Interacting Signaling Pathways that Initiate Squamous Cell Carcinogenesis

引发鳞状细胞癌变的相互作用信号通路

基本信息

批准号：
7964995
负责人：
STUART H. YUSPA
金额：
$ 119.21万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7964995
关键词：
1,2-diacylglycerol Ablation Address Adult Affect Alopecia Anti-Inflammatory Agents Antibodies Benign Binding Biological Models CCL17 gene CCL2 gene CCR CSF3 gene CXCL10 gene CXCR3 gene Cell Line Cell Nucleus Cells Characteristics Chemotactic Factors Collaborations Cultured Cells Cutaneous Cytokine Signaling Dendritic Cells Development Diagnostic Neoplasm Staging Differentiation Antigens Differentiation and Growth Diglycerides Disease Dose-Limiting Effectiveness Elements Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Epidermis Epithelial Epithelium Event Feedback Folliculitis Gene Expression Genes Genetic Granulocyte-Macrophage Colony-Stimulating Factor Harvest Homeostasis Human Human Pathology IL8RB gene ITGAM gene In Vitro Indium Infiltration Inflammation Inflammation Mediators Inflammatory Inflammatory Response Interleukin-1 Receptors Interleukin-1 alpha Interleukin-17 Interleukin-18 Interleukin-6 Knockout Mice Laboratories Lesion Ligands Link MAPK14 gene Malignant Neoplasms Malignant Squamous Cell Neoplasm Mediating Mediator of activation protein Messenger RNA Methods Microarray Analysis Modeling Monitor Mouse Strains Mus Neoplastic Cell Transformation Oncogenes Oncogenic PTPRC gene Pathogenesis Pathway interactions Patients Pattern Pharmaceutical Preparations Phenotype Plasma Pre-Clinical Model Premalignant Procedures Production Property Protein Isoforms Proteins Proto-Oncogene Proteins c-akt Psoriasis RNA Reagent Receptor Activation Regulation Research Resistance Role S100 Proteins S100A7 Signal Pathway Signal Transduction Skin Skin Carcinogenesis Skin Neoplasms Skin Transplantation Skin graft Spleen Squamous Cell Stimulus Systems Biology T-Cell Activation T-Lymphocyte Tamoxifen Testing Tissue Sample Tissues Transcription Factor AP-1 Transducers Transforming Growth Factor beta Transgenes Transgenic Mice Tumor Promotion Tumor stage Tyrosine autocrine cancer initiation cancer therapy carcinogenesis chemokine cytokine in vitro Model in vivo inhibitor/antagonist keratinocyte keratinocyte differentiation lymph nodes macrophage malignant breast neoplasm mast cell member migration neoplastic neoplastic cell prevent progenitor programs receptor receptor expression response skin disorder skin lesion src-Family Kinases therapeutic target tumor tumor progression

项目摘要

In the skin model the clonal benign tumor defines the phenotype of the initiated cell. High proliferation, resistance to terminal differentiation and altered gene expression are hallmarks of that phenotype. Our previous studies have revealed that Ras activation is sufficient to produce this complete phenotype and fully initiate tumor formation. Since oncogenic ras is the most frequently detected oncogene in human cancers and frequently associated with early events in epithelial cancer development, the skin model is particularly suited to study the mechanism of its transforming activity. In skin keratinocytes, oncogenic Ras establishes a positive feedback loop through the epidermal growth factor receptor (EGFR), increasing proliferation and simultaneously activating src kinases to tyrosine phosphorylate and inactivate PKC delta, a critical mediator of keratinocyte terminal differentiation. Ras mediated activation of PKCalpha through increased diacylglycerol production contributes to altered expression of keratinocyte differentiation markers by modifying AP-1 activity. By targeting PKCalpha to the epidermis of genetically altered mice, we have shown that as a consequence of Ras action on EGFR and PKCalpha, NFkB translocates to the nucleus of keratinocytes and upregulates the expression and release of CXCR2 ligands. While producing a strong inflammatory response, these ligands also stimulate keratinocyte migration through an autocrine mechanism. Genetic ablation of CXCR2 on ras transduced mouse keratinocytes inhibits migration and precludes tumor formation. The downstream signals from CXCR2 activation in keratinocytes have focused on the activation of AKT, ERK and Src as transducers of the migration signal. In collaboration with medicinal chemists we are developing and testing topical inhibitors of CXCR2 and Src as anti-tumor and anti-inflammation agents. The connection between ras mediated EGFR activation and NFkB translocation has been clarified in collaboration with members of the Trinchieri Laboratory and the CCR Cancer and Inflammation Program. MyD88 is a crucial intermediate in the signaling from pro-inflammatory receptors. Mice ablated for MyD88 or the IL-1 receptor (IL-1R) are resistant to topical skin carcinogenesis, and their ras transduced keratinocytes form only small