Genetic Alterations in Lung Cancer

肺癌是男性和女性癌症相关死亡的主要原因，并且仍然是一个主要的健康问题。未来一年将有超过16万人死于肺癌，超过乳腺癌、前列腺癌和结肠癌死亡人数的总和。大多数肺癌病例可归因于吸烟，在某些情况下可归因于其他环境风险因素。虽然戒烟者患肺癌的相对风险大大降低，但曾经吸烟的人仍然有患肺癌的风险。最近的几项研究表明，超过50%的新诊断的肺癌发生在前吸烟者中。在前吸烟者中发现的肿瘤中，近50%发生在戒烟5年以上的患者中。据估计，在美国，吸烟者和戒烟者的人数大致相等。由于戒烟是一项重大的公共卫生倡议，前吸烟者在肺癌病例中所占的比例将越来越高。因此，存在两个肺癌高危人群，改善疾病管理对现在和以前的吸烟者都是有益的。此外，肺癌患者的预后非常差，总体5年生存率仅为14%。肺癌患者预后不良的部分原因是由于历史上缺乏有效的早期发现措施。染色体9P上的肿瘤抑制基因：染色体9P缺失和改变在肺癌中早期和经常发生。p16/CDKN2位点位于9p，被怀疑是该肿瘤类型中失活的主要抑癌基因。然而，我们之前已经在染色体9p短臂上的微卫星标记D9S126上发现了一个纯合缺失区域。该区域与p16/CDKN2肿瘤抑制基因（TSG）位点不同，位于近端约2cm处。我们认为该区域含有一个可能在肺肿瘤发生中起重要作用的TSG。为此，我们采用了几种技术来鉴定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）和在D9S126中或其周围定位的基因。此外，我们利用免疫组织化学分析了p16在肺腺癌和鳞状细胞癌中的表达，并利用跨越9号染色体短臂的微卫星标记分析了这些肿瘤亚群的杂合性缺失（LOH）。免疫组化显示p16阳性的肿瘤百分比有显著差异，腺癌的阳性染色百分比高于鳞状细胞癌。杂合性缺失分析表明p16阳性和阴性鳞状细胞癌之间的缺失模式相似。然而，p16阳性腺癌和鳞状细胞癌在某些标志物上的LOH分析存在中度显著差异。在最近的一项研究中，用55个标记筛选了30个非小细胞肺癌和12个小细胞肺癌细胞系，以确定9p染色体上纯合缺失的新区域。检测到三个新的非连续纯合缺失区域，大小从840 Kb到7.4 Mb不等。在D9S126缺失中发现的一个基因TUSC1是一个无内含子基因。多重PCR和Southern blot证实了TUSC1基因的纯合缺失。TUSC1的Northern blot分析显示了两个大约2 kb和1.5 kb的转录本，可能是由可选的多聚腺苷化信号产生的。这两个转录本在几种人体组织中表达，并共享一个开放阅读框，编码209个氨基酸的肽。通过RT-PCR分析细胞系cdna发现，在有或没有纯合缺失的细胞系中，TUSC1表达下调，提示TUSC1可能在肺肿瘤发生中发挥作用。综上所述，这些数据表明染色体9p可能包含其他在肺肿瘤发生中重要的肿瘤抑制基因，并且肺鳞状细胞癌和腺癌之间LOH的不同模式可能指示了它们的位置。肺肿瘤发生中的COT/MAP3K8基因：COT/MAP3K8基因是一种MAP激酶激酶激酶，在多种细胞中表达，包括脾脏、胸腺、肝脏和肺，最初是通过将人甲状腺癌DNA转染到SHOK细胞中发现的。该基因被发现是致癌的，当在3'端改变时，它会被激活。MAP3K8通过转录过表达和突变诱导细胞转化和肿瘤发生。然而，该基因的突变在人类中似乎是罕见的事件，但Cot/MAP3K8表达的改变与多种肿瘤类型有关。Cot/MAP3K8具有激活多种级联的独特特性，包括增殖和凋亡信号转导途径，如MEK-1和SEK-1途径。在使用肺肿瘤DNA的NIH3T3转染检测中，我们的实验室发现Cot/MAP3K8的3'改变与之前的报道相似。因此，我们提出Cot/MAP3K8的致癌激活可能在肺肿瘤的形成和发展中发挥作用。采用聚合酶链反应、单链构象多态性（PCR-SSCP）对40株肺癌细胞系的Cot/MAP3K8开放阅读框进行分析，以确定该序列的突变。在细胞系中发现了一个突变/多态性，但没有导致氨基酸改变。没有发现其他可能激活该基因的点突变。使用3‘ RACE和DNA测序来分析可能在Cot/MAP3K8的3’端有截断或改变的肺肿瘤细胞系，没有发现任何通过这种机制激活该基因的细胞系。我们研究了MAP3K8在肺癌细胞系中的表达，以确定转录畸变是否与肺癌相关。引物延伸、5'RACE和PCR-SSCP鉴定了MAP3K8启动子；real - time PCR和Western blot分析转录和翻译表达；FACS分析检测了MAP3K8对未转化肺细胞细胞周期的影响。我们报道了启动子是一个富含GC的，TATA-less序列，包含至少六个转录起始位点。实时荧光定量PCR显示，7/17非小细胞肺癌（NSCLC）细胞株显著增加了MAP3K8 mRNA的表达，其中3株细胞株比正常肺细胞增加了50倍以上。4/14小细胞肺癌（SCLC）细胞系表达升高，大部分SCLC细胞系表达降低。Western blot分析表明，mRNA和蛋白的表达水平在肿瘤细胞系中不相关，但在转染的非转化肺细胞中相关。出乎意料的是，将野生型或突变型MAP3K8稳定转染到永生化肺细胞系中，以非凋亡的方式减慢了增殖，但只有突变基因在G0/G1期间阻止了细胞。这些数据表明，MAP3K8在肺癌细胞系中的表达发生改变，mRNA和蛋白水平之间存在不一致的关系；而未转化的肺细胞保持mRNA和蛋白表达增加的相关性，并显示MAP3K8活性改变了细胞周期调节。肺癌的遗传易感性：一个多学科研究小组十多年来一直致力于一个项目，以确定高风险肺癌家族的肺癌易感性位点。当我们确定肺癌高危家族，并开始收集家族和必要的生物标本进行基因分型和连锁分析时，这个项目就开始了。我们对52个扩展谱系进行了全基因组连锁分析，这些谱系是通过有几个一级亲属的肺癌先证者确定的。

Lung cancer is the leading cause of cancer related mortality in both men and women and remains a major health issue. More than 160,000 individuals will die from lung cancer in the coming year, more than breast, prostate and colon cancer combined. The majority of lung cancer cases is attributable to tobacco smoking and in some cases other environmental risk factors. Although the relative risk of developing lung cancer declines dramatically in smokers who quit, former smokers remain at risk for the disease. Several recent studies show that greater than 50% of newly diagnosed lung cancers occur in former smokers. Of the tumors detected in former smokers, nearly 50% occurred in patients who had quit for more than five years. It is estimated that there are approximately equal numbers of smokers and former smokers in the United States. Since smoking cessation is a major public health initiative, former smokers will increasingly account for a higher percentage of lung cancer cases. Thus, two high-risk population groups exist for lung cancer and improved disease management can be beneficial to both current and former smokers. Additionally the prognosis for lung cancer patients is very poor, as reflected by an overall, 5-year survival rate of only 14%. The poor prognosis for lung cancer patients is due, in part, to the historical lack of effective early detection measures. TUMOR SUPPRESSOR GENES ON CHROMOSOME 9P: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. However, 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. In a more recent study, 30 