负性共刺激分子B7-H3调控结直肠癌肿瘤干细胞干性的功能及机制研究

Colorectal cancer is one of the most lethal cancers of the gastrointestinal system. The concept of tumor development driven by a unique subpopulation of cancer stem cells (CSCs) may help to explain the high mortality, low response to treatment and tendency of developing multiple tumors in cancer. However, the molecular mecshanim regulated the cancer stem cells are remain to be elucidated. The B7-H3, a kind of B7 family negative costimulatory molecular, mediates tumor evasion of immunosurveillance. Our previous study has found that B7-H3 was abnormally expressed in colorectal cancer (CRC) tissues, correlating to CRC stages and tumor progression; B7-H3 is highly expressed in CD133 positive fractions by flow cytometry; Blocking B7-H3 by siRNAs in CRC cell lines reduced formation of spheroids, expression of stem cell associated genes and surface marker of cancer stem cell; Mechanismly, B7-H3 could bind to c-Met which is an important oncogene by Co-IP and mass spectrometry assay. Knockdown B7-H3 reduced phosphorylation level of Stat3 which was considered as downstream of c-Met signal pathway. Furthermore, Bmi1, which regulated by Stat3 signal and Twist1, is considered as effector gene to maintain stemness of CRC. On this basis, we have analyzed the correlation between B7-H3 and Bmi1 in clinical samples. Tissue IHC analysis of CRC specimens from 197 patients revealed a close correlation between B7-H3 expression and Bmi1 levels. We will further confirm the feature and mechanism of cancer stemness regulation by B7-H3/c-Met/Stat3/Twist1/Bmi1 signal pathway via in vitro and in vivo experiments. In general, the achievement of this program will improve the stemness regulation mechanism research of B7-H3 and provide the new target for clinical tumor immunotherapy.

结直肠癌是常见的恶性肿瘤，其发生发展可由肿瘤干细胞调控，但目前对调控结直肠癌肿瘤干细胞干性的分子及机制尚未阐明。本项目通过分析临床标本及构建多种细胞模型发现：负性共刺激分子B7-H3高表达于结直肠癌肿瘤干样细胞中，且干扰B7-H3后，肿瘤干细胞干性特征显著下降。在机制方面，利用免疫共沉淀及蛋白质谱分析发现，B7-H3通过结合癌基因编码蛋白c-Met，引起下游Stat3磷酸化水平下调，进而通过Twist1蛋白引起干性基因Bmi1表达改变，从而导致干性特征改变。本项目以肿瘤干细胞干性调控为切入点，根据B7-H3与结直肠癌肿瘤干细胞干性的相关性研究，利用临床标本、细胞株及模型动物进行体内外实验论证B7-H3通过c-Met/p-Stat3/Twist1/Bmi1信号维持肿瘤干细胞干性。本项目将从肿瘤干性这个新视点阐明B7-H3在结直肠癌细胞中异常表达的生物学作用及机制，为结直肠癌的治疗提供新思路。

负性共刺激分子B7-H3广泛存在于各种类型的肿瘤中，在正常健康组织中却很少表达。我们将B7-H3在结直肠癌中的表达分为阴性表达，肿瘤细胞表达以及间质细胞表达，分类后统计结果显示B7-H3在肿瘤细胞表达的预后最差，在间质细胞表达的次之，B7-H3阴性表达的病人预后最好，中位生存时间分别是84个月，52个月以及32个月。进而我们检测了结直肠癌组织的B7-H3与干性标志的表达情况，发现B7-H3与三种干性标志都有显著的共表达。而在细胞系中，利用PKH26染色，成球的细胞以及化疗药抵抗的细胞，同样也发现这些富集干性的肿瘤细胞也都高表达B7-H3。这些结果提示我们B7-H3的表达主要定位于肿瘤干样细胞中，并与结直肠癌患者的生存呈负相关。为了进一步分析B7-H3调控干性功能的潜在机制，我们首先通过pull down B7-H3进行质谱分析，找到了可能的B7-H3结合分子c-Met，进而利用Co-IP确认了B7-H3与c-Met的结合，并通过ELISA确认了二者的直接结合。进一步利用HGF以及c-Met的阻断性抗体抑制研究发现B7-H3与c-Met的结合位点可能与c-Met与HGF的结合位点有重合，最后我们利用免疫荧光技术在多种细胞中观察到了二者的共定位。这些数据证实了B7-H3与重要癌基因c-Met的结合，并提示了潜在的结合位点。此外，我们采用自主研发的特异性抗人B7-H3单抗（3E8）成功阻断了B7-H3与c-Met的结合。该抗体对结合的阻断效果呈浓度依赖，并且可以抑制p-Met以及下游p-Stat3的表达。荷瘤裸鼠结果显示，抗体治疗组肿瘤显著小于对照IgG组，将小鼠肿瘤组织进行进一步分析发现抗体治疗组肿瘤区域减小，肿瘤组织的B7-H3与CD133的表达显著减弱。由此表明，B7-H3的阻断性抗体能够阻断B7-H3与c-Met的结合从而抑制肿瘤生长。