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.

一部分 NSCLC、HNSCC、mCRC 和胰腺癌患者对几种针对表皮生长因子受体 (EGFR) 的药物治疗有反应。患者均出现丘疹脓疱性毛囊炎，常伴有脱发、干皮病以及指甲和睫毛的变化。为了在小鼠模型中模拟这种皮疹，在角蛋白 5 驱动的 Cre 重组酶转基因小鼠与 EGFR floxed 小鼠杂交的后代中，表皮中的 EGFR 被消除。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损伤的特征：炎症、瘙痒、皮肤干燥并伴有中性粒细胞性脓疱以及肥大细胞、巨噬细胞和淋巴细胞的浸润。从双转基因小鼠皮肤分离的组织样本含有高水平的炎症介质子集 mRNA，即 TNF-α、IL-18、IL-1β、IL-1ra、G-CSF、iNOS 和 CCL2。在双转基因小鼠的血浆中也测量到了较高水平的这些介质。 EGFR 切除小鼠体温正常，但心率较慢，这与这些动物典型的较小体型形成鲜明对比。从 EGFR 消融小鼠皮肤提取的 RNA 中，TGF-α、双调蛋白和 HB-EGF 下调，而配体上皮调节蛋白则高度上调。通过将 EGFR 切除小鼠与缺少 TNFR1/2 和 iNOS 等重要炎症通路关键调节因子的基因敲除小鼠杂交，或通过施用中和性 G-CSF 抗体来改善皮肤表型的尝试失败了。目前我们正在将双转基因小鼠与 MyD88、CCR2 和 Rag1 敲除小鼠进行杂交。我们获得了 10 名患者在接受抗 EGFR 药物吉非替尼治疗前和治疗 1 个月后收集的血浆样本。我们观察到，接受吉非替尼治疗后，10 名患者中至少有 8 名的炎症介质（CCL11、CCL22、CCL4、IL-1ra、IL-18）升高。检查的其他介质（CCL2、CCL5、CCL17、CCL20、CXCL10、CXCL12、髓过氧化物酶）在所有不同患者中具有不同的结果。此外，治疗患者血浆中的IL-6和CXCL8总体稳定或降低。治疗前后非病变皮肤的冷冻切片用于提取编码细胞因子和趋化因子的RNA。患者体内的表达模式差异很大，并且血浆变化的方向并不总是相同。抗 EGFR 疗法的一个有趣的方面，特别是对于 NSCLC、HNSCC 和 mCRC，是对 RAS 癌基因活性突变体患者的反应较差。去年，我们描述了我们发现的 25 个基因特征，该特征表征了被致癌 ras 转化并通过基因或药理学消除 EGFR 的角质形成细胞。对ras癌基因/EGFR消融特征的独创性分析表明，该特征的56%与一个网络相匹配，并且该网络的中心节点是p38。其他研究表明，p38 在角质形成细胞中被激活，通过基因或药理学消除 EGFR 并用致癌 ras 转化。在 EGFR null/ras 转化的角质形成细胞的肿瘤同种移植物中也观察到 p38 的激活。使用 siRNA，我们发现负责激活的主要 p38 同工型是 p38 α。 p38 的激活和 25 个基因特征中观察到的基因表达变化也出现在经激活的 H-ras 转化的药物处理的人角质形成细胞中，但未见于药物处理的未转化的人角质形成细胞中。对通过基因或药理学消除 EGFR 且未进行 ras 转化的小鼠角质形成细胞进行的额外微阵列分析揭示了 19 个基因特征，其中 8 个基因下调，11 个基因上调。在没有 ras 转化的情况下，对 EGFR 消融特征的 Ingenuity 分析揭示了 57% 的特征基因与一个网络相匹配，中心节点集中于 Erk1/2 和 IL-1beta。这些变化可能与抗 EGFR 治疗期间出现的炎症性皮肤表型有关。在角质形成细胞致癌 ras 转化过程中观察到的可重复表型变化之一是分化过程中基底上角蛋白 K1 和 K10 表达的丧失。我们现在发现这种先前无法解释的反应是由于 MyD88 和 IL-1R 下游的 NF-kappaB 活性所致。从 K1 基因启动子克隆的 2.5kb 片段包含 NFkappaB 共有结合位点。当该序列用于驱动荧光素酶报告基因时，致癌性 ras 会抑制报告基因活性。然而，MyD88 缺陷并不能阻止报告基因活性的减少。 K1 转录物 3UTR 包含 miR203 的高度保守位点。致癌性 ras 在分化条件下反对 miR203 的诱导，但需要进一步的工作来确定这是否是通过 NFkappaB 介导的。 ROCK 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器，参与肌动蛋白-细胞骨架组装以及细胞运动和收缩。为了评估 ROCK 活性在小鼠角质形成细胞分化中的作用，测试了 ROCK 特异性抑制剂 Y-27632 的作用。在 0.05 mM 钙培养基中培养的原代小鼠角质形成细胞中，Y-27632 抑制 ROCK 诱导角质形成细胞分化标志物 K1 和 K10 表达（mRNA 和蛋白质）。 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。向 ras 转化的角质形成细胞中添加 Y-27632 也恢复了 ras 抑制的 K1 和 K10 表达。特异性靶向 ROCK1 和 ROCK2 的 siRNA 降低了 ROCK 蛋白表达并增强了 K1 和 K10 表达。 我们的研究表明，ROCK 活性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非常重要。 在启动促进肿瘤诱导实验中，角质形成细胞中 MyD88 的缺乏会降低肿瘤产量。 MyD88 的缺失可能会影响启动和促进，因为由致癌 ras 转化的角质形成细胞或通过 12-十四烷酰佛波醇-13-乙酸酯 (TPA) 处理所产生的促炎趋化因子和细胞因子会随着 MyD88 的消融而减弱。为了阐明角质形成细胞衍生的炎症对皮肤癌的作用，使用了双转基因小鼠模型（HGF-PKCα或DT）。在基底角质形成细胞中过度表达 PKCα 并在局部 TPA 应用后产生强烈的中性粒细胞皮肤炎症反应的 K5-PKCα 小鼠与在金属硫蛋白启动子下过度表达 HGF 的黑色素瘤倾向 MT1-HGF 小鼠杂交，产生 HGF-PKCα 小鼠及其各自的对照。虽然 PKCalpha 驱动的表皮炎症减少了 DT 小鼠的黑色素瘤生成，但 DT 动物对鳞状癌非常敏感。我们假设在 DT 小鼠中，角质细胞衍生的 HGF 与 PKCα 协同作用，促进肿瘤生长并增加肿瘤生长。来自 HGF 或 DT 小鼠的原代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 角质形成细胞中反式激活，但在 PKCα 或 WT 角质形成细胞中不反式激活。 ras 角质形成细胞培养上清液中 CXCL1 的释放增加，其中 DT ras 角质形成细胞产生最多。这些结果共同表明，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 PKCα 增强肿瘤促进的能力相关。