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

• 批准号: 8348863
• 负责人: STUART H. YUSPA
• 金额: $ 117.89万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8348863
• 关键词: Ablation; Acetates; Actins; Aftercare; Alopecia; Amphiregulin; Animals; Antibodies; Attenuated; Basal Cell; Binding Sites; Biological Models; Body Temperature; CCL17 gene; CCL2 gene; CCL20 gene; CCL22 gene; CCL4 gene; CSF3 gene; CXCL1 gene; CXCL10 gene; CXCL12 gene; Calcium; Cell Culture Techniques; Cell Line; Characteristics; Consensus; Cultured Cells; Cutaneous; Cytoskeleton; DTR gene; Development; Differentiation Antigens; Differentiation and Growth; Disease; Down-Regulation; Epidermal Growth Factor Receptor; Epidermis; Epiregulin; Epithelium; Exanthema; Eyelash; Frozen Sections; Gefitinib; Gene Expression; Genes; Genetic; HRAS gene; Head and Neck Squamous Cell Carcinoma; Heart Rate; Homeostasis; Human; IL8 gene; In Vitro; Indium; Infiltration; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Interleukin-18; Interleukin-6; Keratin; Knockout Mice; Laboratories; Ligands; Luciferases; Lymphocyte; MAPK14 gene; Malignant Neoplasms; Malignant Squamous Cell Neoplasm; Malignant neoplasm of pancreas; Measures; Mediating; Mediator of activation protein; Messenger RNA; Metallothionein; Methods; Modeling; Monitor; Mouse Strains; Mus; NF-kappa B; Nail plate; Neoplastic Cell Transformation; Non-Small-Cell Lung Carcinoma; Oncogenes; Oncogenic; Outcome; Pathogenesis; Pathway interactions; Patients; Pattern; Peroxidases; Pharmaceutical Preparations; Phenotype; Plasma; Premalignant; Procedures; Protein Isoforms; Protein-Serine-Threonine Kinases; Proteins; Pruritus; RANTES; RNA; ROCK1 gene; Rag1 Mouse; Reagent; Receptor Signaling; Regulation; Reporter; Research; Rho-associated kinase; Role; Sampling; Signal Pathway; Signal Transduction; Site; Skin; Skin Cancer; Skin Transplantation; Skin graft; Small Interfering RNA; Squamous Cell; Systems Biology; TNFRSF1A gene; Testing; Tissue Sample; Transcript; Transforming Growth Factor alpha; Transgenic Mice; Transgenic Organisms; Tumor Necrosis Factor-alpha; Tumor Promotion; Up-Regulation; Work; anakinra; cancer initiation; carcinogenesis; cell motility; chemokine; chemotherapy; cytokine; human TNF protein; in vitro Model; in vivo; inhibitor/antagonist; interest; keratin 5; keratinocyte; keratinocyte differentiation; mRNA Expression; macrophage; mast cell; melanoma; mouse model; mutant; neoplastic; overexpression; prevent; promoter; protein expression; ras Oncogene; recombinase; research study; response; rho; skin disorder; skin lesion; therapeutic target; tumor; tumor growth

A subset of patients with NSCLC, HNSCC, mCRC and pancreatic cancer are responding to therapy by several agents directed against the epidermal growth factor receptor (EGFR). Uniformly patients develop a papulopustular follicutis often accompanied by alopecia, xeroderma and changes in nails and eyelashes. To model this skin rash in a mouse model, EGFR was ablated in the epidermis in the litters derived from Keratin 5 driven Cre recombinase transgenic mice crossed with EGFR floxed mice. The skin of double transgenic mice reproduced the hallmarks of the skin lesions of patients undergoing chemotherapy with anti-EGFR agents: inflammation, pruritis, dry skin with neutrophilic pustules and infiltration of mast cells, macrophages and lymphocytes. Tissue samples isolated from skin of double transgenic mice contained high mRNA levels of a subset of inflammatory mediators namely TNF-alpha, IL-18, IL-1beta, IL-1ra, G-CSF, iNOS and CCL2. Higher levels of these mediators were also measured in plasma of double transgenic mice. EGFR ablated mice had normal body temperature but a slower heart rate that is in contrast with the smaller size typical of these animals. TGF-alpha, Amphiregulin and HB-EGF are downregulated in RNA extracted from skin of EGFR ablated mice while the ligand Epiregulin is highly upregulated. Attempts to ameliorate the skin phenotype by crossing EGFR ablated mice with knockout mice that were missing critical regulators of the highlighted inflammatory pathways such as TNFR1/2, and iNOS or by administering neutralizing G-CSF antibodies failed. Currently we are crossing double transgenic mice with MyD88, CCR2 and Rag1 knockout mice. We obtained plasma samples of 10 patients collected before and after one month of treatment with the anti EGFR drug Gefitinib. We observed a subset of inflammatory mediators to be elevated in at least 8 out of 10 patients after treatment with Gefitinib (CCL11, CCL22, CCL4, IL-1ra, IL-18). The other mediators examined (CCL2, CCL5, CCL17, CCL20, CXCL10, CXCL12, myeloperoxidase) have a variable outcome in all the different patients. Moreover IL-6 and CXCL8 are overall stable or decreased in the plasma of treated patients. Frozen sections from nonlesional skin before and after treatment were used to extract RNA encoding cytokines and chemokines. Patterns of expression are very variable within the patients and not always in the same direction of plasma changes. An