tumors in orthotopic grafts. In keratinocytes lacking MyD88 or IL-1R, transduction of ras activates the EGFR loop, but these keratinocytes do not upregulate pro-inflammatory ligands. It is now clear that downstream from ras activation of the EGFR is the release of IL-1alpha, and this is the critical element leading to NFkB translocation and induction of an inflammatory cascade essential for tumor formation. These models are being utilized to dissect the contribution of inflammation and autocrine chemokine/cytokine signaling in benign tumor development and to test the effectiveness of IL-1R inhibitors to prevent or reverse ras mediated tumor development. A second model has been developed to study cutaneous inflammation and tumor development. Previously, we described the pro-inflammatory chemotactic action of two highly homologous human S100 proteins (A7 and A15) originally cloned from inflamed psoriatic lesions where they are highly expressed and genetically linked to psoriasis development. We developed discriminating antibodies that reveal their differential expression pattern in psoriasis indicative of their functional diversity. We discovered that RAGE was the receptor for hS100A7 and a G-coupled protein mediated the chemotactic activity of hS100A15. Each protein is proinflammatory and they potentiate inflammation in-vivo. We then cloned the single mouse gene progenitor (mS100A7A15), generated an antibody to the protein and demonstrated mS100A7A15 conserves human properties. As the human counterparts, the mouse progenitor is regulated with epidermal differentiation and highly expressed in well differentiated epithelial tumors. Similarly, the human S100A7 and S100A15 proteins are both upregulated in epithelial breast cancers. Functionally conserved, mS100A7A15 also binds to RAGE and is a chemoattractant. An inducible mS100A7A15 transgene targeted to the mouse epidermis stimulates infiltration of immunocytes and upregulates inflammatory cytokines and chemokines to potentiate inflammation in response to external stimuli. These mice may serve as a model for psoriasis but also will serve as a preclinical model to examine putative inhibitors of RAGE as potential anti-inflammatory agents to prevent tumor promotion. Tumor progression in mouse and human squamous tissues is associated with amplification of EGFR, and this has become a target for therapy. The major dose limiting adverse consequence of anti-EGFR therapy for cancer treatment is a severe cutaneous folliculitis. To model this adverse condition, we have genetically deleted the EGFR in mice or conditionally in mouse skin. In the absence of EGFR, skin tumors differentiate prematurely and do not grow. Microarray analysis of RNA from ras transduced wildtype, EGFR null or EGFR inhibitor treated keratinocytes reveals a 25 gene signature for EGFR ablation in tumor cells. Activation of p38 is a node in this signature, and phospho-p38 is elevated in EGFR null tumors. In this setting of p38 activation, IP10, a chemokine linked to CXCR3 and migration of T cells, dendritic cells and macrophages, is increased. Three isoforms of p38 are expressed in keratinocytes, and current knockdown studies are directed to determine which isoform is activated in response to the inhibition of EGFR. The relevance of these findings to human pathology is emphasized by the phenotype of mice with skin targeted EGFR ablation. Extensive folliculitis and alopecia characterize the dose-limiting skin lesions of patients on EGFR inhibitor drugs and are common finding in adult mice with epidermal ablation of EGFR. Tissue samples isolated from non lesional skin of adult transgenic mice display increased mRNA levels of a subset of inflammatory mediators namely MIP-2, MIP-3alpha, MCP-1, TNFalpha, IL-6, IL-17, IL-18, TARC, TGFbeta, iNOS and G-CSF. In contrast, cutaneous GM-CSF is decreased in mouse and human epidermis where EGFR is suppressed. Targeted knockout mice also show higher plasma levels of IL-17, IL-6, IP-10, MCP-1, TGFbeta and G-CSF. Higher basal and stimulated infiltration of CD45 + cells is detected in EGFR null mouse skin, and mast cells and macrophages are more abundant. Adult double transgenic mice show a higher level of T cell activation (CD69 positive cells) and an increased percentage of gamma-delta T cells and CD11b/Gr1 positive cells harvested from the lymph nodes and the spleen. Treatment of the mice for 3 weeks with a TNFalpha neutralizing drug, Embrel, did not affect the abundance of these cells while treatment with neutralizing G-CSF antibody normalized the percentage of CD11b/Gr1 positive cells and gamma-delta T cells detected in the spleen and lymph nodes. Tamoxifen-regulated conditional skin targeted EGFR null mice are now under study to address the role of modulating EGFR expression during each stage of tumor induction.

