p53-hdm2-p14ARF調控路徑變異參與台灣肺癌形成之機制探討

Etiological association of alterations in p53-hdm2-p14ARF pathway with lung tumorigenesis in Taiwan

王郁茜(Yu-Chien Wang)

指導教授 ： 王憶卿博士

國立臺灣師範大學/理學院/生物學系/碩士(2002年)

摘要

自民國七十三年以來，肺癌的死亡率在女性與男性分佔癌症死亡率的首位及第二位。由於抑癌基因變異的研究有助於對癌症形成機制的了解，而p53抑癌蛋白參與細胞週期與凋亡的調控，其變異在許多癌症均有發現，所以本研究首先偵測p53抑癌基因與蛋白在113個非小細胞肺癌樣本中變異的情形。研究結果顯示，p53抑癌基因突變的頻率為17％，但卻有48％ p53蛋白過度表達的情形，因此，懷疑這些p53蛋白之異常表達，可能與上游調控蛋白，如p14ARF及hdm2發生變異有關。因此，本研究進一步分析台灣地區之非小細胞肺癌病人，其p53-hdm2-p14ARF調控路徑變異之機率與機制。利用免疫組織染色法分析 p14ARF及hdm2蛋白不表達的頻率，並以反轉錄-聚合酵素鏈反應分析組織細胞中p14ARF及hdm2 mRNA轉錄是否異常，以及偵測p14ARF基因啟動子高度甲基化、異質性喪失，等位基因缺失、及基因發生突變等頻率。本研究結果顯示：在台灣地區非小細胞肺癌病人中，hdm2蛋白不表達頻率為45% (43/95)，mRNA低表達之頻率為41% (35/86)，且發現轉錄編輯錯誤為導致hdm2基因變異之主要機制 (其頻率為63%，22/35)；此外，由於Akt為hdm2上游之磷酸化酵素，與hdm2之活性、及由細胞質轉移至細胞核的能力有關，因此我們亦分析活化態Akt蛋白的表達情形，發現hdm2蛋白未表達且Akt蛋白亦未表達的比例達88% (38/43)。而在p14ARF方面，發現p14ARF蛋白不表達的頻率為34% (35/102)，mRNA低表達頻率為31% (30/96)，而啟動子高度甲基化頻率為30% (27/91)，異質性喪失頻率為24% (19/78)，等位基因缺失頻率為9% (5/55)，基因突變頻率為2% (1/46)；並推論啟動子高度甲基化為導致p14ARF基因變異之主要機制。另外，在38位 wild type p53蛋白過度表達的病人中，有92% (35/38) 病人其hdm2蛋白未表達，而p14ARF蛋白有表達，顯示這些病人p53蛋白的過度表達，的確與上游蛋白—hdm2及p14ARF的調控有關；且發現這些病人多為晚期 (21/35，60%) 且為SQ形式 (22/35，63%) 的肺癌病患。在存活率方面，發現p53蛋白過度表達的病人有較差的存活率 (P=0.013)，故推測p53蛋白的過度表達，應可當作台灣地區肺癌病人的預後指標。
本研究為首篇分析在wild type p53過度表達時，p53、hdm2、p14ARF三者間表達關係之研究；亦為首篇在肺癌中，發現hdm2 mRNA有轉錄編輯錯誤情形之研究。研究結果顯示p53-hdm2-p14ARF調控路徑變異在台灣地區肺癌形成過程中扮演重要的角色，並可作為肺癌之預後分子指標。

關鍵字

p53 ； hdm2 ； p14ARF ； 選擇性轉錄編輯

並列摘要

Lung cancer is the leading and second cause of cancer deaths among women and men in Taiwan, respectively. However, the molecular mechanisms involved in lung tumorigenesis in Taiwan remain poorly defined. There is increasing evidence that alterations in tumor suppressor genes and oncogenes are common in many forms of human cancer including lung cancer. We found that p53 gene mutation frequency was 17% in resected non-small cell lung cancers (NSCLC). However, p53 protein overexpression frequency was 48%. To further identify the molecular basis for this p53 immunohistochemical abnormality, we performed a genetic and epigenetic study of the p53 upstream proteins, p14ARF and hdm2, in NSCLC patients. Specimens of resected NSCLC from 113 patients were recruited in this study. Protein expression and mRNA expression of p14ARF and hdm2 were examined by immunoshitochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Methylation-based PCR assay was conducted to detect promoter methylation of the p14ARF gene. Loss of heterozygosity (LOH), homozygous deletion, and mutation of p14ARF gene were also examined. All data of p14ARF and hdm2 analyses were compared among patients with various clinicopathological parameters and with the p53 protein expression level. The data indicated that 45% and 41% lung cancer patients showed low or absent of hdm2 protein and mRNA expression, respectively. We also found that alternative splicing was the major mechanism which caused hdm2 gene alteration. In addition, we examined the frequency of protein expression of Akt kinase because Akt is known to associate with phosphorylation and nuclear localization of hdm2. The results indicated that the negative Akt kinase protein expression was correlated with negative hdm2 protein expression. With regard to p14ARF analyses, the data indicated that 34% and 31% lung cancer patients showed low or absent of p14ARF protein and mRNA expression, respectively. The frequency of p14ARF promoter hypermethylation, LOH, homozygous deletion, and mutation was 30%, 24%, 9% and 2%, respectively. We suggested that promoter hypermethylation was the major mechanism which caused p14ARF gene alteration. Note that 92% (35/38) patients with p53 overexpression showed absence or low expression of hdm2 protein and near overexpression of p14ARF protein. It indicated that the p53 overexpression was indeed induced by the dysregulation of the upstream proteins, hdm2 and p14ARF. Interestingly, most of the NSCLC patients with dysregulation of the p53-hdm2-p14ARF pathway were suffered late stage and SQ type of cancer. In addition, the patients with p53 overexpression had poor prognosis (P=0.013).
The study was the first report which examines all possible alteration pathways in p53-hdm2-p14ARF gene/protein deregulation in the same series of NSCLC, and examines their relationship with the clinical data of NSCLC. In addition, it was also the first report on the alternative splicing of hdm2 mRNA in lung cancer. In conclusion, the alteration of p53-hdm2-p14ARF regulation pathway plays an important role in tumorigenesis of lung cancer in Taiwan, and could be potentially used as a molecular prognostic marker.

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