microRNA100对乳腺癌干细胞的调控机制研究及其在乳腺癌治疗中的临床意义

There is increasing evidence that many human cancers, including breast cancer, are driven and maintained by a cellular subcomponent that displays stem cell properties. These "cancer stem cells" may also mediate tumor metastasis and contribute to treatment resistance and relapse. Our laboratory has identified cellular markers and developed in vitro and mouse models to isolate and characterize normal and malignant human mammary stem cells. Recent studies have raised a link between dysregulated expression of microRNAs (miRNAs) and carcinogenesis. More recently, some miRNAs (e.g. mir124, mir137, mi34 and Let7) have been reported to regulate cancer stem cell self-renewal and both mir93 and Let7 are highly depleted in mammary stem/progenitor cells isolated with the stem cell marker ALDH. These studies suggest that miRNAs might offer great promise for cancer therapy since they have potential to target the cancer stem cells (CSCs). However, CSC-targeted delivery of miRNA remains a great challenge. Our preliminary data demonstrate that microRNA100 (miR-100) is elevated in more differentiated breast cancer cells, and the induction of miR-100 expression decreased the ALDH-positive population in breast cancer cell line SUM159 in vitro. These studies indicate that miR-100 may hold significant potential as a novel molecular therapy for breast cancer stem cells. We hypothesize that re-introduction of miR-100 into breast cancer cells will impair the cancer stem cell self-renewal capacity to decrease the cancer stem cell population, which eventually render them less tumorigenic and metastatic and more sensitive to chemo treatments. Based on this hypothesis and our preliminary data, we predict that miR-100 will be a good therapeutic target in breast cancer. Our objective is to elucidate fundamental mechanisms by which over-expression of miR-100 in breast cancer cells will impair the tumor growth and metastasis and make the cells more sensitive to chemo treatments. We further intend to develop the anti-CD44-nanovector-miR-100 as a novel molecular therapy targeting breast cancer stem cells and create the potential to greatly increase the efficacy of traditional chemotherapy. Since CSCs may drive tumorigenesis and metastasis as well as contribute to treatment resistance and relapse, the development of novel strategies to target this cell population may lead to improved outcomes for women with advanced breast cancer. Successfully carried out, our studies will provide novel, self-assembled anti-CD44-nanovectors that can deliver miR-100 to breast CSC and inhibit their self-renewal and tumor-initiation, as well as the resistance to chemotherapy. The data from the current proposal will provide an important impetus to develop the anti-CD44-nanovector-miR-100 as a novel and more effective therapy for human breast cancer by modulating breast cancer stem cells.

癌症干细胞（CSCs）是癌症生长、复发、转移及耐药的根源，microRNA对它们的自我更新和分化起着非常重要的调控作用，暗示着microRNA可能是潜在的靶向癌症干细胞的药物。可是，如何将microRNA输送入癌症干细胞还存在着很大的挑战。我们的研究发现，病人乳腺癌组织中miR-100的表达与乳腺癌干细胞标记ALDH的表达成负相关，并且miR-100的表达与病人的总生存时间成正相关。由此推测乳腺癌干细胞受miR-100的调控，并且miR-100有望作为一个靶向癌症干细胞的药物。本项目是要通过体内外实验阐明miR-100调控乳腺癌干细胞的基本分子机制，以及miR-100对乳腺癌生长和转移的功能研究，最后利用我们最新研发的纳米载体结合乳腺癌干细胞标记CD44抗体将miR-100导入乳腺癌干细胞中，促进癌症干细胞的分化，提高化疗药物的疗效。本项目的研究将为恶性肿瘤的治疗提供一种更新颖有效的方案。

乳腺癌是导致妇女死亡的头号杀手，但对导致乳腺癌发病的细胞和分子机制没有足够的理解，目前缺乏有效的治疗药物。肿瘤干细胞（CSCs）是癌症生长、复发、转移及耐药的根源，肿瘤干细胞的发现，为寻找乳腺癌的诊断和治疗的方法提出了新的思路。我们通过对干细胞群体进行基因表达谱和microRNA表达谱的分析鉴定出乳腺肿瘤中存在相互转换的两种状态的干细胞以及不同形态的乳腺肿瘤干细胞受microRNA和相关信号通路分子的调控存在差异。mir-100在ALDH+的乳腺肿瘤干细胞中低表达且通过下调SMARCA5, SMARCD1等的表达来抑制乳腺肿瘤干细胞的自我更新，从而抑制肿瘤发展和转移(Cancer Research 2014)。进一步阐释了miR-100下游NMT1对乳腺癌以及乳腺癌干细胞的调控及机制，为治疗三阴性乳腺癌提供前期临床数据和机制支持(CDDis 2018)。mir-221在CD24-CD44+的乳腺肿瘤干细胞中高表达，可促进肿瘤的生长并调控肿瘤细胞的EMT过程，为临床治疗乳腺肿瘤提供可能的作用靶点（Oncotarget 2015）。同时，我们通过对临床样本等相关数据分析发现，在三阴性乳腺肿瘤干细胞中RAD51的蛋白水平以及活性都显著提高，从而对PARP抑制剂更具有抵抗性。提示靶向抑制RAD51可能会增加PARP抑制剂对三阴性乳腺癌的治疗效果（Clinical Cancer Research 2017）。在肿瘤内部低氧环境下，HIF1α通过上调Notch3来抑制IL-6的表达，从而导致CD24-CD44+肿瘤干细胞的下调。提示IL-6R的阻断剂联合Notch受体的抑制剂治疗Notch3高表达的乳腺癌会有更好的治疗效果(CDDiff 2018)。发现了预测三阴性乳腺癌对紫杉烷类化疗药物疗效的血清标志物CCL20并确定了CCL20通过激活NFkB通路上调ABCB1从而引起肿瘤干细胞化疗耐药的分子机制(Plos Biology 2018)。。这些研究能引导我们针对这些关键的调控节点开发靶向各种肿瘤干细胞的潜在药物，最终为乳腺癌的治疗提供潜在的更有效的干预靶点和联合药物治疗策略。在本项目的资助下，我们以通讯作者身份发表了十二篇SCI论文，申请了一项专利，培养了两名博士研究生、一名硕士研究生，还有一名博士研究生在读。