GalNAc-T6活化MUC1-CD相关通路促进肺腺癌转移的机制研究

The mechanism for the metastasis of lung adenocarcinoma is an important while still pending problem needed to be unraveled. Recent reports show that WNT/β-Catenin signaling pathway plays a key role in the metastasis of lung adenocarcinoma. In this procedure, the phosphorylated cytoplasmic domain of mucin-1(MUC1-CD) is cleaved and shed off from cell membrane into the cytoplasm, where the MUC1-CD can stabilize β-Catenin and promote its transportation to the nuclear. Thus, WNT/β-Catenin signaling pathway is activated. The phosphorylation and the cleavage of MUC1-CD are modulated by mucin-type O-glycosylation of the extracellular domain of MUC1. Polypeptide N-acetylgalactosaminyltransferase 6 (GalNAC-T6) has been proved to be the key catalytic enzyme for mucin-type O-glycosylation of MUC1. For the first time, with clinical paraffin specimens, we found that high expression of GalNAc-T6 in cancer cells significantly correlated with the metastasis of lung adenocarcinoma, and independently predicted shorter disease-free survival. However, the mechanism by which GalNAc-T6 promoted lung adenocarcinoma metastasis was unclear. Therefore, our hypothesis is as follows: GalNAC-T6 regulates WNT/β-Catenin signaling pathway through inducing the phosphorylation and the cleavage of MUC1-CD, and promotes the metastasis of lung adenocarcinoma. Cellular and animal experiments will be done by inducing the overexpression or silence of GalNAc-T6 genes in lung adenocarcinoma cell lines. The results will illustrate the signaling pathway of lung adenocarcinoma metastasis with more insights.

肺腺癌转移涉及多种信号分子参与，WNT/β-连环蛋白（β-Catenin）通路是其中的关键通路。被剪切后脱落的磷酸化的粘蛋白1胞内段（MUC1-CD）可稳定β-Catenin并促进其入核，激活WNT/β-Catenin通路，但该过程的调节机制不明。MUC1-CD的磷酸化和剪切，与其胞外段的粘蛋白型O-糖基化密切相关。而N-乙酰胺基半乳糖转移酶-6（GalNAc-T6）是调节MUC1粘蛋白型O-糖基化的关键酶。我们的原创研究证实，高表达GalNAc-T6与肺腺癌转移，缩短的无病生存和总生存显著相关，且可独立预测无病生存。然而，其促转移机制不清。由此，我们拟建立GalNAc-T6基因过表达或沉默的细胞、动物模型，研究GalNAc-T6可否增加MUC1的磷酸化及游离MUC1-CD的产生，从而激活WNT/β-Catenin通路，促进肺腺癌转移。本研究将对进一步了解肺腺癌转移机制具有重要意义。

GalNAc-Ts糖基转移酶家族是催化粘蛋白型O-糖基化的始动步骤的关键酶。该家族蛋白表达和功能的异常是多种恶性肿瘤共同特征，并与增殖和转移关系密切。然而，单个家族成员，例如GalNAc-T6，在恶性肿瘤的表达和功能还不为人知。本研究着眼于肺腺癌，并延伸到乳腺癌和结肠癌，探索GalNAc-T6在上述癌种的表达，功能和潜在机制。.本研究发现GalNAc-T6是上述恶性肿瘤的预后因子，高表达GalNAc-T6提示预后不良。上述结果经内部验证和利用在线数据集的外部验证证实。基于在线高通量数据，本课题还发现GalNAc-T6高表达与O-糖原合成，Toll样受体，Notch信号通路活化有关；IPA分析显示GalNAc-T6与糖原合成，ER受体等信号有关。在NCI60细胞系发现GalNAc-T6表达与GalNAc-T4、GalNAc-T10,11负相关。基于所有Glycosyltransferases（GTs）的正则化回归结果显示，应用GTs可以区分肺腺癌患者预后。.在细胞系实验中，本研究发现敲除GalNAc-T6后可以抑制上述癌种的增殖，迁移，和侵袭。同时，MUC1-TR、CXCR4、磷酸化EGFR等重要增殖和转移相关蛋白的水平随GalNAc-T6改变，提示GalNAc-T6可以通过调节上述蛋白发挥功能。在乳腺癌MDA-MB-231细胞，本课题组发现GalNAc-T6与上皮间质转化（EMT）表型和相应转录分子密切相关，提示GalNAc-T6与间质表型有关。另外，亲代和瞬时敲除GalNAc-T6的MDA-MB-231细胞株的糖基化组学分析发现，AHSG，角蛋白，α2Μ与CBFA2T2等糖蛋白的O-糖基化水平下调，而nesprin蛋白，piccolo蛋白，D-乳酸脱氢酶粘蛋白型O-糖基化水平上调，提示GalNAc-T6对各个表型的不良影响可能与上述蛋白的O-糖基化改变有关；针对结肠癌的GSEA数据显示，GalNAc-T6高表达与ALDH家族蛋白有关，而利用流式细胞术评价GalNAc-T6对结肠癌HCT-116细胞表面干细胞标志物的影响。结果发现，ALDH1A1与CD44双表达的干细胞亚群比例显著提高。这提示GalNAc-T6与可能结肠癌干细胞的维持和转换有关。.本研究为进一步明确GalNAc-T 影响恶性肿瘤增殖、转移的机制，同时也可能为肺腺癌转移的临床预测等方面提供新的线索。