-RhoGEF 特异性结合，敲低实验表明 p115-RhoGEF 在从凝血酶受体到 Rho 的信号传导中发挥着重要作用。我们提供的证据表明，Pak1 结合 p115-RhoGEF 的 DH-PH 结构域，从而破坏受体依赖性 Rho 信号传导，从而提供这些小 GTP 酶之间的串扰机制。 发育和肿瘤诱导的血管生成的分子基础 Semaphorin 和 Plexins 促进血管生成的分子机制：我们已经证明，Semaphorin 4D (Sema4D) 通过其受体 Plexin-B1 促进 PI3K-Akt 信号通路的激活，从而在内皮细胞中引发促血管生成表型。通过使用受体嵌合方法、Plexin-B1 突变体、敲低策略和显性失活抑制剂，我们发现这种反应依赖于 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，刺激 Pyk2、PI3K、Akt 和 ERK 以及内皮运动。另一方面，我们发现 Sema4D 必须经过处理并从其膜结合形式释放才能以旁分泌方式作用于内皮细胞。我们与 MCU、K. Holmbeck 和 T. Bugge (PTRU) 合作，使用 MMP 的通用和特异性抑制剂以及敲除 MEF 来证明 Sema4D 是膜类型 1-MMP (MT1-MMP) 的新靶点。 MT1-MMP 在非致瘤性上皮细胞中不表达，但存在于头颈鳞状细胞癌 (HNSCC) 细胞中。通过shRNA的慢病毒递送，我们发现MT1-MMP的敲低可以阻止Sema4D从这些细胞中释放为其可溶形式，从而抑制它们在体外和体内的促血管生成反应。这些效应可以通过重新表达具有催化活性的 MT1-MMP 或截短形式的 Sema4D 来挽救。 Sema4D 的蛋白水解可能提供一种新的分子机制，MT1-MMP 通过该机制控制肿瘤诱导的血管生成。 VEGF诱导内皮细胞通透性的分子基础：VEGF是内皮细胞的多效性因子，在血管生成和血管生成中发挥核心作用。失调的 VEGF 表达还通过促进新血管形成而导致大多数实体瘤的异常生长。为了探索 VEGF 刺激内皮细胞增殖、迁移和存活的机制，我们开始研究 VEGF 如何诱导血管渗漏，这是其首次被描述的功能。 在最近的一项研究中，我们发现了一种新的信号传导机制，涉及 Src、Vav2、Rac1 和 Pak1 的顺序激活，VEGF 通过该机制促进关键的细胞间粘附分子 VE-钙粘蛋白从细胞表面快速去除，导致内皮细胞连接的分解和通透性增强。这些发现为治疗许多涉及病理性血管渗漏的人类疾病开辟了新的治疗机会。 艾滋病相关卡波西肉瘤：分子机制 在与艾滋病相关的恶性肿瘤中，卡波西肉瘤（KS）是艾滋病毒感染者中最常见的癌症。人类疱疹病毒 8 型 (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，我们开发了一种高通量体内内皮特异性逆转录病毒基因转移系统，并用它在小鼠体内表达候选KSHV癌基因。令人惊讶的是，在测试的众多 KSHV 基因中，只有一个基因，即组成型活性 GPCR，即 vGPCR，能够促进与人类 KS 病变极为相似的可见血管肿瘤的发展。在研究 vGPCR 用于诱导肿瘤发生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，并且 Akt 及其下游靶标 mTOR 的激活代表了人类 KS 的标志。 vGPCR 通过引起肿瘤抑制蛋白马铃薯蛋白 (TSC2) 的磷酸化和失活来促进 mTOR 的激活。有趣的是，用雷帕霉素对 mTOR 进行药理抑制可防止 vGPCR 肉瘤发生，而 TSC2 单倍体不足导致该通路过度激活，使小鼠易患 KS 样血管肉瘤。这些发现确定了 Akt-mTOR 通路作为 KS 的治疗靶点，并为雷帕霉素及其类似物治疗这种 AIDS 恶性肿瘤的临床评估提供了理论基础。 NF-kB 基因表达特征导致 KSHV vGPCR 诱导的直接和旁分泌肿瘤：使用全基因组微阵列分析，我们发现 vGPCR 通过直接作用于表达 vGPCR 的细胞，或通过直接作用于表达 vGPCR 的细胞，促进内皮细胞基因表达的巨大变化。间接通过可溶性因子的释放。通过使用微阵列数据的基因集富集分析，我们发现 NF-kB 信号传导由 vGPCR 表达以及暴露于 vGPCR 诱导分泌物的细胞中有效触发，从而模拟其旁分泌效应。事实上，我们观察到 vGPCR 有效激活 NF-kB 通路，并且 NF-kB 激活是人类和实验 KS 的一个显着特征。有趣的是，组成型 NF-kB 信号传导不足以促进内皮细胞转化。然而，使用遗传方法来阻断 NF-kB，我们发现该转录因子是 vGPCR 诱导的直接和旁分泌转化所严格需要的。综上所述，这些结果有力地支持了 NF-kB 及其调控基因在 KS 发病机制中的作用。