在皮肤模型中，克隆良性肿瘤定义了起始细胞的表型。高增殖、终末分化抗性和基因表达改变是该表型的标志。我们之前的研究表明，Ras 激活足以产生这种完整的表型并完全启动肿瘤形成。由于致癌ras是人类癌症中最常检测到的致癌基因，并且经常与上皮癌发展的早期事件相关，因此皮肤模型特别适合研究其转化活性的机制。在皮肤角质形成细胞中，致癌 Ras 通过表皮生长因子受体 (EGFR) 建立正反馈回路，增加增殖并同时激活 src 激酶，使酪氨酸磷酸化并使 PKC δ 失活，PKC δ 是角质形成细胞终末分化的关键介质。 Ras 通过增加二酰甘油的产生介导 PKCα 的激活，通过改变 AP-1 活性来改变角质形成细胞分化标记物的表达。通过将 PKCα 靶向基因改造小鼠的表皮，我们发现，由于 Ras 对 EGFR 和 PKCα 的作用，NFkB 易位至角质形成细胞的细胞核，并上调 CXCR2 配体的表达和释放。在产生强烈的炎症反应的同时，这些配体还通过自分泌机制刺激角质形成细胞迁移。 ras 转导的小鼠角质形成细胞上的 CXCR2 基因消融可抑制迁移并阻止肿瘤形成。角质形成细胞中 CXCR2 激活的下游信号集中在 AKT、ERK 和 Src 的激活上，作为迁移信号的转导器。我们与药物化学家合作开发和测试 CXCR2 和 Src 的局部抑制剂作为抗肿瘤和抗炎药物。 ras 介导的 EGFR 激活与 NFkB 易位之间的联系已通过与 Trinchieri 实验室和 CCR 癌症与炎症项目的成员合作得到阐明。 MyD88 是促炎受体信号传导的重要中间体。 MyD88 或 IL-1 受体 (IL-1R) 消融的小鼠对局部皮肤癌具有抵抗力，并且它们的 ras 转导的角质形成细胞在原位移植物中仅形成小肿瘤。在缺乏 MyD88 或 IL-1R 的角质形成细胞中，ras 转导会激活 EGFR 环，但这些角质形成细胞不会上调促炎配体。现在已经清楚，EGFR ras 激活的下游是 IL-1α 的释放，这是导致 NFkB 易位和诱导肿瘤形成所必需的炎症级联的关键因素。这些模型被用来剖析炎症和自分泌趋化因子/细胞因子信号传导在良性肿瘤发展中的作用，并测试 IL-1R 抑制剂预防或逆转 ras 介导的肿瘤发展的有效性。开发了第二种模型来研究皮肤炎症和肿瘤发展。之前，我们描述了两种高度同源的人类 S100 蛋白（A7 和 A15）的促炎趋化作用，它们最初是从发炎的银屑病病变中克隆出来的，它们在发炎的银屑病病变中高度表达，并且与银屑病的发展存在遗传相关性。我们开发了区分性抗体，揭示了它们在银屑病中的差异表达模式，表明了它们的功能多样性。我们发现RAGE是hS100A7的受体，G偶联蛋白介导hS100A15的趋化活性。每种蛋白质都是促炎性的，并且它们会加剧体内炎症。然后，我们克隆了单个小鼠基因祖细胞 (mS100A7A15)，生成了针对该蛋白质的抗体，并证明 mS100A7A15 保留了人类特性。