JAG1經由Notch-independent的路徑促進肺癌惡性度

Abstract

背景: JAG1是Notch訊息傳遞的重要因子，前人研究已經證實Notch訊息傳遞路徑可以用來調控癌症細胞的分化與生長，近期的研究更指出JAG1與癌症的轉移有所關聯，因此我們的目標是要研究在臨床上JAG1在非小細胞肺癌表現量的高低是否與病人的預後有所關聯，以及JAG1是透過什麼機制去造成癌症的進展。 方法: 我們在JAG1表現量較低的肺癌細胞中(CL1-0, A549,以及NCI-H226)過度表現JAG1與抑制JAG1表現量較高CL1-5肺癌細胞中JAG1的表現，藉由觀察體外細胞的不依賴支持物生長能力、生長速度、分解膠質能力、移動與侵襲能力，以及觀察老鼠體內的癌轉移情況。並且利用DNA微陣列晶片與即時定量聚合酶連鎖反應來探討JAG1可能的下游基因，以協助我們釐清JAG1是透過哪些訊息傳遞路徑來影響癌細胞特性的改變。另一方面，我們也藉由即時聚合酶連鎖反應來偵測臨床上90個病人癌症組織中JAG1 mRNA的表現量，利用統計的方法加以分析JAG1 mRNA表現量在正常的組織與癌症的組織是否有所差異，並且分析JAG1 mRNA表現量與臨床上病人的肺癌進展的關連性。 結果: JAG1的過度表現可以增加細胞不依賴支持物生長、細胞移動與侵襲能力，同樣的情況也可以在老鼠的轉移模式中得到證實。藉由分析微陣列晶片的結果，我們發現在過度表現JAG1的時候，會引起heat shock 70kDa protein 2 (HSPA2)表現量的增高，相反地，JAG1的表現受到siRNA抑制時，HSPA2的表現則會降低。進一步分析Notch pathway中的相關基因表現後，我們發現Notch pathway的確存在於我們所使用的細胞株當中，然而改變JAG1的表現量並不影響一般傳統上的Notch pathway下游基因的表現，所以我們推測JAG1是透過Notch-independent的路徑促進肺癌細胞的惡性度。在臨床病人的分析方面，我們發現JAG1的表現量在非小型性肺癌中的鱗狀上皮癌病人這個族群，在腫瘤的部位明顯高於旁邊的正常組織 (14/20 , P= 0.017)。進一步分析也發現JAG1表現量比較高的病人，其存活率顯著的下降 (P=0.04)。然而假使我們不將病人分群而以全部之非小細胞肺癌病人或是非小細胞肺癌病人中的肺腺癌族群加以分析，我們發現JAG1 mRNA表現量高低在癌症部位與旁邊正常的組織並無差異，此外也與病人的存活率並沒有相關性。 結論:在我們的實驗中，我們觀察到JAG1帶有致癌基因的特性，這個特性在臨床上也可以得到證實。在非小細胞肺癌病人中，鱗狀上皮肺癌的病人JAG1表現高，其預後明顯較表現量低的病人差。

Background: JAG-1 is a ligand of Notch signaling pathway and can regulate cell differentiation and proliferation in several cancers. Recent study indicated that JAG1 is a gene associated with tumor invasion. Therefore, we investigated the clinical significance of JAG1 expression in non–small-cell lung cancer (NSCLC) patients and its role in lung cancer progression. Methods: We induced JAG1 overexpression or knockdown in human lung cancer cell lines (CL1-0, CL1-5, A549 and NCI-H226) and analyzed cell anchorage-independent growth, proliferation, cell migration, invasion, and in vivo metastasis, as well as matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. The potential downstream genes of JAG1 were identified by oligonucleotide microarray and were validated by quantitative reverse transcription–polymerase chain reaction (RT-PCR). We measured JAG1 expression in tumors and adjacent normal tissues of 90 NSCLC patients by RT-PCR. Correlation of JAG1 expression and overall survival was determined using the log-rank test and multivariable Cox proportional hazards regression analysis. All statistical tests were two-sided. Results: JAG1 enhances anchorage-independent growth, cell migration, invasion, and in vivo metastasis through upregulating the expression of heat shock 70kDa protein 2 (HSPA2) that is independent of Notch pathway. JAG1 expression was higher in tumors than in adjacent normal tissue in 14 of 20 patients of subtype squamous carcinoma of NSCLC patients studied (P=0.017). Subtype squamous carcinoma of NSCLC patients with high JAG1 expressing tumors had shorter overall survival (HR = 2.87; 95% CI = 0.99 to 8.33; P = 0.04) than those with low-expressing JAG1. However, in clinical analysis, JAG1 expression was not associated with overall survival in either NSCLC or subtype adenocarcinoma of NSCLC patients. Conclusion: JAG1 showed an oncogenic characteristic in Subtype squamous carcinoma of NSCLC, and high JAG1 expression is associated with reduced survival of subtype squamous cell carcinoma patients.