内质网应激相关E2F4泛素化调控PI3K-AKT通路在胰腺癌吉西他滨耐药中的机制研究

Dysregulation of some signaling pathways is one of the leading causes of chemoresistance which is also the research hotspot currently. Previously, we have found that activation of PI3K-AKT pathway via downregulating of E2F4 could promote gemcitabine resistance in pancreatic cancer cells. Further study indicated that compared to naive cancer cells, the protein level of E2F4 obviously decreased in gemcitabine resistant cells while the mRNA level was not. Based on bioinformatics analysis and preliminary results, we hypothesize that E2F4 downregulation is induced by ER stress associated ubiquitin-dependent protein degradation, which would further change cell cycle profile and activate PI3K-AKT pathway via specific cyclin-dependent kinase (CDK) that finally cause chemoresistance. With commercial and primary PDAC cell lines, genetically engineered KPC mice and patient-derived tumor xenografts mouse models, the project will go depth in the molecular mechanisms of E2F4 degradation by ER stress and how this regulates the activation of PI3K-AKT pathway in the process of chemoresistance in vitro and in vivo. Furthermore, we will evaluate the combined efficacy of gemcitabine and related specific inhibitors in PDAC mouse models. The prospective results of this study would not only help to explore the molecular mechanisms of chemoresistance, but also provide experimental and theoretical basis on novel therapeutic strategies and chemotherapy regimens against pancreatic cancer.

信号通路的异常调控是化疗耐药的主要原因之一，也是现阶段研究的热点。我们前期发现E2F4的降低可活化PI3K-AKT通路从而增加胰腺癌细胞对吉西他滨的抵抗；而较亲本细胞，耐药细胞株中E2F4的蛋白水平显著下降而mRNA水平无明显变化。基于进一步的生物信息学分析及预实验结果，我们提出如下假说：吉西他滨通过启动内质网应激相关蛋白泛素化降解途径导致E2F4表达降低；进而通过活化特定周期依赖性蛋白激酶，引发细胞周期的改变及PI3K-AKT通路的活化，最终导致耐药。本项目拟借助商品化及原代胰腺癌细胞系、KPC转基因工程及PDX模型鼠，从体内、体外及活体动物水平深入探索耐药进程中E2F4泛素化降解的原因及其活化PI3K-AKT通路的分子机制，进而评价吉西他滨联合相关小分子化合物抑制剂在模型鼠中的疗效。本课题不仅能够提高对胰腺癌化疗耐药机制的认识，也将为发现新的治疗靶点及化疗方案提供实验基础和理论依据。

在研究进行中本研究我们根据实际情况调整研究计划，结合前期对E2F4的研究，我们针对其上游靶点CDKN2A展开深入研究，因该位点在胰腺癌中的突变率达到70-90%。我们应用CDKN2A-pRB-CDK4/6-E2F通路中靶向CDK4/6的特异性抑制剂PD-0332991进行实验，证实CDK4和CDK6在胰腺癌中均高表达，并与生存负相关；而利用PD-0332991单纯抑制CDK4/6仅有轻微的抑瘤作用，但反而促进肿瘤细胞的迁徙及侵袭能力；进一步的机制研究表明，CDK4/6通过活化Wnt/β-catenin通路从而引发上皮-间质转化（EMT），最终导致肿瘤侵袭和转移，而Wnt通路的活化则是依赖于TGFβ/Smad介导的GSK3β Ser9位点的磷酸化。BET抑制剂JQ1能够抑制GSK3β Ser9的磷酸化，进而阻断由TGFβ/Smad导致的Wnt/β-catenin活化，从而与PD-0332991产生协同作用，并抑制肿瘤细胞的侵袭及迁移。我们该项研究证实联合PD-0332991与JQ1在体内外均具备明显的抑瘤作用，为制定胰腺癌新的治疗方案提供理论基础(第一作者，Journal of Cellular and Molecular Medicine，修回)。在该课题延申的基础上，我们还发现EMT相关的关键转录因子Snail在胰腺癌原发灶及转移灶中存在差异表达，且与巨噬细胞极化具备显著相关性，因此我们的研究认为由于巨噬细胞的极化在转移灶中为肿瘤细胞的定植创造合理条件，通过诱导Snail的表达，从而使得肿瘤细胞转移（第一作者，Journal of Oncology，接收）。在内质网应激方面，尽管我们没有在胰腺癌化疗耐药中取得相关成果，但在肝癌中鉴定出急性期蛋白ORM1与肝癌索拉菲尼耐药性显著相关（第一作者，Bosnian Journal of Basic Medical Science，见刊）。

内容获取失败，请点击重试