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: 8552562
• 负责人: STUART H. YUSPA
• 金额: $ 116.3万
• 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/8552562
• 关键词: Ablation; Acute; Aftercare; Alopecia; Architecture; Attenuated; B-Lymphocytes; Backcrossings; Biochemical; Biochemistry; Biological Assay; Biological Models; Birth; Blood Cell Count; CCL11 gene; CCL2 gene; CCL22 gene; CXCL1 gene; Cell Line; Cells; Cellularity; Characteristics; Collaborations; Cultured Cells; Cutaneous; Dermal; Development; Dichloromethylene Diphosphonate; Differentiation Antigens; Differentiation and Growth; Disease; Environment; Eosinophilia; Eotaxin; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Epidermis; Epithelium; Exanthema; Exhibits; Eyelash; Family; Gefitinib; Genes; Genetic; Growth Factor; Guanosine Triphosphate; Hair follicle structure; Head and Neck Squamous Cell Carcinoma; Hepatocyte Growth Factor; Homeostasis; Human; Hyperplasia; In Vitro; Incidence; Indium; Infiltration; Inflammation; Inflammation Mediators; Inflammatory; Interleukin-1; Interleukin-18; Keratin; Laboratories; Lesion; Ligands; Liposomes; Lymphocyte; Lymphopenia; Malignant Neoplasms; Malignant Squamous Cell Neoplasm; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Maps; Measures; Mediating; Methods; Modeling; Monitor; Mouse Strains; Mus; Mutation; Nail plate; Neoplastic Cell Transformation; Neutrophilia; Non-Small-Cell Lung Carcinoma; Oncogenic; Papilloma; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phorbol Esters; Plasma; Platelet Count measurement; Population; Premalignant; Procedures; Pruritus; RANTES; Rag1 Mouse; Reaction; Reagent; Regulation; Reporting; Research; Role; Series; Severities; Signal Pathway; Signal Transduction; Skin; Skin Neoplasms; Skin Transplantation; Skin graft; Squamous Cell; Squamous Cell Papillomas; Subcutaneous Injections; Systems Biology; TNFRSF1A gene; Transactivation; Transgenic Mice; Tumor Promoters; Tumor Promotion; anakinra; autocrine; base; cancer initiation; carcinogenesis; chemokine; chemokine receptor; chemotherapy; cytokine; dimethylbenzanthracene; experience; granulocyte; in vitro Model; in vivo; interest; keratin 5; keratinocyte; macrophage; mast cell; mouse model; neoplastic; overexpression; paracrine; prevent; recombinase; repaired; response; skin disorder; skin lesion; therapeutic target; trend; tumor

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 (EGFR KO) 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. In previous reports we detailed the temporal biochemical changes in chemokines and cytokines that are upregulated in KO skin starting 3 days after birth and the phenotypic consequences of this biochemistry starting 7 days after birth. We also showed similar changes in plasma cytokine/chemokine levels apparently emanating from the skin. A characteristic hematological change was identified with neutrophilia, eosinophilia and reduction in lymphocytes. Cutaneous inflammation preceded changes in the hair follicle phenotype and resulted in I aberrant expression of differentiation markers in the epidermis. The first infiltrating cells are macrophages and mast cells. In an attempt to prevent the skin inflammation, we crossed the KO mice with mice deficient in major pathways leading to an inflammatory reaction. Backcrosses of the epidermal ablated EGFR KO mice with mice genetically ablated for iNOS, TNFR1/R2, MyD88, CCR2 and Rag1 micemice retained the skin phenotype and the increased recruitment of macrophages in the skin. This genetic approach revealed a lack of a primary role for the IL-1/TLR family, CCL2 and T/B cells in the development of the lesions. In a second approach we successfully reduced macrophage infiltration in the skin by subcutaneous injection of clodronate/liposomes. Clodronate treatment partially repaired the architecture of epidermal EGFR KO skin with more parallel growing hair follicles, less dermal cellularity, fewer keratin plugs, and reduced levels of a subset of cytokines, chemokines and chemokine receptors in both FVB/N and C57Bl/6 background. Clodronate treatment also reduced infiltrating mast cells and aberrant expression of several epidermal differentiation markers in the KO skin. In collaboration with Elise Kohn and Seth Steinberg we examined the plasma collected from 10 patients participating in a Gefitinib trial for ovary cancer. Examining a series of changes in circulating cytokines and chemokines before and one month after treatment we determined that inflammatory mediators levels were related to rash grade and pruritus. Most circulating factors examined increased after treatment, but unexpectedly, patients whose pretreatment