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.

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（EGFR KO）的皮肤再现了接受抗EGFR药物化疗的患者皮肤病变的特征：炎症、瘙痒、皮肤干燥、中性粒细胞脓疱以及肥大细胞、巨噬细胞和淋巴细胞浸润。在之前的报告中，我们详细介绍了 KO 皮肤中从出生后 3 天开始上调的趋化因子和细胞因子的时间生化变化，以及从出生后 7 天开始这种生化的表型后果。我们还发现明显来自皮肤的血浆细胞因子/趋化因子水平的类似变化。发现特征性血液学变化为中性粒细胞增多、嗜酸性粒细胞增多和淋巴细胞减少。皮肤炎症先于毛囊表型的变化，并导致表皮中分化标记物的异常表达。第一个浸润细胞是巨噬细胞和肥大细胞。为了预防皮肤炎症，我们将 KO 小鼠与缺乏导致炎症反应的主要途径的小鼠杂交。表皮去除的 EGFR KO 小鼠与 iNOS、TNFR1/R2、MyD88、CCR2 和 Rag1 基因去除的小鼠回交保留了皮肤表型，并且皮肤中巨噬细胞的募集增加。这种遗传方法揭示了 IL-1/TLR 家族、CCL2 和 T/B 细胞在病变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/脂质体成功减少了皮肤中的巨噬细胞浸润。氯膦酸盐治疗部分修复了表皮 EGFR KO 皮肤的结构，在 FVB/N 和 C57Bl/6 背景中，毛囊平行生长更多，真皮细胞结构更少，角蛋白栓更少，并且细胞因子、趋化因子和趋化因子受体子集的水平降低。氯膦酸盐治疗还减少了 KO 皮肤中浸润性肥大细胞和几种表皮分化标记物的异常表达。我们与 Elise Kohn 和 Seth Steinberg 合作，检查了从参加吉非替尼卵巢癌试验的 10 名患者身上收集的血浆。通过检查治疗前和治疗后一个月循环细胞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等级和瘙痒有关。大多数检测到的循环因子在治疗后有所增加，但出乎意料的是，治疗前这些因子水平最低的患者在治疗后往往出现更严重的皮疹和瘙痒。吉非替尼治疗前后 Eotaxin/CCL11 和 IL-18 水平降低与皮疹和瘙痒加重相关。治疗后较低水平的 IL-1ra 和 MDC/CCL22 与更强的皮疹显着相关，而只有较低水平的 IL-1ra 显示出与瘙痒相关的趋势。相反，治疗开始前较高水平的 RANTES/CCL5 与瘙痒相关。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历的显着关联。与小鼠模型一样，患者在治疗时表现出中性粒细胞增多和淋巴细胞减少。血细胞计数中粒细胞（较高）和淋巴细胞（较低）百分比之间的差异（治疗后减去治疗前）与皮疹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。小鼠研究表明，巨噬细胞或肥大细胞可能是皮疹表型的根本原因，并且可以根据治疗开始前的循环趋化因子/细胞因子水平来预测人类对抗 EGFR 药物的不良皮肤反应。在之前的研究中，我们已经证明 MyD88 的消融可减少小鼠皮肤肿瘤的诱导。使用由致癌 Ras 转化的培养角质形成细胞，我们绘制了 MyD88 依赖性途径，并发现激活剂是 IL-1a，它在 EGFR 激活的下游上调。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 缺陷型角质形成细胞不会在同种移植物中形成肿瘤，但矛盾的是，IL-1R 缺陷型角质形成细胞却会在同种移植物中形成肿瘤。这表明 IL-1R 可能对肿瘤促进剂（例如佛波酯、TPA）有不同的反应。 MyD88-/- 小鼠中的急性 TPA 反应减弱（皮肤中 MPO 和趋化因子表达降低），并且在表皮特异性删除 MyD88 的小鼠中观察到类似的表型。然而，IL-1R 缺乏（遗传或药理学阻断）不会改变对 TPA 的急性反应。这一结果表明，MyD88 对急性 TPA 介导的 PMN 募集的贡献大部分独立于 IL-1 信号传导。当用 TPA 处理时，缺乏 MyD88 的原代角质形成细胞未能上调 CXCL1 表达。然而，在 IL-1R 缺陷的角质形成细胞中，TPA 对 CXCL1 的诱导作用得以维持。这提供了两种菌株体内肿瘤形成差异的机制。在 MyD88 完全缺乏的小鼠中，重复 TPA 应用 (4x) 引起的表皮增生有所减少，但在角质形成细胞特异性删除 MyD88 的小鼠中，表皮增生得到部分维持。该结果表明非表皮群体有助于与肿瘤促进相关的过度增殖。另一种对皮肤肿瘤诱导感兴趣的生长因子是肝细胞生长因子（HGF）。我们一直在与 Glenn Merlino 合作研究过度表达 HGF 的小鼠皮肤肿瘤的形成。这些小鼠已与皮肤中过度表达 PKCα 的小鼠杂交，这种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）。 DMBA和TPA后DT小鼠皮肤肿瘤形成的发生率大大增加。经过 16 周的肿瘤促进（在没有之前的 DMBA 的情况下），100% 的 DT 小鼠出现鳞状乳头状瘤。在这些 DT 乳头状瘤中未发现 ras 突变。这些结果表明，异常的 HGF 表达可能通过 EGFR 的反式激活来补偿起始的缺乏。它提供了在没有 ras 突变的情况下发生 SCC 的潜在机制。 K5-PKCα 和 DT 小鼠体内对 TPA 更敏感，微脓肿形成、趋化因子表达、Cox-2 诱导和表皮增生证明了这一点。与 WT 或 K5-PKCa 角质形成细胞相比，在没有激活 ras 的情况下，HGF 和 DT 角质形成细胞中的活化 ras 水平（ras-GTP 下拉测定）较高。在没有 ras 转导的情况下，HGF 和 DT 原代角质形成细胞中角质形成细胞表达的所有 EGFR 配体均上调。使用 EGFR 抑制剂 (PD168393) 治疗可减弱 HGF 和 DT 角质形成细胞的基线 Ras 样表型。这些结果共同表明肿瘤环境中 HGF 的高表达可以替代 ras 的激活。