Signal Transduction by Tyrosine Phosphorylation

 描述（由申请方提供）：胰腺导管腺癌（PDA）是癌症死亡的第四大原因。这些研究的总体目标是更深入地了解胰腺星状细胞（PSC）（胰腺基质细胞的一个小亚群）在胰腺导管腺癌（PDA）进展中的生物学功能，并确定星状细胞活化及其与胰腺肿瘤细胞相互作用的分子机制。胰腺癌细胞（PCC）和PSC之间通过细胞因子和细胞外基质蛋白的相互信号传导导致PSC的持续活化，并且还促进肿瘤进展和转移。为了研究人PSC和人PCC之间的旁分泌相互作用，使用MS来定义两种细胞类型的分泌组。发现LIF IL 6家族细胞因子是PSC分泌的刺激PCC的主要因子，并且相反地，PDGF被鉴定为刺激PSC的主要PCC分泌因子，建立了相互刺激环。将评估LIF在人PDA肿瘤样品中的表达以验证细胞培养结果，并且将使用原位异种移植物评估LIF在PDA中的作用，所述原位异种移植物具有与PSC共注射的亲本或LIF受体缺失的PCC。将研究PDGF诱导的途径，该途径涉及诱导PSC中LIF表达的UBASH 3B/STS 1衔接蛋白。已经发现人PCC系的子集对MEK抑制剂具有抗性，即使它们表达通过MEK驱动ERK MAP激酶途径活化的突变KRAS。通过用来自抗性PCC系的条件培养基处理，使MEK抑制剂敏感性PCC系对MEK抑制具有抗性。通过MS分析和耐药因子的部分表征，GDF-15，一种变体TGF β家族成员，被鉴定为候选者，并且将通过反向遗传操作和使用中和抗体来评估GDF 15在MEK抑制剂耐药中的可能作用。或者，通过常规方法纯化抗性因子。为了研究体内PDA进展与PSC活化之间的关系，已经制作了基因工程小鼠模型，其中荧光素酶（Luc）和反向四环素反式激活因子（rtTA）在活化的星状细胞中特异性表达，使得能够生物发光标记和多西环素诱导的胰腺中活化PSC的操纵。通过将PDCA小鼠与KPflC PDA模型小鼠杂交，将研究PDA进展过程中的PSC活化。（c）tetO-DTA;将产生其中白喉毒素A链（DTA）可通过多西环素喂养诱导的KPf 1C小鼠，并将其用于评估在肿瘤发生开始后的限定时间通过DTA灭活PSC对PDA进展的后果。最后，KPFLC;将产生LIFRfl/fl小鼠，并与KPflC小鼠进行比较，以评估肿瘤细胞中特异性LIFR信号传导的缺乏如何影响PDA进展。通过施用可溶性显性阴性Fc-LIFR ECD融合蛋白，在KPfIC小鼠模型中评估LIF阻断对PDA治疗的功效。

 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.