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

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

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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启动细胞的表型。高增殖，对末端分化和基因表达的改变是该表型的标志。我们以前的研究表明，RAS激活足以产生这种完整的表型并完全启动肿瘤形成。由于致癌性RA是人类癌症中最常检测到的癌基因，并且经常与上皮癌发展中的早期事件有关，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，致癌性RA通过表皮生长因子受体（EGFR）建立了阳性的反馈回路，增加了增殖并同时激活SRC激酶以磷酸化和抑制PKC Delta，这是Keratinocycyte术语的重要层。 RAS通过增加的二酰基甘油的产生通过增加角质形成细胞分化标记的表达来改变了PKCALPHA的激活，从而改变了AP-1活性。通过将PKCALPHA靶向遗传改变的小鼠的表皮，我们表明，由于RAS对EGFR和EGFR和PKCALPHA的作用，NFKB将NFKB转移到角质形成细胞的核并上调CXCR2 Ligands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。 CXCR2在RAS转导的小鼠角质形成细胞上的遗传消融抑制了迁移并排除肿瘤的形成。角质形成细胞中CXCR2激活的下游信号集中在Akt，ERK和SRC作为迁移信号的换能器中的激活。我们与药物学家合作，我们正在开发和测试CXCR2和SRC的局部抑制剂作为抗肿瘤和抗炎药。 RAS介导的EGFR激活与NFKB易位之间的联系已与Trinchieri实验室成员与CCR癌症和炎症计划合作阐明。 MyD88是来自促炎受体的信号传导中的关键中间。用于MyD88或IL-1受体（IL-1R）的小鼠对局部皮肤致癌作用具有抗性，其RAS转导的角质形成细胞仅形成原位移植物中的小肿瘤。在缺乏MYD88或IL-1R的角质形成细胞中，RAS的转导会激活EGFR回路，但这些角质形成细胞不会上调促炎的配体。现在很明显，EGFR的RAS激活下游是IL-1Alpha的释放，这是导致NFKB易位的关键元素，并引起了炎症性级联反应对肿瘤形成必不可少的。这些模型被用来剖析炎症和自分泌趋化因子/细胞因子信号在良性肿瘤发育中的贡献，并测试IL-1R抑制剂预防或反向RAS介导的肿瘤发育的有效性。已经开发出第二种模型来研究皮肤炎症和肿瘤的发展。以前，我们描述了两种高度同源的人类S100蛋白（A7和A15）的促炎性趋化作用，该蛋白（A7和A15）最初是从发炎的银屑病病变中克隆的，在这些损伤中，它们高度表达并与牛皮癣的发展有遗传相关。我们开发了歧视抗体，这些抗体揭示了其在牛皮癣中的差异表达模式，表明其功能多样性。我们发现愤怒是HS100A7的受体，G偶联蛋白介导了HS100A15的趋化活性。每种蛋白质都是促炎性的，并且在体内会增强炎症。然后，我们克隆了单小鼠基因祖细胞（MS100A7A15），该基因产生了对蛋白质的抗体，并证明了MS100A7A15保留了人类特性。作为人类对应物，小鼠祖细胞受表皮分化的调节，并在分化良好的上皮肿瘤中高度表达。同样，人类S100A7和S100A15蛋白都在上皮乳腺癌中上调。在功能上保守的，MS100A7A15也与愤怒结合，是趋化因子。针对小鼠表皮的诱导型MS100A7A15转基因刺激免疫细胞的浸润，并上调炎症细胞因子和趋化因子和趋化因子，从而响应外部刺激，从而增强炎症。这些小鼠可以作为牛皮癣的模型，但也将作为临床前模型，以检查愤怒的推定抑制剂，作为潜在的抗炎药，以防止肿瘤促进。小鼠和人类鳞状组织中的肿瘤进展与EGFR的扩增有关，这已成为治疗的靶标。抗EGFR治疗对癌症治疗的主要限制不良后果是严重的皮肤毛囊炎。为了建模这种不良条件，我们在遗传上删除了小鼠或小鼠皮肤中的EGFR。在没有EGFR的情况下，皮肤肿瘤过早区分并且不会生长。 RAS中RNA的微阵列分析转导的WildType，EGFR NULL或EGFR抑制剂处理的角质形成细胞揭示了肿瘤细胞中EGFR消融的25个基因特征。 p38的激活是该特征中的一个节点，而EGFR无效肿瘤中的磷酸p38升高。在这种p38激活的情况下，IP10增加了与CXCR3相关的趋化因子以及T细胞，树突状细胞和巨噬细胞的迁移。 p38的三种同工型在角质形成细胞中表达，当前的敲低研究被指示以确定响应于EGFR的抑制而激活的同工型。这些发现与人类病理的相关性是由小鼠的表型与皮肤靶向EGFR消融的相关性。广泛的卵泡炎和脱发是EGFR抑制剂药物中患者的剂量限制皮肤病变的特征，并且在EGFR表皮消融的成年小鼠中很常见。从成年转基因小鼠的非病变皮肤中分离出的组织样品显示出炎症介质的子集的mRNA水平，即MIP-2，MIP-3Alpha，MCP-1，TNFALPHA，IL-6，IL-6，IL-17，IL-17，IL-18，IL-18，IL-18，IL-18，TARC，TARC，TGFBETA，INOS，INOS和G-CSF。相反，在抑制EGFR的小鼠和人类表皮中，皮肤GM-CSF降低。靶向敲除小鼠还显示出较高的血浆水平IL-17，IL-6，IP-10，MCP-1，TGFBETA和G-CSF。在EGFR无效的小鼠皮肤中检测到CD45 +细胞的较高基础和刺激的浸润，肥大细胞和巨噬细胞更丰富。成年双转基因小鼠显示出更高水平的T细胞激活（CD69阳性细胞），并增加了从淋巴结和脾脏收获的CD11b/GR1阳性细胞的伽马 - 二烷T细胞和CD11b/GR1阳性细胞的增加。用TNFALPHA中和药物的治疗小鼠3周，在用中和用中和的G-CSF抗体治疗CD11b/GR1阳性细胞和脾脏和淋巴结中检测到的CD11b/Gr1阳性细胞的百分比，并不影响这些细胞的丰度。他莫昔芬调节的条件皮肤靶向EGFR无效小鼠现在正在研究中，以解决调节在肿瘤诱导的每个阶段调节EGFR表达的作用。