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

胰腺癌具有侵袭性生长和早期转移的特点，预后极差。此外，胰腺癌细胞对化疗具有广泛的抗药性。为了改善胰腺癌患者的预后，必须阐明胰腺癌的恶性特征，以便随后设想新的高效抗癌治疗方法。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，CDK9的高表达与生存受损有关。有趣的是，选择性抑制CDK9抑制了胰腺癌细胞的生长，并增强了化疗药物的作用。此外，我们发现选择性CDK9抑制是一种非常有效的策略，可以使癌细胞而不是正常细胞对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导的细胞凋亡敏感。在我们的建议中，我们开始研究CDK9在胰腺癌肿瘤进展、转移和化疗耐药中的作用，并破译潜在的分子机制。为了达到这一目的，我们将产生可诱导CDK9-shRNA和CDK9-过表达的胰腺癌细胞，并从体外增殖、迁移、侵袭和上皮-间充质转化(EMT)以及体内肿瘤生长、转移和化疗耐药等方面对其进行表征。为了揭示潜在的分子机制，将进行RT-PCR、免疫组织化学、Western blotting、BH3分析和基因表达分析。此外，还将使用原位患者来源的异种移植(PDX)小鼠模型。在这个模型中，手术切除后，小块的人类肿瘤组织和周围的间质组织将直接植入NOD SCID伽玛鼠的皮下。肿瘤建立后，该组织将在2-4代小鼠身上进行扩增，以产生足够数量的肿瘤组织，以便能够通过外科手术将肿瘤原位植入治疗组所需的足够小鼠体内。在这个模型中，将测试CDK9抑制剂diaciclib和TRAIL的新组合。肿瘤的大小和转移将通过小鼠MRI和组织病理学分析来量化。此外，将从这些肿瘤建立原代细胞系，并随后对其进行鉴定。利用上述技术，将表征耐药性的机制，以便在药物上克服或先发制人对该疗法的耐药性。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.