Cyclin-Dependent Kinase (CDK)9 in pancreatic cancer: role for tumor progression, metastasis and chemoresistance of pancreatic and evaluation of novel TRAIL-/CDK9-inhibitory-based therapies

胰腺癌的预后极差，因为它具有浸润性生长和早期转移。此外，胰腺癌细胞对化疗具有广泛的抗性。为了改善胰腺癌患者的预后，必须阐明这种肿瘤实体的恶性特征，以便随后设想新的高效抗癌疗法。最近，我们发现细胞周期蛋白依赖性激酶（CDK）9在胰腺癌组织中过表达。此外，高CDK 9表达与受损的存活率相关。有趣的是，选择性CDK 9抑制抑制胰腺癌细胞的生长，并增强化疗药物的作用。此外，我们确定了选择性CDK 9抑制作为一种非常有效的策略，使癌细胞而不是正常细胞对TNF相关的凋亡诱导配体（TRAIL）诱导的凋亡敏感。在我们的提案中，我们着手研究CDK 9在胰腺癌的肿瘤进展、转移和化疗耐药性中的作用，并解码潜在的分子机制。为了实现这一点，将产生具有诱导型CDK 9-shRNA和CDK 9过表达的胰腺癌细胞，并在体外增殖、迁移、侵袭和上皮-间质转化（EMT）以及体内肿瘤生长、转移和化疗抗性方面进行表征。为了揭示潜在的分子机制，将进行RT-PCR、免疫组织化学、蛋白质印迹、BH 3谱和基因表达谱分析。此外，将使用原位患者来源的异种移植物（PDX）小鼠模型。在该模型中，在手术切除后，将具有周围基质组织的小块人肿瘤组织直接皮下植入NOD scid γ小鼠中。肿瘤建立后，将在2-4代小鼠中扩增组织，以产生足够量的肿瘤组织，从而能够在治疗组所需的足够小鼠中原位手术植入肿瘤。在该模型中，将测试CDK 9-抑制剂Dinaciclib和TRAIL的新组合。将通过小鼠MRI和组织病理学分析定量肿瘤大小和转移。此外，将从这些肿瘤建立原代细胞系并随后表征。利用上述技术，将表征耐药机制，以避免克服或预先消除对该疗法的耐药性。因此，我们着手建立一种新的，高效的胰腺癌治疗策略。

Prognosis of pancreatic cancer is extremely poor due to invasive growth and early metastasis. Moreover, pancreatic cancer cells are widely resistant to chemotherapy. To improve the outcome of pancreatic cancer patients, the malignant characteristics of this tumor entity has to elucidate to subsequently envisage novel and highly effective anti-cancer therapies. Recently, we identified Cyclin-dependent kinase (CDK)9 being overexpressed in pancreatic cancer tissue. Moreover, high CDK9 expression was associated with impaired survival. Interestingly, selective CDK9-inhibition suppressed the growth of pancreatic cancer cells and augmented the effect of chemotherapeutic agents. Moreover, we identified selective CDK9-inhibtion as an extremely potent strategy to sensitize cancer cells, but not normal cells, to TNF related apoptosis inducing ligand (TRAIL)-induced apoptosis. In our proposal we set out to investigate the role of CDK9 for tumor progression, metastases and chemoresistance of pancreatic cancer and to decode underlying molecular mechanism. To achieve this pancreatic cancer cells with inducible CDK9-shRNA and CDK9-overexpression will be generated and characterized in terms of proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in vitro as well as tumor growth, metastasis and chemoresistance in vivo. To uncover underlying molecular mechanisms RT-PCR, immunohistochemistry, western blotting, BH3-profiling and well as gen-expression profiling will be performed. In addition, an orthotopic patient derived xenograft (PDX) mouse model will be used. In this model, small pieces of human tumor tissue with surrounding stroma tissue will be implanted subcutaneously in NOD scid gamma mice directly upon surgical resection. After tumor establishment the tissue will be amplified over 2-4 mouse generations to generate sufficient amount of tumor tissue to be able to surgically implant tumors orthotopically in enough mice required for the treatment groups. In this model the novel combination of the CDK9-inhihitor Dinaciclib and TRAIL will be tested. The tumor size and metastases will be quantified by mouse MRI and histopathological analysis. Moreover, primary cell lines will be established from these tumors and subsequently characterized. Utilizing above described techniques, mechanisms of resistance will be characterized, in order to pharmacologically overcome or preempt resistance to this therapy. Hereby, we set out to establish a novel and highly effective therapeutic strategy for pancreatic cancer.