Invasive Behavior of Tumor Cells Producing Collagenase-1

产生胶原酶 1 的肿瘤细胞的侵袭行为

• 批准号: 7028998
• 负责人: CONSTANCE E BRINCKERHOFF
• 金额: $ 24.59万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-15 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028998
• 关键词: athymic mouse; biomarker; cell line; clinical research; collagen; collagenase; extracellular matrix; fibroblast growth factor; fibroblasts; gene expression; genetic transcription; genetically modified animals; host neoplasm interaction; human tissue; laboratory mouse; melanoma; mitogen activated protein kinase; neoplasm /cancer genetics; neoplasm /cancer invasiveness; neoplasm /cancer transplantation; protooncogene; single nucleotide polymorphism; stromal cells; tissue /cell culture; athymic mouse; biomarker; cell line; clinical research; collagen; collagenase; extracellular matrix; fibroblast growth factor; fibroblasts; gene expression; genetic transcription; genetically modified animals; host neoplasm interaction; human tissue; laboratory mouse; melanoma; mitogen activated protein kinase; neoplasm /cancer genetics; neoplasm /cancer invasiveness; neoplasm /cancer transplantation; protooncogene; single nucleotide polymorphism; stromal cells; tissue /cell culture

DESCRIPTION (provided by applicant): Major factors in the morbidity and mortality of malignant melanoma are tumor invasion and metastasis, which are mediated by Matrix Metalloproteinases (MMPs). MMPs degrade the extracellular matrix, and are associated with invasive cancers. Degradation of interstitial collagens (types I and III) is needed for invasion, and is accomplished by the ubiquitously expressed collagenase-1 (MMP-1). A single nucleotide polymorphism (SNP; 2G allele) at -1607 bp (5'-GGAA-3') in the MMP-1 promoter creates a binding site for the Ets family of transcription factors and, compared to the 1G allele (5'-GAA-3'), enhances MMP-1 transcription. This 2G SNP is associated with the incidence/progression of seven malignancies, including melanoma. Levels of Basic Fibroblast Growth Factor (bFGF) correlate with invasive melanoma, and bFGF induces MMP-1 expression. In addition, an activating mutation in BRAF found in 80% of melanomas in the signaling cascade of Ras/Raf/Mek/Erk also induces MMP-1. We hypothesize that: (a) the 2G allele increases MMP-1 expression and melanoma invasion, (b) bFGF and the activating mutation in BRAF orchestrate an invasive phenotype in the tumor cells by preferentially increasing MMP-1 expression through the 2G allele, and (c) host/tumor/matrix interactions target the 2G allele in stromal cells adjacent to the tumor to further enhance MMP-1 expression and tumor invasion. We will, therefore, (1) Generate transgenic mice harboring 4 kb of the human MMP-1 promoter, containing either 1G or 2Gs at -1607 bp, linked to the beta-galactosidase reporter. Transgenes will be inserted as a single copy at the hprt locus to provide information on the transcriptional activity of MMP-1 in stromal cells. Mice will be challenged with B16 melanoma and expression of the transgenes, which reflects collagenolytic potential, will be examined in stromal cells adjacent to the tumor; (2) Convert a diploid 1G homozygous melanoma cell line to 2G homozygous, and conversely, a diploid 2G homozygous melanoma cell line to 1G homozygous, to compare effects of the 1G and 2G alleles on MMP-1 promoter and enzyme activities, and on tumor invasion in vitro and in vivo in nude mice. We will determine how bFGF and mutant BRAF target the 2G allele to enhance the invasive phenotype of melanoma cells. These studies will provide novel mechanisms for studying MMP-1 gene expression in mouse models, and contribute to our understanding of how this human interstitial collagenase contributes to the pathology of invasive melanoma.

描述（由申请人提供）：恶性黑色素瘤发病率和死亡率的主要因素是肿瘤侵袭和转移，这些因素是由基质金属蛋白酶（MMP）介导的。 MMP降解细胞外基质，并与浸润性癌症有关。 浸润需要间质胶原蛋白（I型和III）的降解，并且通过无处不在表达的胶原酶-1（MMP-1）来实现。 MMP-1启动子中的单个核苷酸多态性（SNP； 2G等位基因）在-1607 bp（5'-ggaa-3'）处为转录因子的ETS家族创建一个结合位点，并且与1G等位基因（5'-GAA-3'）相比，增强了MMP-1转录。 该2G SNP与包括黑色素瘤在内的七种恶性肿瘤的发生率/进展有关。 碱性成纤维细胞生长因子（BFGF）的水平与浸润性黑色素瘤相关，而BFGF诱导MMP-1表达。 此外，在RAS/RAF/MEK/ERK的信号级联中，在80％的黑色素瘤中发现了BRAF中的激活突变也会诱导MMP-1。 We hypothesize that: (a) the 2G allele increases MMP-1 expression and melanoma invasion, (b) bFGF and the activating mutation in BRAF orchestrate an invasive phenotype in the tumor cells by preferentially increasing MMP-1 expression through the 2G allele, and (c) host/tumor/matrix interactions target the 2G allele in stromal cells adjacent to the tumor to further enhance MMP-1表达和肿瘤入侵。 因此，（1）生成具有4 kb人类MMP-1启动子的转基因小鼠，其中包含-1g或2gs的-1607 bp，与β-半乳糖苷酶报告剂有关。 转基因将被插入HPRT基因座的单个副本，以提供有关MMP-1在基质细胞中转录活性的信息。 将在反映胶原式潜能的转基因的B16黑色素瘤和转基因的表达中挑战小鼠，将在与肿瘤相邻的基质细胞中进行检查。 (2) Convert a diploid 1G homozygous melanoma cell line to 2G homozygous, and conversely, a diploid 2G homozygous melanoma cell line to 1G homozygous, to compare effects of the 1G and 2G alleles on MMP-1 promoter and enzyme activities, and on tumor invasion in vitro and in vivo in nude mice. 我们将确定BFGF和突变BRAF如何靶向2G等位基因以增强黑色素瘤细胞的侵入性表型。 这些研究将为研究小鼠模型中的MMP-1基因表达提供新的机制，并有助于我们理解这种人间质胶原酶如何有助于浸润性黑色素瘤的病理学。