levels of these factors were the lowest tended to have greater rash and pruritus following treatment. Lower Eotaxin/CCL11 and IL-18 levels before and after treatment with Gefitinib wereas associated with stronger rash and pruritus. Lower levels of IL-1ra and MDC/CCL22 after the treatment were significantly associated with stronger rash while only lower levels of IL-1ra showed a trend to be associated with pruritus. In contrast, higher levels of RANTES/CCL5 before the treatment initiates were associated with pruritus. We measured the blood cell counts of eight patients and analyzed for trends or significant association with the rash grade or the pruritus experience. As in the mouse model, patients exhibited neutrophilia and lymphopenia on treatment. The difference (after treatment minus before treatment) between the percentage of granulocytes (higher) and lymphocytes (lower) in the blood cell counts was significantly associated with rash severity and pruritus. Moreover, larger differences in the absolute number of platelets are significantly associated with pruritus. The mouse studies suggest that macrophages or mast cells may be fundamentally causative in the rash phenotype and human adverse skin response to anti-EGFR drugs may be predicted based on circulating chemokine/cytokine levels before treatment begins. In previous studies we have demonstrated that ablation of MyD88 reduces skin tumor induction in mice. Using cultured keratinocytes transformed by oncogenic Ras, we have mapped the MyD88 dependent pathway and discovered that the activator is IL-1a that is upregulated downstream from activation of EGFR. This leads to NFkB activation, expression of multiple pro-inflammatory factors and both autocrine and paracrine consequences important to tumor induction. Ras transformed MyD88 deficient keratinocytes do not form tumors in orthografts but paradoxically IL-1R deficient keratinocytes do form tumors in orthografts. This suggested that IL-1R may respond differently to tumor promoters, such as the phorbol ester, TPA. Acute TPA response is attenuated in MyD88-/- mice (lower MPO and chemokine expression in the skin) and a similar phenotype is observed in mice with an epidermal specific deletion of MyD88. However IL-1R deficiency (genetic or pharmacological blockade) does not alter the acute response to TPA. This result suggests that MyD88 contribution to acute TPA mediated recruitment of PMNs is mostly independent of IL-1 signaling. Primary keratinocytes deficient for MyD88 failed to upregulate CXCL1 expression when treated with TPA. However, the induction of CXCL1 by TPA in IL-1R deficient keratinocytes is maintained. This provides a mechanism for the difference observed in tumor formation among the two strains in vivo. The epidermal hyperplasia caused by repeated TPA application (4x) is reduced in MyD88 totally deficient mice but is partially maintained in mice with keratinocyte specific deletion of MyD88. This result suggests that a non epidermal population contributes to the hyperproliferation associated with tumor promotion. Another growth factor of interest for skin tumor induction is Hepatocyte Growth Factor (HGF). We have been studying skin tumor formation in mice overexpressing HGF in collaboration with Glenn Merlino. These mice have been crossed to mice overexpressing PKCalpha in skin, a model that leads to induced acute neutrophilic inflammation when activated topically. These are double transgenic mice (DT). DT mice have a greatly increased incidence of skin tumor formation after DMBA and TPA. After 16 weeks of tumor promotion (in the absence of prior DMBA), squamous papillomas appear in 100% of DT mice. No ras mutations have been found in those DT papillomas. These results suggest that aberrant HGF expression can compensate for the lack of initiation, likely through the transactivation of EGFR. It provides a potential mechanism for the development of SCC in the absence of ras mutations. K5-PKCalpha and DT mice are more sensitive to TPA in vivo as evidenced by microabscess formation, chemokines expressions, Cox-2 induction and epidermal hyperplasia. Levels of activated ras (ras-GTP pull down assay) are higher in HGF and DT keratinocytes in the absence of activated ras when compared to WT or K5-PKCa keratinocytes. All EGFR ligands expressed by keratinocytes are upregulated in HGF and DT primary keratinocytes in the absence of ras transduction. Treatment with an EGFR inhibitor (PD168393) attenuates the baseline Ras-like phenotype of HGF and DT keratinocytes. Together these results suggest that high expression of HGF in the tumor environment could substitute for ras activation.

