MFAP5通过缺氧微环境诱导的HIF-1α/Notch通路促进口腔鳞癌转移的机制研究

Metastasis is mainly accounts for treatment prognosis of oral cancer. Occult cervical lymphatic metastasis is nearly 30% in oral squamous cell carcinoma with early stage (cT1-2N0M0). The key point is to seek out biomarker to distinguish the groups who can benefits from the treatment. The biomarker should explain the mechanism of metastasis before clinical application. Our research group has completed comparative transcriptomics for tumor and normal tissues from patients with or without occult cervical lymphatic metastasis. The results showed that microfibrillar-associated protein 5(MFAP5) is obvious highly expressed in tumor tissue of occult cervical lymphatic metastasis group and also in cell line which has been considered with highly metastatic potential. All this results have been validated in mRNA and protein levels, and MFAP5 is correlation with prognosis and metastasis. Hypoxia micro-environment can elevate the expression of MFAP5 in oral cancer cells, which was potential for further mechanism research of its up-regulation and metastasis. It has been reported that Notch signaling can be activated by up-regulated HIF-1α during hypoxia-environment and also mediate intracellular function of MFAP5 binding with its ligand or receptor. Thus we hypothesize that a positive feedback loop may be present between MFAP5 and HIF-1α/Notch pathway in hypoxia environment, which promoting metastasis. Based on our previous results, we main to focus on the HIF-1α/Notch signal pathway which was induced in hypoxia environment and to explore the molecular mechanism of up-regulation and metastasis promoting ability of MFAP5 in oral squamous cell carcinoma by in-vivo and in-vitro studies and further to validate it in patients. Our research will provide an experimental evidence for the selection of early stage metastasis biomarkers in oral squamous cell carcinoma.

颈淋巴转移是影响口腔癌预后的主要因素，课题组临床试验发现近30%的早期口腔癌出现隐匿性颈转，亟需能从机制上解释的生物标志物提供早期预警进而有效治疗。全转录组芯片筛选发现微丝相关蛋白5（MFAP5）在转移患者的原发癌组织中高表达，并在mRNA和蛋白水平证实与转移和生存预后相关；缺氧培养可使口腔癌细胞系中MFAP5表达上调，是探究其表达升高及促进转移分子机制的可能切入点。查阅文献Notch通路可由缺氧相关因子HIF-1α激活，也可介导MFAP5发挥胞内功能。因此我们提出口腔癌中MFAP5可能与HIF-1α/Notch通路形成正反馈环路，上调表达并促进转移。本研究拟通过功能性拯救实验与裸鼠模型，研究MFAP5对口腔癌细胞侵袭转移能力的影响，以缺氧诱导的HIF-1α/Notch通路为切入点，探讨MFAP5上调及促进转移的分子机制，与临床样本验证相结合，提供其作为口腔癌转移早期预警标记物的实验依据。

课题组前期通过转录本芯片等研究已经在RNA、蛋白水平证明肿瘤细胞运动相关的微丝相关蛋白5（MFAP5）在口腔颌面头颈鳞癌样本中高表达，且与患者的不良预后及颈淋巴结转移密切相关，缺氧是恶性肿瘤的重要特征之一，但对肿瘤缺氧微环境与MFAP的关系的机制尚不清楚，课题组结合前期结果推测，缺氧微环境在MFAP5促进口腔颌面头颈鳞癌侵袭转移过程中发挥重要作用。通过细胞学的功能拯救实验发现：MFAP5过表达导致口腔颌面头颈鳞癌细胞侵袭、迁移能力的显著增强，缺氧状态可促进MFAP5的过表达。小鼠的体内实验证实了MFAP5过表达可以显著提高肿瘤细胞肺转移的能力。在其相关下游机制的研究中：MFAP5表达的升高可以引起AKT通路的激活，磷酸化的AKT进入细胞核重要元件Snail的表达，引起侵袭、迁移相关蛋白如金属基质蛋白酶2（MMP2），金属基质蛋白酶9（MMP9）和波形蛋白（Vimentin）等的表达升高，引起了转移的标志事件上皮间充质转化，同时这一过程可被AKT磷酸化抑制剂所逆转。临床样本的免疫组织化学染色也证实了MFAP5的表达水平与缺氧标志物HIF-1α及侵袭相关蛋白Vimentin的表达密切相关。通过本课题的研究，我们首次在头颈鳞癌内证明了缺氧与MFAP5之间的关系，发现了MFAP5在缺氧诱导的不良预后中扮演的关键作用并揭示了其相关的机制。本课题的研究结果丰富了对头颈鳞癌细胞侵袭转移过程中分子事件的认识，为开发以MFAP5靶点蛋白提供了重要的依据。项目实施以来，共发表SCI论文9篇，其中第一标注5篇，国内外会议交流2次。