雷洛昔芬对胰腺癌干细胞IL-6/gp130信号途径的抑制效应及其抗肿瘤新机制的研究

There is a small part of cancer cells, called cancer stem cells, which is self-renewal, playing an important role in the tumor recurrence, infiltration, migration and drug resistance. The Signal Transducer and Activator of Transcription3 (STAT3) was demonstrated to be activated in many tumors including panncreatic cancer cells, and involved in the tumor growth, proliferation, invasion and migration. Importantly, STAT3 is activated by pro-inflammatory factors such as interleukin-6 that has also been linked to increased cancer risk and poorer prognosis. IL-6 first binds to IL-6R, then recruits gp130 to form the IL-6/IL-6R/gp130 complexes. Furthermore homodimerization of gp130 occurs by interactions between IL-6 of one IL-6/IL-6R/gp130 and the gp130 of the other IL-6/IL-6R/gp130. The reciprocal homodimerization of the IL-6/IL-6R/gp130 triggers a signaling cascade of phosphorylation of Janus (JAK) kinase and a downstream effector STAT3, followed by reciprocal dimerization of the phosphorylated STAT3. It is highly desirable to design and identify novel, small molecule drugs to disable the dimerization of the IL-6/IL-6R/GP130 heterotrimers, which offer new options for cancer therapy. Using a novel computational strategy for fragment-based drug design by combining MLSD and drug repositioning, we demonstrated that Raloxifene has a new function against IL-6/GP130 protein-protein interface. Raloxifene primarily target human Estrogen Receptor (ER), which involves estrogenic actions on bone but anti-estrogenic actions on uterus and breast. They are commonly used in the prevention of osteoporosis and breast cancer. In the present study, we will examine the inhibitory effects of Raloxifene in pancreatic caner stem cells in vitro and in vivo. We hope to demonstrate that Raloxifene has more potent activity against IL-6/gp130 and lower IC50 than a reported gp130 inhibitor, MDL-A, in pancreatic cancer cells. We will separate pancreatic cancer stem cells by flow cytometry using stem cell markers ALDH, CD44,and CD24, and examine the STAT3 activity in ALDH+ and CD44+/CD24+ pancreatic cancer stem cells (PCSC). Then, we will test the inhibitory efficacy of Raloxifene as IL-6/gp130 inhibitors in pancreatic cancer stem cells in Vitro. Raloxifene will also be further tested for efficacy on tumorigenesis in pancreatic tumor model using PCSC from established pancreatic cancer cell lines and primary pancreatic cancer cells from pancreatic cancer patients. The results will provide an innovative strategy for pancreactice cancer therapy by targeting IL-6/gp130/STAT3 signaling in PCSC using Raloxifene. Raloxifene can also serve as a drug lead for optimization to speed up the development of novel compunds, which might be more potent and with desirable drug properties to disrupting IL-6/gp130/STAT3 in pancreatic cancer stem cells

肿瘤干细胞是肿瘤组织中很少一部分具有自我更新能力的细胞，在肿瘤复发，转移及抗药性中起着十分重要的作用。白介素-6（IL-6）通过结合其受体gp130,激活信号传导与转录激活因子3（STAT3），参与多种肿瘤包括胰腺癌的发生发展、浸润和转移。我们采用计算机模拟分析和多配体同步对接技术发现雷洛昔芬（一种FDA批准上市的雌激素受体调节剂)可能具有抑制IL-6/gp130结合的效应。本研究旨在进一步证实雷洛昔芬对IL-6/gp130/STAT3的抑制效应；以ALDH和CD44、CD24为胰腺癌干细胞标记物，分选出胰腺癌干细胞，检测其IL-6/gp130/STAT3水平；检测雷洛昔芬对胰腺癌干细胞生长，STAT3活性及鼠胰腺癌干细胞移植模型的抑制效应。为雷洛昔芬用于防治胰腺癌提供实验基础，为以IL-6/gp130/STAT3为靶点开发新的胰腺癌干细胞分子靶向治疗药物提供重要的理论依据和模板药物。

肿瘤干细胞是肿瘤组织中很少一部分具有自我更新能力的细胞，在肿瘤复发,转移及抗药性中起着十分重要的作用。已有研究报道STAT3在胰腺癌组织中呈激活状态，但IL-6/gp130/STAT3 在胰腺癌干细胞中是否处于激活状态，目前尚无人报导。因此,本研究拟以 ALDH,CD44,CD24作为干细胞标志物，用流式细胞仪筛选出胰腺癌干细胞,并检测其IL-6/gp130/STAT3 水平,探讨IL-6/gp130/STAT3 信号通路在胰腺癌干细胞生长、增殖、浸润以及自我更新等生物学行为中的作用；同时，进一步验证雷洛昔芬作为一种新发现的IL-6/gp130 抑制剂对胰腺癌细胞以及胰腺癌干细胞 IL-6/gp130/STAT3 信号途径是否具有抑制效应。本研究证明了ALDH+CD24+/CD44+胰腺癌干细胞中IL-6/gp130/STAT3处于激活状态，ALDH+ CD44+/CD24+胰腺癌干细胞磷酸化STAT3表达水平较ALDH-/CD44-/CD24-细胞明显升高,小分子STAT3抑制剂LLL12, Stattic, FLLL32,能有效抑制ALDH+ CD24+/CD44+胰腺癌干细胞STAT3磷酸化水平,降低STAT3下游靶基因Bcl-xL, survivin的表达，抑制肿瘤干细胞活性并抑制其Tumorsphere形成能力。雷洛昔芬能抑制胰腺癌细胞STAT3的磷酸化,而且能特异性的抑制IL-6诱导的STAT3的激活,对LIF, IFN-α诱导 的p-STAT3的升高无影响。对IL-4, IFN-γ分别诱导激活磷酸化STAT6，STAT1的升高也无影响。在细胞水平上，雷洛昔芬能增加胰腺癌细胞cleaved caspase-3表达及活性，促进胰腺癌细胞凋亡。用免疫荧光方法检测胰腺癌细胞内STAT3的位置，结果证明雷洛昔芬能抑制IL-6诱导胰腺癌细胞核转位，进一步说明其通过IL-6/gp130/STAT3信号途径发挥抗肿瘤作用。此外，我们发现雷洛昔芬对肝癌细胞的生长也具有抑制作用，通过细胞实验及裸鼠成瘤模型验证了雷洛昔芬能抑制肝癌细胞的生长、增殖及迁移。总之,本研究首次报道IL-6/gp130/STAT3 信号通路在胰腺癌干细胞中呈激活状态，雷洛昔芬能抑制IL-6/gp130/STAT3 信号途径并发挥抗肿瘤作用，为肿瘤干细胞分子靶向治疗的提供新靶点.