Genetic Alterations in Lung Cancer

肺癌是男性和女性癌症相关死亡的主要原因。染色体9 p缺失和改变发生在肺癌的早期和经常发生。p16/CDKN 2位点位于9 p，被怀疑是该肿瘤类型中失活的主要抑癌基因。肺癌患者的预后非常差，总体5年生存率仅为14%。预后不良主要是由于历史上缺乏有效的早期检测措施。我们以前已经确定了一个区域的纯合性缺失染色体9 p的短臂上的微卫星标记D9 S126。该区域与p16/CDKN 2肿瘤抑制基因（TSG）位点不同，位于近端约2 cM处。我们提出，该地区窝藏TSG可能是重要的肺肿瘤发生。为此，我们采用了几种技术来确定该染色体区域内的基因。目前，我们已经分离出几个表达序列标签（EST）和基因定位在D9 S126或周围。此外，我们用免疫组化分析了肺腺癌和鳞状细胞癌中p16的表达，并用跨越9号染色体短臂的微卫星标记分析了这些肿瘤的亚群的杂合性丢失（洛）。免疫组化结果显示，腺癌p16蛋白阳性率高于鳞癌。杂合性丢失分析表明，p16阳性和阴性鳞状细胞癌之间的丢失模式相似。然而，在p16阳性的腺癌和鳞状细胞癌之间的洛分析中，在某些标志物上存在中度显著差异。总之，这些数据表明，染色体9 p可能含有其他肿瘤抑制基因在肺肿瘤的发生和肺鳞癌和腺癌之间的不同模式的洛可能表明他们的位置。 Cot基因最初是通过将人甲状腺癌DNA转染到SHOK细胞中来鉴定的。发现该基因在3'端改变时是致癌的，组成性激活蛋白质。测序分析显示，野生型Cot是一种MAP激酶，在多种细胞中表达，包括脾、胸腺、肝和肺。Cot具有激活多个级联的独特特性，包括增殖和凋亡信号转导途径，例如分别为MEK-1和SEK-1途径。在利用肺肿瘤DNA的NIH 3 T3转染测定中，我们的实验室鉴定了与先前报道类似的Cot的3'改变。因此，我们认为Cot的致癌激活可能在肺肿瘤的形成和发展中起作用。应用聚合酶链反应-单链构象多态性（PCR-SSCP）分析技术，对40株肺癌细胞株的Cot全开放阅读框架进行了分析，以确定Cot序列内的突变。在细胞系中鉴定出一种突变/多态性，但未导致氨基酸变化。没有发现可能激活该基因的其他点突变。使用3' RACE和DNA测序来分析可能在Cot的3'末端具有截短或改变的肺肿瘤细胞系，没有鉴定出通过该机制具有基因激活的任何细胞系。然而，通过RT-PCR对肺肿瘤细胞系的表达分析确实证明了Cot基因在小细胞肺癌细胞系和非小细胞肺癌细胞系中的下调。事实上，两个小细胞肺肿瘤细胞系似乎不表达任何Cot基因转录本。因此，我们目前正在对这些细胞系进行Southern和Western印迹分析，以确定基因组重排或蛋白质改变。这些实验将有助于理解Cot的调节及其在细胞凋亡、增殖和致瘤潜力中的作用。

Lung cancer is the leading cause of cancer related mortality in both men and women. Chromosome 9p deletions and alterations occur early and often in lung cancer. The p16/CDKN2 locus, located on 9p, is suspected to be the major tumor suppressor gene inactivated in this tumor type. The prognosis for lung cancer patients is very poor, as reflected by an overall, 5-year survival rate of only 14%. The poor prognosis is largely due to a historical lack of effective early detection measures. We have previously identified a region of homozygous deletion on the short arm of chromosome 9p at the microsatellite marker D9S126. This region is distinct from the p16/CDKN2 tumor suppressor gene (TSG) locus and lies approximately 2 cM proximal. We proposed that the region harbors a TSG that may be important in lung tumorigenesis. To this end we have employed several techniques to identify genes within this chromosomal region. Currently, we have isolated several expressed sequence tags (ESTs) and genes mapping in or around D9S126. Additionally, we have used immunohistochemistry to analyze the expression of p16 in adenocarcinomas and squamous cell carcinomas of the lung and subsets of these tumors were analyzed for loss of heterozygosity (LOH) with microsatellite markers spanning the short arm of chromosome 9. The immunohistochemistry revealed a significant difference in the percent of tumors positive for p16 with the adenocarcinomas having a higher percentage of positive staining than the squamous cell carcinomas. Loss of heterozygosity analysis demonstrated that the pattern of loss was similar between p16 positive and negative squamous cell carcinomas. However, there were moderately significant differences in the LOH analysis between p16 positive adenocarcinomas and squamous cell carcinomas at some of the markers. Taken together, these data suggest that chromosome 9p may contain other tumor suppressor genes important in lung tumorigenesis and that the different patterns of LOH between squamous cell carcinomas and adenocarcinomas of the lung may indicate their locations. The Cot gene was originally identified by transfection of a human thyroid cancer DNA into SHOK cells. The gene was found to be oncogenic when altered at the 3' end, constitutively activating the protein. Sequencing analysis revealed that wild-type Cot is a MAP kinase kinase kinase expressed in a variety of cells including spleen, thymus, liver, and lung. Cot possesses the unique characteristic of activating multiple cascades, including both proliferative and apoptotic signal transduction pathways such as the MEK-1 and SEK-1 pathways, respectively. In NIH3T3 transfection assays utilizing lung tumor DNA, our lab identified a 3' alteration of Cot similar to the previous reports. We therefore proposed that oncogenic activation of Cot might play a role in lung tumor formation and development. The complete open reading frame of Cot was examined by polymerase chain reaction, single strand conformational polymorphism (PCR-SSCP) analysis in 40 lung tumor cell lines to identify mutations within the sequence. One mutation/polymorphism was identified in a cell line but did not result in an amino acid change. No other point mutations were found which might activate the gene. Using 3' RACE and DNA sequencing to analyze lung tumor cell lines that might harbor truncations or alterations at the 3' end of Cot did not identify any cell lines with activations of the gene through this mechanism. However, expression analysis by RT-PCR from lung tumor cell lines did demonstrate down-regulation of the Cot gene in small cell lung cancer cell lines and non-small cell lung cancer cell lines. In fact, two small cell lung tumor cell lines do not appear to express any Cot gene transcripts. Therefore, we are currently conducting Southern and Western blot analysis with these cell lines to identify genomic rearrangements or protein alterations. These experiments should help to understand the regulation of Cot and its role in cellular apoptosis, proliferation, and tumorigenic potential.