non-small cell lung cancer and 12 small cell lung cancer cell lines were screened with 55 markers to identify new regions of homozygous deletion on chromosome 9p. Three novel non-contiguous homozygously deleted regions were detected and ranged in size from 840 Kb to 7.4 Mb. One gene identified in the deletion at D9S126, TUSC1, is an intronless gene. Multiplex PCR and Southern blot confirmed the homozygous deletion of TUSC1. Northern blot analysis of TUSC1 demonstrated two transcripts of approximately 2 and 1.5 kb that are likely generated by alternative polyadenylation signals. Both transcripts are expressed in several human tissues and share an open reading frame encoding a peptide of 209 amino acids. Analyzing cell line cDNAs by RT-PCR demonstrated down regulation of TUSC1 in cell lines with or without homozygous deletions suggesting TUSC1 may play a role in lung tumorigenesis. 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/MAP3K8 GENE IN LUNG TUMORIGENESIS:The Cot/MAP3K8 gene is a MAP kinase kinase kinase expressed in a variety of cells including spleen, thymus, liver, and lung and was originally identified by transfection of a human thyroid cancer DNA into SHOK cells. The gene was found to be oncogenic and constitutively activate when altered at the 3' end. MAP3K8 induces cellular transformation and tumorigenesis by transcriptional over-expression and mutation. However, mutation of the gene appears to be a rare event in humans, but altered Cot/MAP3K8 expression is associated with multiple tumor types. Cot/MAP3K8 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/MAP3K8 similar to the previous reports. We therefore proposed that oncogenic activation of Cot/MAP3K8 might play a role in lung tumor formation and development. The complete open reading frame of Cot/MAP3K8 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/MAP3K8 did not identify any cell lines with activations of the gene through this mechanism. We investigated MAP3K8 expression in lung cancer cell lines to determine if transcriptional aberrations are associated with lung cancer. Primer extension, 5'RACE, and PCR-SSCP characterized the MAP3K8 promoter; Realtime PCR and Western blot analyzed transcriptional and translational expression; and FACS analysis examined effects of MAP3K8 on cell cycling in non-transformed lung cells. We reported that the promoter is a GC rich, TATA-less sequence containing at least six transcription initiation sites. Realtime PCR demonstrated 7/17 non-small cell lung cancer (NSCLC) cell lines significantly increased MAP3K8 mRNA expression, with 3 cell lines over 50-fold greater that of normal lung cells. While 4/14 small cell lung cancer (SCLC) cell lines increased expression, the majority of SCLC cell lines decreased expression. Western blot analysis demonstrated that mRNA and protein expression levels did not correlate in the tumor cell lines but did correlate in transfected non-transformed lung cells. Unexpectedly, stable transfection of either a wild type or mutant MAP3K8 into an immortalized lung cell line slowed proliferation in a non-apoptotic manner, but only the mutant gene arrested cells during G0/G1. These data suggest MAP3K8 expression is altered in lung cancer cells lines with a discordant relationship existing between mRNA and protein levels; whereas non-transformed lung cells maintain a correlation between increased mRNA and protein expression and demonstrate altered cell cycle regulation from MAP3K8 activity.GENETIC SUSCEPTIBILITY TO LUNG CANCER:A multidisciplinary team of researchers has been working on a project for more than a decade to identify lung cancer susceptibility loci in high-risk lung cancer families. The project began when we identified high-risk lung cancer families and began to collect the families and necessary biospecimens for genotyping and linkage analysis. We conducted a genome-wide linkage analysis of 52 extended pedigrees ascertained through lung cancer probands who had several first degree relatives.