白血病抑制因子（LIF）通过调控结肠癌细胞CD44v6表达介导结肠癌耐药的分子机制研究

The relapse of colon cancer is believed to be partly correlated with the failure of chemotherapy which was always caused by drug resistance in cancer cells. The persistence of cancer stem cells (CSC) has been one of the reasons for the development of drug resistance and the relapse of cancer. Based on the reports, it was observed that LIF could stimulate the proliferation of cancer stem cells as well as promote the chemoresistance of cancer cells. But the detailed mechanism is not clear. It was shown in our preliminary study that LIF was overexpressed in colon cancer tissues. Exogenous LIF addition was observed to be able to promote the proliferation as well as to enhance the chemoresistance against 5-FU in colon cancer cells. It was observed that LIF could enhance the expression of CD44v6, a recently identified colon cancer stem cell marker. Meanwhile, LIF was also observed to be able to increase the expression level of PEA3, a member of ETS transcription factor family. Interestingly, it had been revealed that there were potential binding sits for ETS transcription factor family in CD44 promoter region. Moreover, it was observed that ERK1/2, a kind of protein kinase involved in the MAPK/ERK signaling pathway which had already been proved to be able to regulate the expression of PEA3, could be activated by exogenous LIF addition. Hence in this study, we proposed the following hypothesis. Through the regulation of MAPK/ERK/PEA3 signaling pathway, LIF could promote the expression of CD44v6 and thus enhance the stem cell properties of colon cancer cells, which could result in the chemoresitance against 5-FU in colon cancer cells. In this study, we will determine whether the expression of CD44v6 could be regulated by LIF. We will also determine the stem cell property changes in response to CD44v6 expression in colon cancer cells. The regulation effects of PEA3 on the expression of CD44v6 will be confirmed. In addition, factors affecting CD44v6 alternative splicing will also be determined. Meanwhile, we will combine the analysis results obtained from the animal experiment and from the cancer patients' survey to further confirm our hypothesis. Finally, the correlation between LIF and CD44v6 expression levels and the colon cancer patients' prognosis will be defined. The purpose of this study is to understand the role and molecular mechanisms of LIF in the progression of colon cancer disease as well as to provide the research basis for further novel treatments development.

结肠癌复发与癌细胞耐药所致化疗失败相关，肿瘤干细胞（CSC）是耐药性产生及肿瘤复发原因之一。研究表明LIF可促进CSC增殖并增强肿瘤耐药性，但具体机制尚无报道。我们的研究显示结肠癌组织高表达LIF；LIF可促进结肠癌细胞增殖、增强其对5FU耐受并促进CSC标记分子CD44v6表达；CD44启动子存在ETS转录因子家族结合位点，而LIF可促进该家族成员PEA3表达并激活其上游信号通路MAPK/ERK。由此我们认为LIF可通过MAPK/ERK/PEA3通路促进CD44v6表达以维系结肠癌细胞干性特征并增强其对5FU耐受。本项目评估LIF促进CD44v6表达及对结肠癌细胞干性特征的影响，检测PEA3对CD44v6表达的调控作用并确定其他CD44v6剪切调节因子，同时用动物模型和临床病例进行验证并评估LIF、CD44v6与病人预后的关系，阐明LIF对结肠癌疾病进程的作用机制，为结肠癌治疗提供依据。

结肠癌复发与癌细胞耐药所致化疗失败相关，肿瘤干细胞（Cancer stem cells, CSCs）是耐药性产生及肿瘤复发原因之一。我们通过生物信息学和原位杂交的方法对比结肠癌病人的肿瘤组织和癌旁组织中白血病抑制因子（Leukemia inhibitory factor, LIF）的表达水平，发现结肠癌组织中LIF表达水平升高，应用LIF过表达及LIF敲除的结肠癌细胞模型，证明LIF可促进结肠癌细胞增殖、克隆形成、迁移并增强结肠癌细胞成球能力，进一步研究结果提示LIF可促进结肠癌细胞干性能力增强，并升高干细胞标志物如CD44的表达水平。同时LIF可促进CD44不同剪接体如CD44v2表达水平升高，与CD44剪接相关的酶的表达水平也出现相应变化。我们在研究中还发现化疗药物处理也可导致结肠癌细胞CD44表达水平升高，其机制可能在于ETS转录因子家族成员之一的ELF3相关，而LIF可能通过上调转录因子ELF3增强结肠癌细胞CD44的表达，体外添加LIF阻断抗体可逆转上述所观察到的表型。此外，我们在本研究的过程中还发现姜黄素可通过结合CD44分子从而特异杀伤CD44+的结肠癌细胞；LncRNA-H19可能通过AKT-myc信号通路参与LIF对结肠癌细胞干性调节；LIF可通过结合结肠癌病人γδT细胞上高表达的LIFR激活STAT3信号通路，从而降低结肠癌病人血液中γδT细胞的细胞毒作用，在结肠癌肿瘤免疫微环境中发挥重要作用。最后我们还发现LIF/LIFR信号通路同胰腺癌的干性特征增强有关，应用LIF阻断抗体可阻抑LIF/LIFR信号通路，抑制胰腺癌细胞的增殖并可能参与调节胰腺癌的肿瘤微环境。总之，我们的研究揭示了LIF可促进肿瘤干细胞的形成，并可能有助于营造免疫抑制微环境，在结肠癌和胰腺癌的发生发展中具有重要作用，因此应用阻断LIF抗体阻断LIF/LIFR信号通路可能是一种行之有效的胰腺癌及结肠癌的治疗措施。