PI3K催化亚单位调控AKT活化影响非小细胞肺癌脑转移的机制研究

Brain metastasis (BM) is a common complication of non-small cell lung cancer (NSCLC)and the survival time after the diagnosis of BM remain poor. PI3K/AKT is one of the key signaling pathways that affects tumor metastasis.We found that brain metastasesdeveloped more often in patients with the AG/AA genotype of PIK3CA: rs2699887 than in patients with the GGgenotypes. Our research also confirmed that the AKT activation level are closely related with brain metastases in NSCLC patients. As is known to all, the AKT activation is regulated by the PI3K. We assume that the activation of PI3K/AKT pathway for NSCLC patients with PIK3CA: rs2699887 AG/AA genotype is regulated by a particular PI3K catalytic subunit, the activation level is higher, and the incidence of brain metastases is increased. On this basis, this study will further verify the previous find in larger samples. We will also use the molecular transfection technology to build two A549 lung cancer cell lines which express two kinds of different genotypes of PIK3CA: rs2699887. The pAKT and downstream protein expression will be detected to find the differences in cells and animals. And the inhibitors of PI3K catalytic subunits will be used separately to suppress two subunits (p110a and p110 beta), which in order to make clear the role of two subunits in brain metastasis of NSCLC. This will provide theoretical basis to clarify the molecular mechanism of brain metastases in NSCLC, provide new molecular targets for the intervention, which is expected to improve the treatment efficacy of NSCLC.

非小细胞肺癌（NSCLC）脑转移很常见，而预后很差。PI3K/AKT是影响肿瘤转移的关键信号通路之一。申请者在前期研究中发现PIK3CA:rs2699887 AG/AA较GG基因型NSCLC患者的脑转移发生风险更高。我们还证实，AKT活化水平与NSCLC脑转移相关。而AKT活化受PI3K调控。我们假设AA基因型患者PI3K通路的活化受特定PI3K催化亚基调控，因而活化水平增高，脑转移风险增加。在此基础上，本研究除了在更大样本中验证前期发现外，还将利用分子转染技术建立表达PIK3CA:rs2699887两种不同基因型的肺癌细胞系，在细胞和动物水平探讨pAKT及下游蛋白表达差异。且通过PI3K催化亚基抑制剂分别抑制两种亚基（p110a和p110β），明确两种亚基在NSCLC脑转移中所起的作用。为阐明NSCLC脑转移分子机制提供理论基础，为对其干预提供新的分子靶点，有望提高NSCLC的治疗疗效。

项目背景及主要研究内容：非小细胞肺癌患者脑转移很常见，而预后很差。PI3K/AKT是影响肿瘤转移的关键信号通路之一。申请者在前期研究中发现PIK3CA:rs2699887 AG/AA基因型较GG基因型患者的脑转移发生风险更高。我们的研究还证实，AKT活化水平与非小细胞肺癌（NSCLC）患者脑转移密切相关，而AKT活化受PI3K调控。我们假设PIK3CA:rs2699887AG/AA基因型NSCLC患者PI3K通路的活化受特定的PI3K催化亚基调控，因而活化水平增高，进而脑转移发生率增加。在此基础上，本研究除了在更大样本中进一步验证前期发现外，还将利用分子转染技术建立表达PIK3CA:rs2699887两种不同基因型的A549肺癌细胞系，在细胞和动物水平探讨pAKT及下游蛋白表达差异。且通过PI3K催化亚基抑制剂分别抑制两种亚基（p110a和p110β），明确两种亚基在NSCLC脑转移中所起的作用。另外，查找PI3K/AKT下游影响NSCLC脑转移的基因，构建不同基因型细胞及脑转移动物模型，研究不同基因型对脑转移的影响。.重要结果及关键数据：（1）在既往323例NSCLC脑转移的样本基础上，新增272例样本。（2）在组织样本中验证PIK3CA:rs2699887基因多态性及pAKT蛋白水平对NSCLC脑转移的预测价值。（3）发现PI3K/AKT下游基因——自噬相关基因的遗传变异可能影响NSCLC患者的脑转移(BM) 。我们发现3个SNPs, ATG10:rs10036653,ATG16L1:rs2241880,ATG12:rs26532与BM发生风险相关。ATG10:rs10036653(P = 0.063)和AG / GG基因型ATG16L1:rs2241880(P = 0.014)的BM发生率较低。ATG12型AC / CC基因型患者的BM发生率较高:rs26532(P = 0.015)。（4）构建含不同基因型质粒，并稳定转染肺癌A549细胞。（5）构建了不同基因型A549细胞裸鼠的脑转移瘤动物模型。发现A549– 300T组(42.9%)中BM的百分比高于A549 – 300A组(14.2%)和A549-MOCK组(12.5%)。.科学意义：在此基础上建立了结合临床和遗传学因素的脑转移预测模型，改变了国际上仅用临床因素预测脑转移发生风险的模式。为阐明NSCLC脑