Investigation of genes induced at the invasive front of lung adenocarcinomas

我们构建了组织微阵列（TMA），包括5例细支气管-肺泡癌（BAC）， 23例含有BAC成分的混合型腺癌和20例纯腺癌。利用这种TMA，我们研究了(A)细胞周期调节因子（p16、p21、p27、cyclin D1和cyclin E）， (B)肿瘤抑制因子（p53、E-cadherin、PTEN、TSLC1、Smad3和Smad4）的表达，并比较了BAC、混合腺癌和纯腺癌的结果。我们发现(1)与其他两组相比，p16、p27、PTEN、E-cadherin和TSLC1在纯腺癌中的表达明显降低；(2)细胞周期蛋白D1在BAC和混合腺癌中的表达比在纯腺癌中的表达更频繁地升高；(3)Smad4在BAC中的表达保持不变，但在混合腺癌和纯腺癌中的表达降低。我们对混合腺癌周围和中心区域的癌细胞进行显微解剖（分别为7例和5例），并进行全面的基因表达分析。正常肺组织（n=3）也进行分析比较。基因富集分析表明，上皮-间质转化（epithelial-mesenchymal transition， EMT）和缺氧诱导的基因组在混合性腺癌的中央区和外周区过表达。为了研究缺氧对癌细胞细胞特性的影响，我们将肺腺癌细胞A549置于缺氧环境下，通过寡核苷酸阵列分析其基因表达谱。结果表明，缺氧诱导的基因主要与细胞周期、DNA修复、细胞外基质合成和血管生成有关。我们还注意到EGFR和CXCR4的上调，这可能解释了缺氧诱导的运动表型。事实上，EGFR的抑制完全消除了缺氧处理引起的运动性增加。

We constructed tissue microarray (TMA) that consisted of 5 cases of bronchiolo-alveolar carcinoma (BAC), 23 cases of mixed type adenocarcinoma with BAC component, and 20 cases of pure adenocarcinomas. Using this TMA, we investigated the expressions of (A) cell cycle regulators (p16, p21, p27, cyclin D1, and cyclin E), (B) tumor suppressors (p53, E-cadherin, PTEN, TSLC1, Smad3, and Smad4), and the results were compared between BAC, mixed adenocarcinoma, and pure adenocarcinomas. We found that (1) expressions of p16, p27, PTEN, E-cadherin, and TSLC1 were significantly decreased in pure adenocarcinomas compared to the other two groups, (2) expression of cyclin D1 was more frequently elevated in BAC and mixed adenocarcinomas than in pure adenocarcinomas, and (3) expression of Smad4 was maintained in BAC, but decreased in mixed and pure adenocarcinoms.We microdissected cancer cells from peripheral and central areas of mixed adenocarcinomas (n=7 and 5, respectively), and performed comprehensive gene expression analysis. Normal lung tissues (n=3) were also analyzed for comparison. Gene enrichment analysis demonstrated that gene sets induced by epithelial-mesenchymal transition (EMT) and hypoxia were overexpressed in the central vs. peripheral areas of mixed adenocarcinomas.To investigate the influence of hypoxia on cellular properties of cancer cells, we subjected lung adenocarcinoma cell line A549 to hypoxia, and gene expression profiles were analyzed by oligonuleotide arrays. The results showed that among the genes induced by hypoxia were those related to cell cycle, DNA repair, extracellular matrix synthesis, and angiogenesis. We also noted upregulation of EGFR and CXCR4, which may explain the motile phenotype that was induced by hypoxia. In fact, inhibition of EGFR completely abrogated the increase of motility caused by hypoxic treatment.