肺鳞癌候选预后标志物SLC1A3的研究

Metastasis is the major cause of lung cancer death. Although lymph node status at surgery is one of important factors for prognosis, patients with or without lymph node metastasis could be either at high (die within 2 years) or low (survive more than five years) risk, inferring that lymph node status alone cannot be an accurate predictor for lung cancer patients’ survival. Therefore identification and application of the biomarkers of metastasis and prognosis is essential for selecting the best treatment strategy. Using array-CGH and bioinformatics analysis, our previous study constructed a prognosis-predicting model with 41 chromosome fragments, which could accurately separate squamous cell lung cancer (SCC) patients at high (50 cases) or low risk (50 cases). Among 41 fragments, genomic gain or loss of 5p13.2 was correlated with good or poor prognosis, respectively (P＜0.001). In 45 independent SCC samples, the association of genomic alteration of 5p13.2 with prognosis was confirmed using real-time PCR analysis (P＜0.05). SLC1A3 was the only gene located in 5p13.2 regions. Using immunohistochemistry (IHC) analysis，the expression of SLC1A3 protein was not shown in normal lung tissue. However in 37% of SCC cases, SLC1A3 protein was overexpressed and the overexpression of the protein was correlated with poor prognosis (P＜0.01). It indicated that the SLC1A3 might be similar to TP53, of which the mutations result in the synthesis of nonfunctional proteins. This project aims to identify if the gene SLC1A3 is the target gene, of which the amplification or deletion can predict SCC metastasis and prognosis. Firstly, we will apply array-CGH analysis to compare copy number variation of SLC1A3 in normal and cancer tissue, in order to identify if the amplification of SLC1A3 in cancer tissue was actually a copy number variation in germline DNA. Secondly, Fluorescent in situ Hybridization (FISH) and real-time PCR analysis will be applied to evaluate the incidence of SLC1A3 amplification (or deletion). The correlation of SLC1A3 amplification (or deletion) with recurrence, metastasis and prognosis will also be analyzed. The sequencing analysis will be applied to examine the gene mutation status in the SCC cases with high expression of the SLC1A3 protein. The gene function of SLC1A3 will be studied in SCC cell lines and 3D organotypic Human Bronchial Epithelial Cell air liquid interface culture system. The SLC1A3 gene function will be finally studied in nude mice. Eventually we will evaluate the effectiveness of SLC1A3 as a prognosis marker for SCC.

淋巴结转移状态不能准确预测肺鳞癌预后，在转移和非转移病例中均有高危(＜2年内死亡)和低危（＞5年生存）患者。本课题组前期研究采用array-CGH和生物信息学分析，构建了由41个染色体片段组成的预后预测模型准确区分高危和低危肺鳞癌病例。其中5p13.2区段扩增或缺失与良好或不良预后显著相关(P＜0.001)。SLC1A3是该区段唯一基因，提示可能是候选抑癌基因。免疫组化分析显示SLC1A3在正常肺组织中阴性表达，在37%的肺鳞癌病例中高表达且与不良预后显著相关(P＜0.01)，提示SLC1A3可能与TP53相似，即基因突变造成失活蛋白质高表达。本研究旨在验证SLC1A3是否为5p13.2区段的靶标，即其扩增或缺失（拷贝数变化）能够作为判断预后的依据。此外，我们将检测SLC1A3蛋白高表达病例中基因突变情况并对SLC1A3基因功能在2D和3D细胞水平以及裸鼠动物模型中进行研究。

约80%的肺腺癌由于具有治疗靶点可以采用靶向治疗，但是肺鳞癌却因缺乏有效的治疗靶点，预后较差。本研究主要目的是为了发现肺鳞癌中的可靶向的基因以及预后预测指标。本研究采用二代测序检测，在35例样本中8例样本肿瘤细胞中(22.8%) 检测到BRCA1、BRCA2、TSC2基因致病性突变或ROS1基因融合，是临床Level2B级的靶向治疗证据；另外5例样本(14.3%)检测到FGFR1拷贝数增高，为临床Level3A级的靶向治疗证据；其次PIK3CA拷贝数增高或致病性突变出现在16例肿瘤组织中(45.7%)，为临床level4级的靶向治疗证据。在总体的突变中，频率较高的基因分别为TP53，CDKN2A,BRCA1,TSC2,NOTC1等; 拷贝数变异频率较高的基因包括PIK3CA,MYC,FGFR1,CCND1等目前BRCA1、BRCA2、TSC2致病性突变和ROS1基因融合在文献中鲜有报道，尚不明确是否为中国I期肺鳞癌患者特有的特征。目前本研究在进一步扩大样本验证中，如果证实肺鳞癌确实存在较高频率的BRCA1、BRCA2等基因突变，那么可以在临床中采用靶向治疗，将对临床有推动作用。