interesting aspect of anti-EGFR therapy, particularly for NSCLC, HNSCC and mCRC, is the poor response in patients with an active mutant of the RAS oncogene. Last year we described our discovery of a 25 gene signature that characterizes keratinocytes transformed by oncogenic ras and either genetically or pharmacologically ablated for EGFR. Ingenuity analysis of the ras oncogene/EGFR ablation signature indicates 56% of this signature matches to one network, and the center node of the network is p38. Additional studies indicated that p38 is activated in keratinocytes genetically or pharmacologically ablated for EGFR and transformed with oncogenic ras. Activation of p38 was also seen in tumor orthografts of EGFR null/ras transformed keratinocytes. Using siRNA we found that the primary p38 isoform responsible for the activation is p38 alpha. Activation of p38 and gene expression changes seen in the 25 gene signature are also seen in drug treated human keratinocytes transformed by activated H-ras but not in drug treated non-transformed human keratinocytes. Additional microarray analyses of mouse keratinocytes genetically or pharmacologically ablated for EGFR without ras transformation revealed a 19 gene signature, of which 8 genes were down regulated and 11 genes were up-regulated. The Ingenuity analysis of the EGFR ablation signature in the absence of ras transformation reveals 57% of the signature genes that match to one network, with center nodes focusing on Erk1/2 and IL-1beta. These changes could be relevant for the inflammatory skin phenotype that develops during anti-EGFR therapy. One of the reproducible phenotypic changes observed during oncogenic ras transformation of keratinocytes is the loss of expression of suprabasal keratins K1 and K10 during differentiation. We now find that this previously unexplained response is due to NF-kappaB activity downstream of MyD88 and IL-1R. A 2.5kb fragment cloned from the K1 gene promoter contains an NFkappaB consensus binding site. When this sequence is used to drive a luciferase reporter, oncogenic ras inhibits reporter activity. However, MyD88 deficiency does not prevent this reduction in reporter activity. The K1 transcript 3UTR contains a well conserved site for miR203. Oncogenic ras opposes the induction of miR203 in differentiating conditions, but further work will be required to determine if this is mediated through NFkappaB. ROCK or Rho-associated kinase, a serine/threonine kinase, is an effector of Rho-dependent signaling and is involved in actin-cytoskeleton assembly as well as cell motility and contraction. To evaluate the role of ROCK activity in mouse keratinocyte differentiation, the effect of Y-27632, a ROCK-specific inhibitor, was tested. Inhibition of ROCK by Y-27632 induced keratinocyte differentiation markers K1 and K10 expression (mRNA and protein) in primary mouse keratinocytes cultured in 0.05 mM calcium medium. Y-27632 also enhanced the expression of K1 and K10 in keratinocytes grown in 0.12 mM calcium. Addition of Y-27632 to ras transformed keratinocytes also restored ras suppressed K1 and K10 expression. siRNA specifically targeting ROCK1 and ROCK2 reduced ROCK protein expression and enhanced K1 and K10 expression. Our study indicates that ROCK activities are important for maintaining the basal cell phenotype in cultured mouse primary keratinocytes. MyD88 deficiency in keratinocytes reduces the tumor yield in an initiation-promotion tumor induction experiment. Both initiation and promotion may be influenced by loss of MyD88 since the proinflammatory chemokines and cytokines elaborated by keratinocytes transformed by oncogenic ras or by treatment with 12-tetradecanoylphorbol-13-acetate (TPA) are attenuated with MyD88 ablation. To elucidate the role of keratinocyte-derived inflammation on skin cancers, a double transgenic mouse model (HGF-PKCalpha or DT) was used. K5-PKCalpha mice which overexpress PKCalpha in basal keratinocytes and develop a strong neutrophilic cutaneous inflammatory response upon topical TPA application were crossed with melanoma-prone MT1-HGF mice which overexpress HGF under a metallothionein promoter to create HGF-PKCalpha mice and their respective controls. While PKCalpha driven epidermal inflammation reduces melanomagenesis in DT mice, DT animals are very sensitive to squamous carcinogenesis. We hypothesize that in DT mice, keratinocyte-derived HGF synergizes with PKCalpha to drive tumor promotion and increase tumor growth. Primary keratinocytes derived from HGF or DT mice display upregulation of keratin 8 (K8) and downregulation of K1 and K10 mRNAs. The EGFR is transactivated in HGF and DT keratinocytes but not in PKCalpha or WT keratinocytes. The release of CXCL1 is augmented in ras-keratinocyte cell culture supernatants with DT ras-keratinocytes producing the most. Together these results suggest that the sensitivity of DT transgenic skin to squamous tumor induction is associated with the ability of HGF to transactivate EGFR signaling and PKCalpha to enhance tumor promotion.

