LKB1信号通路对细胞周期和细胞极性的协同调控机制研究

Cancer can develop in virtually any of the body's tissues, and the basic processes that produce cancer are quite similar in all forms of the disease. Loss of growth control and disruption of epithelial polarity are two hallmarks of cancer cells. After years of extensive scientific discovery, valuable information has been learned about the networks regulating cell growth and cell polarity, but the underlying molecular mechanisms, especially the crosstalk between them, are still elusive. LKB1 is a critical tumor suppressor, which regulates numerous biological pathways including apoptosis, cell proliferation, epithelial polarity, cell migration, and so on. In our previous study we have shown that endogenous LKB1 knockdown in two normal cell lines accelerates cell cycle progression through G1/S checkpoint by inhibition of p53 and p16 pathways. In addition, we have found reintroduction of LKB1 into LKB1 deficient Hela cells inhibited cell growth significantly and changed Hela cells into small, spindle-shaped cells, suggesting LKB1 may coordinate the cell growth and cell polarity pathways. In the present study, with molecular techniques, we ablate endogenous LKB1 expression in human normal epithelial cells and express exogenous LKB1 protein in human cancer cells. Based on the three-dimensional cell-culture models, we explore the mechanisms involved in LKB1 modulation of cell growth and cell polarity, and try to reveal the crosstalk between these two pathways. Our findings will extend earlier observations in single signaling pathway. A better understanding of these pathways and how their deregulation impact on cancer development will lead to the discovery of novel prognostic factors, novel chemotherapeutic targets and fundamental insights in to epithelial tumor biology and cancer progression.

增殖失控和极性紊乱是上皮细胞恶性肿瘤的两大特征，虽然近年来相关研究取得不少进展，但其信号转导机制仍不十分明确，特别是二者在肿瘤发生中的交互作用更知之甚少。抑癌基因LKB1在细胞增殖和极性调控中发挥着重要作用。我们前期研究发现，抑制正常细胞LKB1表达，p53和p16下调，细胞周期进程加快；恢复Hela细胞LKB1表达，细胞生长减慢，体积变小，形态变梭形，提示LKB1调控细胞周期和细胞极性的信号通路紧密联系、协同作用，共同决定细胞的转归。本课题拟采用分子生物学技术，建立LKB1基因表达沉默的人上皮细胞实验模型与LKB1基因表达恢复的人癌细胞实验模型，结合体外三维细胞培养技术，从细胞水平和分子水平研究LKB1基因对细胞周期和细胞极性的调控机制，解析两大通路间的协同作用和交联对话，以进一步揭示肿瘤发生的分子信号网络，为肿瘤防治提供新思路和理论依据。

增殖过度和极性紊乱是上皮细胞恶性肿瘤的两大主要特征，其相关的分子机制仍不完全明确，尤其是二者在肿瘤发生中的交联对话更知之甚少。抑癌基因LKB1与恶性肿瘤发生发展密切相关，参与调控细胞增殖、细胞极性等多条肿瘤相关信号通路。我们既往研究发现抑制正常细胞LKB1表达，p53和p16下调，细胞周期进程加快；恢复Hela细胞LKB1表达，细胞生长减慢，体积变小，形态变梭形，提示LKB1调控细胞周期和细胞极性的两条信号通路紧密联系、协同作用。微管亲和力调节激酶(microtubule affinity-regulating kinase, MARK)在细胞极性调控中发挥着关键性作用，研究已证实LKB1通过磷酸化并活化MARK激酶活性。本课题在表达内源性LKB1蛋白的三个细胞系中，外源性激活LKB1/AMPK通路，结果发现p53、p21、p16上调，细胞周期停滞在G1期，上述作用依赖于LKB1的激酶活性。我们进一步利用重组慢病毒体系，在LKB1表达缺陷的人宫颈癌细胞株Hela细胞中外源性增强MARK激酶4个亚型（MARK1、 MARK2、MARK3、MARK4）的表达，在人正常前列腺上皮细胞株RWPE-1中抑制内源性MARK1、 MARK2、MARK3、MARK4的表达，结果发现外源性激活MARK2能够重建Hela细胞的细胞极性，通过增强AMPK的磷酸化，促进细胞周期抑制因子p21及p16的表达，使细胞周期停滞在G1期，抑制细胞生长及增殖；抑制内源性MARK2、MARK3表达后RWPE-1细胞极性减弱，通过抑制AMPK的磷酸化，减少p53、p21及p16的表达，促进细胞周期从G1期进入S期，使细胞生长加快、增殖增强。我们的结果表明，LKB1/MARK通路，通过调控AMPK的活性，调节其下游p53、p21、p16的表达，从而协同调节细胞周期及细胞极性的两条信号通路。我们的结果进一步完善了肿瘤发生的分子信号网络，为肿瘤防治提供新思路和理论依据。