CD44v16对乳腺癌干细胞耐药性的影响及其应用

In our previous study, a new variant of CD44 gene, named as CD44v16, has been successfully cloned from multidrug resistant breast cancer cell line, MCF-7/Adr in our laboratory. The nucleotide sequence of CD44v16 has been deposited GenBank under accession number FJ216964. The preliminary functional analyses of CD44v16 gene found that it was related to multidurg resistance of breast cancer stem cells. This study will be divided into the following four parts: ①In part I, the doxorubicin-resistant breast cancer stem cells will be identified from the MCF-7 cells and breast cancer tissue cells. ②In the second part, CD44v16 will be confirmed as a specific surface marker of breast cancer stem cells, and effects of CD44v16 on breast cancer stem cell self-renewal, proliferation and resistance characteristics will be examined. ③Furthermore, the molecular mechanism of chemosensitivity modulation by CD44v16 in breast cancer stem cells will be explored from the aspect of transcriptional regulation and signaling transduction pathway of drug-resistant gene. ④Finally, the CD44v16 antigen loaded dendritic cells in vitro will stimulate cytotoxic T lymphocyte, by which killing efficiency to breast cancer stem cells will be examined. This study could look for a specific marker of cancer stem cells, and provide target for gene therapy that might help to reverse drug resistance of breast cancer stem cells. It is important for further improve the efficacy of chemotherapy in clinical practice.

我们前期研究在乳腺癌耐药细胞株MCF-7/Adr中发现了一条新的人CD44剪接变异体，命名为CD44v16，GenBank登录号：FJ216964，初步功能研究发现，CD44v16与乳腺癌干细胞耐药密切相关。本课题拟进行四个方面的后续研究：一是从乳腺癌MCF-7细胞和乳腺癌组织原代培养细胞中分离耐药的乳腺癌干细胞并进行鉴定；二是证实CD44v16是乳腺癌干细胞的一种特异性表面标志物，并且对乳腺癌干细胞自我更新、增殖及耐药特征有影响；三是从耐药基因转录调控、信号转导环节研究CD44v16调节乳腺癌干细胞耐药的分子机制；四是以CD44v16作为抗原负载树突状细胞体外激发细胞毒性T淋巴细胞反应，探索其对乳腺癌（干）细胞的杀伤效率。这一研究对于寻找特异性的肿瘤干细胞标志物，开发针对乳腺癌干细胞的新一代高特异性免疫药物及生物疗法，克服乳腺癌干细胞多药耐药具有重要意义和潜在的临床应用价值。

我们前期研究在乳腺癌耐药细胞株MCF-7/Adr中发现了一条新的人CD44剪接变异体，命名为CD44v16，GenBank登录号：FJ216964，初步功能研究发现，CD44v16与乳腺癌干细胞耐药密切相关。本课题主要研究内容包括：一是从乳腺癌MCF-7细胞和乳腺癌组织原代培养细胞中分离耐药的乳腺癌干细胞并进行鉴定；二是证实CD44v16是乳腺癌干细胞的一种特异性表面标志物，并且对乳腺癌干细胞自我更新、增殖及耐药特征有影响；三是从耐药基因转录调控、信号转导环节研究CD44v16调节乳腺癌干细胞耐药的分子机制；四是以CD44v16作为抗原负载树突状细胞体外激发细胞毒性T淋巴细胞反应，探索其对乳腺癌（干）细胞的杀伤效率。实验结果表明：(1)通过化疗药物结合无血清培养法分离富集耐药乳腺癌干细胞，可诱导获得乳腺癌干细胞微球体;(2)CD44v16过表达促进细胞增殖和干性，抑制细胞凋亡，影响乳腺癌耐药性；(3)CD44v16增强干性转录因子OCT4、SOX2和c-Myc以及降低KLF4基因的表达来影响乳腺癌细胞的干性，还可以通过自噬途径调控乳腺癌细胞的耐药性，以及通过JNK 及ERK1/2 双重信号转导通路的作用促进MMP7 的过表达，影响乳腺癌的迁移及侵袭能力；(4)乳腺癌干细胞抗原致敏的DC疫苗对荷瘤小鼠存在免疫治疗作用，能够诱导靶向肿瘤干细胞的抗肿瘤免疫反应，抑制乳腺癌的发生发展，降低了乳腺癌细胞的侵袭能力，延长生存时间，在乳腺癌的免疫治疗方面具有潜在的优势。这一研究对于寻找特异性的肿瘤干细胞标志物，开发针对乳腺癌干细胞的新一代高特异性免疫药物及生物疗法，克服乳腺癌干细胞多药耐药具有重要意义和潜在的临床应用价值。