NSCLC，HNSCC，MCRC和胰腺癌患者的一部分正在对针对表皮生长因子受体（EGFR）的几种药物作出反应。统一的患者会出现丘疹肠foll虫，通常伴随着脱发，静脉皮以及指甲和睫毛的变化。为了模拟小鼠模型中的这种皮疹，EGFR在源自角蛋白5驱动的CRE重组酶转基因小鼠的表皮中烧了，与EGFR Floxed小鼠交叉。双转基因小鼠的皮肤再现了接受抗EGFR药物化疗的患者皮肤病变的标志：炎症，瘙痒，伴有嗜中性脓肿的皮肤干燥皮肤以及肥大细胞，巨噬细胞和淋巴细胞的浸润。从双转基因小鼠皮肤中分离出的组织样品含有高度mRNA水平的炎症介质，即TNF-Alpha，IL-18，IL-1Beta，IL-1Beta，IL-1BET，IL-1RA，G-CSF，INOS和CCL2。在双转基因小鼠的血浆中，还测量了这些介体的较高水平。 EGFR消融小鼠的体温正常，但心率较慢，与这些动物的典型尺寸较小相反。 TGF-Alpha，Amphiregulin和Hb-EGF在从EGFR消融小鼠皮肤中提取的RNA中下调，而配体环蛋白含量高度上调。试图通过将EGFR消融的小鼠与敲除小鼠横断的小鼠来改善皮肤表型，这些小鼠缺少突出显示的炎症途径的关键调节剂，例如TNFR1/2，iNOS或通过对中和中和G-CSF抗体进行中和失败。目前，我们正在使用MYD88，CCR2和RAG1敲除小鼠穿越双转基因小鼠。我们获得了在用抗EGFR药物吉非替尼治疗一个月之前和之后收集的10例患者的血浆样本。我们观察到在用吉非替尼治疗后，至少有10例患者中，至少有8例炎症介质（CCL11，CCL22，CCL4，CCL4，IL-1RA，IL-18）升高。其他检查的介质（CCL2，CCL5，CCL17，CCL20，CXCL10，CXCL12，骨髓过氧化物酶）在所有不同患者中都有变化的结果。此外，在治疗患者的血浆中，IL-6和CXCL8总体稳定或减少。处理前后的非常规皮肤的冷冻切片用于提取编码细胞因子和趋化因子的RNA。表达模式在患者中的变化很大，并且并不总是与等离子体变化的相同方向。抗EGFR疗法的一个有趣方面，特别是对于NSCLC，HNSCC和MCRC，是RAS癌基因活跃突变体的患者的反应较差。去年，我们描述了我们发现了一个25个基因特征，该基因特征是角质形成细胞，该角质形成细胞由致癌性Ras转化，并且在遗传学上或药理学上为EGFR烧蚀。 RAS癌基因/EGFR消融签名的Ingenity分析表明，此签名的56％与一个网络匹配，网络的中心节点为p38。其他研究表明，p38在遗传或药理学上为EGFR烧蚀并用致癌性RAS转化的p38被激活。在EGFR NULL/RAS转化的角质形成细胞的EGFR NULL/RAS的肿瘤正交移植物中也观察到p38的激活。使用siRNA，我们发现负责激活的主要p38同工型为p38 alpha。在由活化的H-RAS转化的药物治疗的人角质形成细胞中，在25个基因特征中看到的p38和基因表达变化也可以看到，但在药物治疗的未经转化的人角质形成细胞中却没有。在没有RAS转化的EGFR上对小鼠角质形成细胞进行的小鼠角质形成细胞的其他微阵列分析显示，有19个基因特征，其中8个基因受到了调节，并上调了11个基因。在没有RAS转换的情况下，EGFR消融签名的创造性分析揭示了与一个网络匹配的签名基因的57％，中心节点的重点是ERK1/2和IL-1Beta。