ROS/PKC/MAPK调控EMT促进高糖环境下胰腺癌侵袭转移的实验研究

Pancreatic cancer (PC) is one of the most lethal malignant diseases and has a dismal prognosis.Although surgery remains the only way to cure this disease,almost 80% patients are not suitable for surgical resection at the time of initial diagnosis because of locally deteriorated or metastatic disease.Diabetes mellitus has long been considered as a potential risk factor for pancreatic cancer.Approximately 85% pancreatic cancer patients are suffered from glucose intolerance or even frank diabetes.In the latest decades, accumulating data and studies have started to examine the relationship between epithelial-mesenchymal transition and hyperglycemia in the normal cells.Through the preliminary studies, we demonstrated that high glucose could increase the production of reactive oxygen species (ROS) and the ability of invasion in pancreatic cancer cells .We speculate that high glucose could regulate the oxidative stress and promote the generation of ROS and further activate the PCK / MAPK signaling pathway which modulate the downstream gene expression. As a result,the tumor cells transform from the epithelial-like cells to mesenchymal-like cells with the enhanced ability to pass through the basement membrane and extracellular matrix and eventually lead to distant metastasis of pancreatic cancer. In this study, we utilize a variaty of ways,including the clinical pathology testing, in vitro cancer cell biological dynamics observation and diabetic nude mice invasion and metastasis model construction, transmission electron microscopy, flow cytometry, Western blot and RT-PCR to observe the effects of high glucose on pancreatic cancer from the histological and molecular levels. Ultimately，we block oxygen stress to observe the tumor metastatic potential changes.Our research provide new direction for cancer treatment that managing hyperglycemia or inhibiting the production of ROS may prevent tumor recurrence not only locally but also at distant sites.

胰腺癌恶性度极高，约80%的患者在确诊时已发生远处转移。糖尿病被认为是诱发胰腺癌的重要危险因素。高达85%的胰腺癌患者存在糖耐量异常甚或糖尿病。课题组前期研究首次发现高糖状态可以促进胰腺癌细胞侵袭能力。然而这种高糖微环境调控肿瘤侵袭转移的具体机制及中间环节，迄今国内外尚无报道。我们推测高糖可以通过调控氧应激，促进活性氧簇的产生，进而激活PCK/MAPK信号通路及下游基因转录表达，诱发肿瘤细胞从上皮样细胞向间质样细胞转型(EMT)，最终导致胰腺癌远处转移。本研究应用临床病理检测、体外癌细胞生物动力学动态观察、糖尿病裸鼠侵袭转移模型构建等关键手段，通过透射电镜观察、流式细胞技术、Western blot等方法从组织细胞学、分子生物学等水平观察高糖对胰腺癌产生的影响；随后通过阻断氧应激，反向观察肿瘤转移能力变化，从正反两方面验证我们的假设，为胰腺癌转移机制和治疗研究提供新方向。

1. 项目背景：.胰腺癌是最常见的消化系统恶性肿瘤之一，由于其早期易发生侵袭、转移，大多数患者在确诊时已处于不可逆转的晚期，无法进行手术治疗。确定并控制胰腺癌发生的病因及危险因素对疾病的预防及治疗起到尤为关键的作用。糖尿病与胰腺癌之间的关系一直以来都是人们所关注的热点问题。高达85%的胰腺癌患者存在糖耐量异常甚或糖尿病。然而，关于高糖状态对胰腺癌浸润转移的影响及其具体机制尚不明确。.2. 项目主要研究内容：.该项目主要分为临床、体外和体内实验三部分。.1）通过临床资料分析，初步揭示高糖-氧应激-肿瘤转移之间内在联系； .2）通过体外细胞学实验，动态观察肿瘤细胞在高糖环境中EMT变化并验证可能涉及的信号通路； .3）通过裸鼠体内实验，进一步验证体外实验结果，并通过抑制氧应激反向观察侵袭转移的程度。.3. 项目重要结果：.1）高糖微环境可以提高胰腺癌总ROS及H2O2的产生，高糖环境主要通过调节SOD2的表达与活力影响氧化应激；.2）高糖微环境可以提高胰腺癌增殖及淋巴结转移能力。高糖微环境可以促进胰腺癌细胞侵袭、迁移、uPA表达；.3）H2O2作为重要的调控因素，在高糖与肿瘤侵袭转移之间起到桥梁作用； .4）高糖微环境及过氧化氢可以促进胰腺癌细胞EMT的发展；.5）高糖微环境及H2O2可以活化MAPK信号通路并促进其下游转录因子NF-κB和c-Jun的表达。高糖微环境所激活的MAPK信号通路是通过其所诱导产生的H2O2所介导。高糖微环境所促进的胰腺癌细胞侵袭、迁移能力是由ERK及p38 MAPK信号通路所调控。.4. 关键数据：.1）高糖微环境主要通过调节SOD2的表达与活力影响胰腺癌细胞H2O2水平； .2）高糖微环境可通过提升H2O2水平，直接激活ERK及p38 MAPK信号通路并诱导其下游转录因子NF-κB和c-Jun的活化；.3) 高糖借助于H2O2激活MAPK通路，可进一步影响肿瘤EMT发生及侵袭、转移能力。.5. 科学意义：.近年来，EMT被认作是肿瘤转移的重要机制之一。然而目前对EMT的研究仅限于肿瘤本身，是否包括糖尿病在内的内分泌系统疾病可以促进肿瘤转移及EMT的发生报道颇少。通过本项目的研究，证实高糖微环境可以通过调控氧化应激促进胰腺癌EMT发生。该项目不仅填补了国内外的研究空白，也拓宽人们对糖尿病及肿瘤EMT的认识，为后续研究奠定了理论和实验基础。