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（EGFR KO）的皮肤再现了接受抗EGFR剂化学疗法的患者的皮肤病变的标志：炎症，瘙痒症，伴有嗜中性脓肿的皮肤干燥的皮肤和乳腺细胞的浸润，巨噬细胞，巨噬细胞和淋巴细胞。在先前的报告中，我们详细介绍了趋化因子和细胞因子的时间生物化学变化，这些变化从出生后3天开始在KO皮肤上上调，以及该生物化学的表型后果从出生后7天开始。我们还显示出显然来自皮肤的血浆细胞因子/趋化因子水平的类似变化。用嗜中性粒细胞，嗜酸性粒细胞和淋巴细胞降低来鉴定出特征性的血液学变化。皮肤炎症先于毛囊表型的变化，并导致表皮中分化标记的异常表达。第一个浸润细胞是巨噬细胞和肥大细胞。为了防止皮肤炎症，我们越过KO小鼠，小鼠在主要途径中缺乏导致炎症反应的主要途径。表皮消融的EGFR KO小鼠的反向杂交，其遗传烧蚀用于iNOS，TNFR1/R2，MYD88，CCR2和RAG1 MICEMICE，保留了皮肤表型，并增加了皮肤中巨噬细胞的募集。这种遗传方法表明，IL-1/TLR家族，CCL2和T/B细胞在病变的发展中缺乏主要作用。在第二种方法中，我们通过皮下注射氯膦酸盐/脂质体成功地减少了皮肤中巨噬细胞的浸润。克罗德膦酸盐的治疗部分修复了表皮EGFR KO皮肤的结构，其毛囊生长较大，皮肤细胞较少，角蛋白塞的较少以及降低的FVB/N和C57BL/6背景中的细胞因子，趋化因子和趋化因子和趋化因子受体的水平降低。克罗德膦酸盐处理还减少了浸润性肥大细胞和KO皮肤中几个表皮分化标记的异常表达。与Elise Kohn和Seth Steinberg合作，我们检查了参加卵巢癌试验的10名患者收集的血浆。在治疗之前和一个月之前，检查了循环细胞因子和趋化因子和趋化因子的一系列变化，我们确定炎症介质水平与皮疹级和瘙痒有关。在治疗后检查的大多数循环因素都增加了，但出乎意料的是，这些因素预处理水平的患者最低的是治疗后的皮疹和瘙痒更大。在用吉法替尼治疗之前和之后，eotaxin/ccl11和IL-18水平与皮疹和瘙痒相关。治疗后IL-1RA和MDC/CCL22的水平较低，与皮疹更强，而仅IL-1RA水平较低，与瘙痒相关的趋势。相比之下，在治疗开始之前，较高水平的rantes/ccl5与瘙痒有关。我们测量了八名患者的血细胞计数，并分析了趋势或与皮疹等级或瘙痒经验的显着关联。与小鼠模型一样，患者在治疗时表现出嗜中性粒细胞和淋巴细胞减少症。血细胞计数中粒细胞（较高）和淋巴细胞（较低）的百分比之间的差异（在治疗前减去治疗）与疹的严重程度和瘙痒显着相关。此外，血小板绝对数量的较大差异与瘙痒显着相关。小鼠的研究表明，巨噬细胞或肥大细胞在皮疹表型中可能是基本原因是基于循环趋化因子/细胞因子水平在治疗开始之前，可以预测对抗EGFR药物的人类不良皮肤反应。在先前的研究中，我们已经证明MyD88的消融可减少小鼠皮肤肿瘤的诱导。使用培养的角质形成细胞由致癌Ras转化，我们绘制了MyD88依赖性途径，并发现激活剂是IL-1A，它因EGFR的激活而下游上调。这导致NFKB激活，多种促炎因子的表达以及对肿瘤诱导很重要的自分泌和旁分泌后果。 RAS转化的MyD88缺陷角质形成细胞不会在正交移植物中形成肿瘤，而是矛盾的IL-1R缺乏的角质形成细胞确实会在正交移植中形成肿瘤。这表明IL-1R对肿瘤启动子的反应不同，例如Phorbol酯TPA。急性TPA反应在MyD88 - / - 小鼠（皮肤中的MPO和趋化因子表达较低）中减弱，并且在MyD88表皮特异性缺失的小鼠中观察到类似的表型。但是IL-1R缺乏（遗传或药理阻滞）不会改变对TPA的急性反应。该结果表明，MyD88对PMN的急性TPA介导的募集的贡献主要独立于IL-1信号传导。用TPA处理时，原发性角质形成细胞缺乏MyD88的表达无法上调CXCL1。但是，TPA在IL-1R缺乏的角质形成细胞中诱导CXCL1。这为体内两种菌株中肿瘤形成中观察到的差异提供了一种机制。由重复的TPA应用引起的表皮增生（4X）在MyD88完全不足的小鼠中降低，但在具有角质形成细胞特定的MyD88的小鼠中部分维持。该结果表明，非表皮种群有助于与肿瘤促进相关的过度增殖。皮肤肿瘤诱导的另一个生长因素是肝细胞生长因子（HGF）。我们一直在研究与Glenn Merlino合作过表达HGF的小鼠的皮肤肿瘤形成。这些小鼠已被横断到过表达皮肤的PKCALPHA的小鼠，该模型在局部激活时会导致诱发急性中性粒细胞炎症。这些是双转基因小鼠（DT）。 DT小鼠DMBA和TPA后皮肤肿瘤形成的发生率大大增加。肿瘤促进16周后（在没有先验DMBA的情况下），鳞状乳头瘤中出现在100％的DT小鼠中。在那些DT乳头状瘤中未发现RAS突变。这些结果表明，异常的HGF表达可以通过EGFR的反式激活来补偿缺乏起始。在没有RAS突变的情况下，它为SCC的发展提供了潜在的机制。 K5-PKCALPHA和DT小鼠对体内TPA的敏感性更高，这是微观鳞状形成，趋化因子表达，COX-2诱导和表皮增生的证明。与WT或K5-PKCA角化形成细胞相比，在没有活化的Ras的情况下，HGF和DT角质形成细胞的活化RA（RAS-GTP下拉测定）的水平较高。在没有RAS转导的情况下，HGF和DT原代角质形成细胞中的所有EGFR配体在HGF和DT原代角质形成细胞中均上调。用EGFR抑制剂（PD168393）治疗HGF和DT角质形成细胞的基线Ras样表型。总之，这些结果表明，HGF在肿瘤环境中的高表达可以代替RAS激活。