肺癌干细胞通过上调Stat3介导mTOR抑制剂耐药的机制研究

Lung cancer is the first leading cause of cancer death due to its high incidence, low surgical resection rate, and cancer cells resistance to chemotherapy. There is therefore an urgent need for development of new targeted therapies which have less side effects and more efficacy. mTOR (mammalian target of rapamycin) signal pathway is dysregulated in lung cancer cells and promotes cancer genesis and progression, thus emerging as a hot target for cancer therapy. Most recent studies indicated that mTOR dual inhibitor (AZD8055) indeed shows encouraging efficacy for growth inhibition and cell apoptosis in serum-cultured cell lines in vitro, but the clinical data is not satisfactory. According to this confused difference, in our earlier studies we find the tumor tissues of lung cancer patients usually contain both general cancer cells and cancer stem cells, we also find that AZD8055 as mTOR inhibitor blocking mTOR signal pathway effectively in both cancer cells and cancer stem cells, however it enrich lung cancer stem cells and induce the overactivation of Stat3 molecule, which play an important role in cell proliferation and survival. In this proposal, we will therefore test the hypothesis that the resistance of lung cancer stem cells occurs through the upregulation of Stat3 activity induced by mTOR inhibitor, and the cure of lung cancer requires simultaneous eradication of lung cancer stem cells. First we will employ RNA interfering, lentivirus infection, MTS assay, flow cytometry, sphere formation assay, Western blot and qRT-PCR approaches in analysis of AZD8055 treated lung cancer stem cells (LCSCs). Then we will generate lung cancer xenografts by injection of CD133 positive LCSCs in mice and assess whether the combination therapy of mTOR inhibitor AZD8055 and Stat3 inhibitor Niclosamide can inhibit the tumor growth and relapse. Thus, determining whether Stat3 is the effective target to reverse the resistance of stem cells to AZD8055 and clarify the mechanism of Stat3 upregulation induced by AZD8055 in lung cancer stem cells. This proposal will provide new proof and therapeutic strategy in treating human lung cancers.

肺癌发病率高，切除率低，传统放化疗效果差，因此靶向药物成为改善肺癌治疗的研究热点。肺癌细胞内mTOR通路异常激活，mTOR双重抑制剂（AZD8055，AZD）在体外实验可有效抑制血清培养条件下一般肺癌细胞的增殖，但临床治疗效果不显著。针对该问题，本课题组经前期研究验证肿瘤干细胞普遍存在于肺癌组织中，并发现AZD在有效抑制肺癌细胞和肺癌干细胞中mTOR通路的同时，却诱导肺癌干细胞中维持细胞生存和增殖的Stat3分子激活上调，然而肺癌干细胞中Stat3的激活上调是否是介导AZD临床治疗失败的关键以及具体的分子机制尚不清楚。本课题拟在前期工作基础上，明确以Stat3为靶点改善肺癌干细胞及其移植瘤对AZD耐药的作用，并阐明AZD诱导肺癌干细胞中Stat3激活上调的分子机制，从而为以Stat3为靶点逆转肺癌干细胞对AZD的耐药，为结合肿瘤干细胞的治疗解决肿瘤耐药的临床治疗方案提出新的思路和理论依据。

肺癌发病率高，切除率低，死亡率高，吉西他滨作为一线治疗方案广泛应用于临床，但耐药性严重影响临床治疗效果，而相关耐药靶点的缺乏也给临床医生制定理想的治疗方案带来巨大困扰，因此，阐明介导耐药的关键分子及其作用机制是亟待解决的问题。我们的研究以外泌小体为切入点，探讨耐药细胞是否通过外泌小体传递蛋白或miRNAs以介导更多的细胞耐药并促进肿瘤的发展。我们的研究发现肺癌吉西他滨耐药细胞株分泌的外泌小体参与诱导敏感受体细胞恶性表型的改变，而其携带的miRNA在这一过程中扮演重要的角色。外泌小体主要通过小窝蛋白和脂筏依赖的方式进入受体细胞，并传递大量的miR-222-3p；外泌小体-miRNA-222-3p在受体细胞中与内源miRNA的功能相类似，通过结合于靶基因SOCS3，抑制SOCS3的表达，并释放Jak2/Stat3信号通路的持续激活，进而促进敏感受体细胞的耐药、侵袭迁移以及抗失巢凋亡；同时，我们结合临床患者资料，发现患者外周血外泌小体中miR-222-3p含量与肿瘤转移呈正相关，与患者对吉西他滨的治疗效果呈负相关，miR-222-3p含量较高的患者常显示较差的预后。该研究为改善吉西他滨的耐药提供新的靶点，也为提前预测吉西他滨的疗效和预后提供新的靶点和理论依据。