单磷酸腺苷活化蛋白激酶羧基端S/T-区域磷酸化负调节及其在肿瘤转移中的作用

AMP-activated protein kinase (AMPK) has emerged as a metabolic tumor suppressor. Recent studies have shown that AMPK is suppressed in advanced stages of several different cancers, which may constitute a premise for cancerous epithelial to mesenchymal transition and formation of cancer stem cells. However, little is known about the mechanism underlying AMPK suppression in cancer cells and few reports have documented the negative regulation of AMPK via phosphorylation of the carboxyterminal residue S485/S491 (equivalent sites in 1 and 2, respectively). There is no investigation on the phosphorylation status of these sites in human cancer. Thus, we carried out preliminary studies and showed that phosphorylation of S485 was significantly increased in pancreatic cancer specimens, which exhibited an opposite change, as compared to adjacent normal tissues. Furthermore, we identified several other phosphorylation sites on the carboxyterminal domain in cancer cells which might exert negative impact on AMPK1. Therefore, we hypothesize that phosphorylation of these sites on the S/T-Loop suppresses AMPK activation, leading to cancer progression and metastasis. To test this hypothesis, we will carry out the following specific aims: (1) screen a panel of cancer cell lines containing high and low phosphorylation of S485 and identify kinases responsible for the phosphorylation, (2) elucidate the mechanism by which phosphorylation of S485 and other sites identified by us inhibits AMPK, (3) explore AMPK activators used in human that give rise to optimal activation of AMPK in the context of high phosphorylation of S/T-Loop, (4) determine the role for phosphorylation of these negative sites in cancer metastasis. The proposed studies will hopefully provide important insights into the mechanism underlying the phosphorylation of the carboxyterminal S/T-Loop in the regulation of AMPK activation and the role of AMPK in cancer progression and metastasis. They will also provide us clues regarding the potential of using AMPK in cancer therapy.

AMPK为代谢性肿瘤抑制因子。在肿瘤晚期，AMPK活性受抑制，可能为肿瘤上皮细胞向间充质细胞转化和干细胞形成创造了条件。但目前对AMPK活性被抑制的机制缺乏认识，AMPK的负性调控仅限于其羧基端S485/S491磷酸化的少数报道，且尚不知该位点在肿瘤中的磷酸化状态。我们的前期研究发现AMPK S485磷酸化在胰腺癌中显著升高，与活化磷酸化位点T172呈反向变化；并发现其它可能起负调节作用的磷酸化位点。因此，我们假设S/T-区域的磷酸化抑制AMPK活性，从而促进肿瘤的进展和转移。为此，我们将开展以下研究：(1)在肿瘤细胞中，寻找S485的磷酸化激酶;(2)确立S485和S/T-区域位点磷酸化抑制AMPK活性的机制；(3)探索这些位点高磷酸化时，有效的AMPK激活剂；(4)确立这些位点磷酸化对肿瘤转移的影响。该研究对解析AMPK在肿瘤细胞中的抑制机制、转移中的作用和提供治疗方案具有重要意义。

AMPK为代谢性肿瘤抑制因子。在肿瘤晚期，AMPK活性受抑制，可能为肿瘤上皮细胞向间充质细胞转化和干细胞形成创造了条件。但目前对AMPK活性被抑制的机制缺乏认识，AMPK的负性调控仅限于其羧基端S485/S491磷酸化的少数报道，且尚不知该位点在肿瘤中的磷酸化状态。本研究旨在揭示AMPK在肿瘤细胞中的抑制机制，为此本研究围绕AMPK S485位点开展了一系列的研究。本课题组首次发现，与癌旁组织相比较，胰腺癌组织中AMPK α1 S485的磷酸化水平明显增加，且强阳性表达患者，其生存率显著低于p-AMPKα1 S485的阴性表达者；AMPK α1 S485的磷酸化水平受PI3K/PKB和PKC的调控；我们的研究显示，在基础状态下，肿瘤细胞中AMPK S485的磷酸化受PKC抑制剂的阻遏，而在胰岛素刺激下，它的磷酸化受PI3K和PKB抑制剂的抑制，提示S485受多种癌基因激活蛋白激酶调节。当AMPKα1 S485突变成A的去磷酸化突变体后，AMPK-T172 的磷酸化水平增加，癌细胞增殖和迁移能力下降。这些结果提示，AMPK的S/T-区域的S485位点的磷酸化抑制AMPK的活性，从而促进肿瘤的进展和转移。鉴于Ras和PI3K活性突变和PTEN失活性突变见于许多肿瘤，我们的结果提示AMPK S485磷酸化可以作为一种广谱的肿瘤标志物，也可以作为对肿瘤化疗药物敏感性的衡量标准。因此，本研究为解析AMPK在肿瘤细胞中的抑制机制、转移中的作用和肿瘤防治提供了重要的理论和实验依据。我们将进一步将肿瘤细胞中AMPK S485的磷酸化水平与Ras、PI3K、PTEN突变基因联系在一起，进行突变位点序列分析，确立它们之间的关系。