Signal Transduction by Tyrosine Phosphorylation

通过酪氨酸磷酸化进行信号转导

基本信息

批准号：
9085210
负责人：
TONY R. HUNTER
金额：
$ 67.43万
依托单位：
SALK INSTITUTE FOR BIOLOGICAL STUDIES
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9085210
关键词：
Accounting Adaptor Signaling Protein Affect Biological Process Cancer Etiology Cancer cell line Cell Culture Techniques Cells Cessation of life Chimeric Proteins Coculture Techniques Complement Conditioned Culture Media Development Diphtheria Toxin Dominant-Negative Mutation Doxycycline Event Extracellular Matrix Proteins Extracellular Signal Regulated Kinases Family Family member GDF15 gene Genetically Engineered Mouse Goals Growth Health Human IL6 gene Injury KRAS2 gene LIF gene LIFR gene Label Lead Luciferases MEK inhibition MEKs Malignant Neoplasms Molecular Mus Myofibroblast Neoplasm Metastasis Outcome Oxidative Stress PDGFRB gene Pancreas Pancreatic Ductal Adenocarcinoma Paracrine Communication Pathway interactions Patients Phenotype Phosphorylation Physiological Platelet-Derived Growth Factor Process Production Proteins Proteomics Protocols documentation Resistance Role Route STAT3 gene Sampling Set protein Signal Pathway Signal Transduction Stromal Cells Tetracyclines Time Tissues Trans-Activators Transforming Growth Factor beta Tyrosine Phosphorylation Variant Xenograft procedure base cell growth cell type cytokine diphtheria toxin fragment A feeding genetic manipulation in vivo inhibitor/antagonist insight knock-down leukemia inhibitory factor receptor mouse model mutant neoplastic cell neutralizing antibody novel therapeutic intervention outcome forecast pancreatic cancer cells pancreatic neoplasm pancreatic tumorigenesis paracrine phosphoproteomics resistance factors response reverse genetics screening stellate cell tumor tumor progression tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer death. The overall goal of these studies is to gain a deeper understanding of the biological functions of pancreatic stellate cells (PSCs), a small subpopulation of pancreatic stromal cells, in the progression of pancreatic ductal adenocarcinoma (PDA), and to define molecular mechanisms underlying stellate cell activation and their interactions with pancreatic tumor cells. Reciprocal signaling via cytokines and extracellular matrix proteins between pancreatic cancer cells (PCCs) and PSCs leads to sustained activation of PSCs and also promotes tumor progression and metastasis. To investigate paracrine interactions between human PSCs and human PCCs, MS was used to define the secretomes of the two cell types. The LIF IL6 family cytokine was found to be the major factor secreted by PSCs that stimulates PCCs, and, conversely, PDGF was identified as a major PCC-secreted factor that stimulates PSCs, setting up a reciprocal stimulatory loop. The expression of LIF in human PDA tumor samples will be assessed in order to validate cell culture results, and the role of LIF in PDA will be assessed using orthotopic xenografts with parental or LIF receptor-depleted PCCs co-injected with PSCs. The PDGF-induced pathway involving the UBASH3B/STS1 adaptor protein that induces LIF expression in PSCs will be investigated. A subset of human PCC lines has been found to be resistant to MEK inhibitors, even though they express mutant KRAS that drives activation of the ERK MAP kinase pathway through MEK. MEK inhibitor sensitive PCC lines are rendered resistant to MEK inhibition by treatment with conditioned medium from resistant PCC lines. By MS analysis and partial characterization of the resistance factor, GDF-15, a variant TGFß family member, was identified as a candidate, and the possible role of GDF15 in MEK inhibitor resistance will be evaluated by reverse genetic manipulation and use of neutralizing antibodies. Alternatively, the resistance factor will be purified by conventional means. To study the relationship between PDA progression and PSC activation in vivo, a genetically engineered mouse model, SLT, has been made, in which luciferase (Luc) and reverse tetracycline transactivator (rtTA) are specifically expressed in activated stellate cells, enabling both bioluminescent labeling and doxycycline-inducible manipulation of activated PSCs in the pancreas. By crossing SLT mice with KPflC PDA model mice, PSC activation in the course of PDA progression will be studied. SLT;tetO-DTA;KPflC mice, in which diphtheria toxin A chain (DTA) is inducible by doxycycline feeding, will be generated and used to evaluate the consequences of PSC inactivation by DTA on PDA progression at defined times after the initiation of tumorigenesis. Finally, KPflC;LIFRfl/fl mice wll be generated, and comparison with KPflC mice will be used to evaluate how deficiency of LIFR signaling specifically in tumor cells affects PDA progression. The efficacy of LIF blockade for PDA therapy will be assessed in the KPflC mouse model by administration of soluble dominant-negative Fc-LIFR ECD fusion protein.

描述（由适用提供）：胰腺导管腺癌（PDA）是癌症死亡的第四个主要原因。 The overall goal of these studies is to gain a deeper understanding of the biologic functions of pancreatic stellate cells (PSCs), a small subpopulation of pancreatic stromal cells, in the progression of pancreatic ductal adenocarcinoma (PDA), and to define molecular mechanisms underlying stellate cell activation and their interactions with pancreatic Tumor cells.通过细胞因子和胰腺癌细胞（PCC）和PSC之间的细胞因子和细胞外基质蛋白的相互信号传导可导致PSC的持续激活，并促进肿瘤进展和转移。为了研究人PSC与人PCC之间的旁分泌相互作用，使用MS来定义两种细胞类型的秘密组。发现LIF IL6家族细胞因子是刺激PCC的PSC分泌的主要因素，相反，PDGF被确定为刺激PSC的主要PCC分泌因子，建立了互相刺激的循环。为了验证细胞培养结果，将评估人类PDA肿瘤样品中LIF的表达，并将使用与父母或LIF受体贫血的PCCS一起注入PSC的原位异种移植物来评估PDA的作用。 PDGF诱导的途径涉及影响PSC中LIF表达的UBASH3B/STS1衔接蛋白。人们发现，人类PCC系的一部分对MEK抑制剂具有抗性，尽管它们表达了突变的KRAS，该突变体可以通过MEK激活ERK MAP激酶途径。 MEK抑制剂敏感的PCC系通过耐药PCC线的条件培养基处理对MEK抑制具有抗性。通过MS分析和电阻因子的部分表征，将变体TGFß家族成员GDF-15确定为候选者，并且将通过反向遗传操作和使用中和抗体的使用来评估GDF15在MEK抑制剂抗性中的可能作用。或者，电阻因子将通过常规手段纯化。 To study the relationship between PDA progression and PSC activation in vivo, a genetically engineered mouse model, SLT, has been made, In which luciferase (Luc) and reverse tetracycline transactivator (rtTA) are specifically expressed in activated stellate cells, enabling both bioluminescent labeling and doxycycline-inducible manipulation of activated PSCs in the pancreas.通过将SLT小鼠与KPFLC PDA模型小鼠跨越，PSC在PDA进程过程中的激活将进行研究。 SLT; TETO-DTA; KPFLC小鼠将产生并使用Diphtheria毒素A链（DTA）是可诱导的强力霉素喂养，并用于评估DTA在肿瘤造成肿瘤造成后在定义的时间下DTA对PSC失活的后果。最后，生成KPFLC; LIFRFL/FL小鼠WLL，并将与KPFLC小鼠的比较用于评估肿瘤细胞中特异性LIFR信号的缺乏如何影响PDA的进展。 LIF封锁对PDA治疗的效率将在KPFLC小鼠模型中通过施用固体显性fc-LIFR ECD融合蛋白进行评估。