这些变化可能与抗EGFR治疗期间发生的炎症性皮肤表型有关。角质形成细胞的致癌性RAS转化期间观察到的可重复的表型变化之一是分化过程中表达性角蛋白K1和K10的表达丧失。现在，我们发现这种以前无法解释的响应是由于MyD88和IL-1R下游的NF-kappab活动引起的。从K1基因启动子克隆的2.5kb片段包含NFKAPPAB共识结合位点。当使用此序列驱动荧光素酶报告基因时，致癌性RAS会抑制报告基因活性。但是，MYD88缺乏并不能阻止记者活动的减少。 K1转录本3UTR包含一个MiR203的保守位点。致癌性RA反对在分化条件下诱导miR203的诱导，但是需要进一步的工作才能确定这是否是通过NFKAPPAB介导的。岩石或Rho相关激酶是一种丝氨酸/苏氨酸激酶，是Rho依赖性信号传导的效应因子，参与肌动蛋白 - 细胞骨架组件以及细胞运动和收缩。为了评估岩石活性在小鼠角质形成细胞分化中的作用，测试了Y-27632（一种岩石特异性抑制剂）的作用。 Y-27632抑制岩石在0.05 mM钙培养基中培养的原代小鼠角质形成细胞中诱导角质形成细胞分化标记K1和K10表达（mRNA和蛋白）。 Y-27632还增强了在0.12 mm钙中生长的角质形成细胞中K1和K10的表达。在RAS中添加Y-27632转化的角质形成细胞也恢复了RAS抑制的K1和K10表达。 siRNA专门针对岩石1和岩石2降低了岩石蛋白的表达，并增强了K1和K10的表达。 我们的研究表明，岩石活动对于维持培养的小鼠原代角质形成细胞中的基底细胞表型很重要。 角质形成细胞的MyD88缺乏可在开始促进肿瘤诱导实验中降低肿瘤产量。启动和促进都可能受到MyD88的损失的影响，因为致癌性RAS转化的角质形成细胞阐述的促炎性趋化因子和细胞因子或细胞因子通过致癌性RAS转化或用12-四核氯酚-13-乙酸（TPA）治疗MyD88 Ablation。为了阐明角质形成细胞衍生的炎症在皮肤癌中的作用，使用了双转基因小鼠模型（HGF-PKCALPHA或DT）。在局部TPA施用时在局部TPA时过度表达PKCALPHA的K5-PKCALPHA小鼠在局部TPA时会产生强烈的中性粒细胞发炎反应，黑色素瘤prone mT1-HGF小鼠在高表达HGF的HGF下，在金属蛋白下在金属蛋白上产生HGF-PKCCCCCCCCCCCLAPHA MICESENIVE HGF，他们的HGF与他们的HGF-Pkcccalpha Miese和他们的分配相关。尽管PKCALPHA驱动的表皮炎症会降低DT小鼠的黑色素作用，但DT动物对鳞状癌的发生非常敏感。我们假设在DT小鼠中，角质形成细胞衍生的HGF与PKCALPHA协同促进肿瘤促进并增加肿瘤的生长。衍生自HGF或DT小鼠的初级角质形成细胞表现出角蛋白8（K8）的上调以及K1和K10 mRNA的下调。 EGFR在HGF和DT角质形成细胞中进行反式激活，但在PKCALPHA或WT角质形成细胞中不进行。 CXCL1的释放在Ras-keratinocyte细胞培养物中的释放量增强，DT Ras-keratinopytes产生的最多。总之，这些结果表明，DT转基因皮肤对鳞状肿瘤诱导的敏感性与HGF反式激活EGFR信号传导和PKCalpha的能力有关增强肿瘤促进。