与人类对应物一样，小鼠祖细胞受到表皮分化的调节，并在分化良好的上皮肿瘤中高度表达。同样，人类 S100A7 和 S100A15 蛋白在上皮性乳腺癌中均上调。 mS100A7A15 功能保守，也与 RAGE 结合，是一种化学引诱剂。针对小鼠表皮的诱导型 mS100A7A15 转基因刺激免疫细胞浸润并上调炎症细胞因子和趋化因子，从而增强对外部刺激的炎症反应。这些小鼠可以作为牛皮癣模型，也可以作为临床前模型来检查假定的 RAGE 抑制剂作为潜在抗炎剂以防止肿瘤生长。小鼠和人类鳞状组织中的肿瘤进展与 EGFR 的扩增有关，这已成为治疗的目标。用于癌症治疗的抗EGFR疗法的主要剂量限制不良后果是严重的皮肤毛囊炎。为了模拟这种不利条件，我们从小鼠体内或有条件地删除了小鼠皮肤中的 EGFR。在缺乏 EGFR 的情况下，皮肤肿瘤会过早分化并且不会生长。对来自 ras 转导的野生型、EGFR 缺失或 EGFR 抑制剂处理的角质形成细胞的 RNA 进行微阵列分析，揭示了肿瘤细胞中 EGFR 消融的 25 个基因特征。 p38 的激活是该特征中的一个节点，并且磷酸化 p38 在 EGFR 缺失肿瘤中升高。在 p38 激活的情况下，IP10（一种与 CXCR3 和 T 细胞、树突细胞和巨噬细胞迁移相关的趋化因子）增加。 p38 的三种同工型在角质形成细胞中表达，当前的敲除研究旨在确定哪种同工型因 EGFR 的抑制而被激活。皮肤靶向 EGFR 消融小鼠的表型强调了这些发现与人类病理学的相关性。广泛的毛囊炎和脱发是使用 EGFR 抑制剂药物的患者的剂量限制性皮肤损伤的特征，并且在 EGFR 表皮消融的成年小鼠中很常见。从成年转基因小鼠非病变皮肤中分离的组织样本显示，炎症介质子集的 mRNA 水平升高，即 MIP-2、MIP-3α、MCP-1、TNFα、IL-6、IL-17、IL-18、TARC、 TGFbeta、iNOS 和 G-CSF。相反，在 EGFR 受到抑制的小鼠和人类表皮中，皮肤 GM-CSF 减少。靶向敲除小鼠还表现出较高的血浆 IL-17、IL-6、IP-10、MCP-1、TGFbeta 和 G-CSF 水平。在 EGFR 缺失的小鼠皮肤中检测到更高的基础和刺激的 CD45 + 细胞浸润，并且肥大细胞和巨噬细胞更丰富。成年双转基因小鼠表现出更高水平的 T 细胞激活（CD69 阳性细胞），并且从淋巴结和脾脏收集的 γ-δ T 细胞和 CD11b/Gr1 阳性细胞的百分比增加。用 TNFα 中和药物 Embrel 治疗小鼠 3 周，并没有影响这些细胞的丰度，而用中和 G-CSF 抗体治疗使脾脏中检测到的 CD11b/Gr1 阳性细胞和 γ-δ T 细胞的百分比正常化和淋巴结。目前正在研究他莫昔芬调节的条件性皮肤靶向 EGFR 缺失小鼠，以探讨在肿瘤诱导的每个阶段调节 EGFR 表达的作用。