K-ras致癌基因在正常腎上腺皮質細胞中之功能性分析極其診斷晶片之應用研發

論文總摘要 K-ras基因是所有腫瘤中突變頻率最高之致癌基因(oncogene)，在胰臟癌突變率高達75﹪、在大腸直腸癌高達50﹪、在肺癌也有高達40%的突變率、甚至在乳癌、胃癌、子宮頸內膜癌等多種癌症中都有顯著的突變現象存在；針對K-ras基因在人類功能性腎上腺腫瘤的突變情形，本實驗室長期以來有一系列的研究，確定K-ras致癌基因在功能性腎上腺皮質腫瘤有高達44%的突變率，且其突變位多集中在Codon 15,16,18及31；此外更透過人類功能性腎上腺皮質腫瘤突變K-ras蛋白生化特性分析，確定人類腎上腺皮質腫瘤的突變K-Ras蛋白中，僅在胺基酸15、16、18和31發生突變的突變K-Ras蛋白會影響其對GAP的敏感性(sensitivity)；一連串的研究中，我們也成功建立突變K-ras基因穩定轉染之人類腎上腺皮質細胞株，作為之後探討突變K-ras基因功能的主要研究材料。首先，利用此細胞株進行相關研究，我們證實K-ras基因不僅影響正常腎上腺皮質細胞的增生同時也影響腎上腺皮質細胞的類固醇荷爾蒙生合成反應。以前段所提之豐富研究成果為基礎，為了更深入探討突變K-ras基因在正常腎上腺皮質細胞中，調控腎上腺腫瘤生成及其類固醇荷爾蒙生合成的分子調控機轉，並希望能將相關研發成果推廣至臨床應用，吾人將本研究分為兩大部分，第一部分:主要探討在腎上腺皮質細胞中K-ras基因所調控之類固醇生合成及腫瘤生成相關基因表現與作用途徑。此部分，吾人利用傳統分子生物技術，加入微矩陣列分析、生物資訊等多項基因體學之新興技術，針對突變K-ras基因在腎上腺皮質細胞之調控因子及作用機轉進行一系列研究探討。首先”探討K-ras致癌基因在腎上腺皮質細胞中調控類固醇生合成反應之分子機制”，吾人利用類固醇生合成相關基因之表現及其他多項蛋白分析實驗的進行，確認在K-ras突變基因穩定轉染的人類腎上腺皮質細胞珠中，K-ras致癌基因透過Ras-to-MAPK pathway調控腎上腺之類固醇生合成反應。接著”探討腎上腺皮質細胞中K-ras致癌基因所調控之基因表現及其作用機轉”，承接第一部分確認突變K-ras基因調控類固醇生合成之途徑後，吾人希望更深入探討此致癌基因在腎上腺皮質細胞中，所調控之基因表現與作用途徑，藉此了解此基因之下游調控基因在腎上腺皮質細胞之作用；吾人利用微矩陣列分析技術找出所有因為突變K-ras基因活化而產生顯著差異性表現之基因群，深入探討其基因表現與參與機轉。在此部分研究，吾人得到94個受K-ras基因影響而產生特異表現的基因，經由深入探討其中具意義之基因，證實了pRB在腎上腺皮質細胞中之活化機轉，當突變K-ras基因被IPTG誘導活化後，pRB明顯被磷酸化。最後”探討腎上腺皮質細胞中K-ras致癌基因調控細胞增生及癌化之作用因子及其作用途徑”，為了解答腎上腺皮質腫瘤之腫瘤生成反應，吾人針對上一部分所得之94特異表現基因，利用生物資訊軟體分析，找尋其中與細胞增生及腫瘤生成相關之基因群，並繪出其作用機轉，再以實際實驗進行確認生物資訊分析結果。此部分研究成果，吾人獲得一群K-ras致癌基因所調控與腫瘤生成相關之基因群，同時在針對這些基因表現分析的同時，吾人確認了Ets1這個與癌化最相關的基因在腎上腺皮質細胞中活化作用的途徑。此外，這部分的研究更確立生物資訊軟體在腫瘤研究中之可行性及其效益。第二大部分:”K-ras致癌基因診斷新工具的研發”，為了能將研究成果實際應用於臨床，吾人針對目前臨床最迫切需要之部分進行研發。K-ras致癌基因是目前被發現分佈最廣泛的致癌基因，在大腸癌、肺癌、乳癌等多種癌症皆可見其突變情形。雖然陸續有針對此基因變異進行治療之藥物被研發，但卻始終缺乏一完善診斷系統。吾人著手研發一全新K-ras致癌基因診斷系統，盼能解決以上問題，以達早期診斷早期治療之效。又生物晶片實驗結果精確，又可解決傳統診斷侵略性、檢體取得不易等問題，故吾人著手進行K-ras致癌基因診斷晶片之研發。首先，吾人針對前部分研究中微矩陣列分析結果所得的94個顯著差異表現基因，更深入利用Go miner 、DAVID等相關生物軟體分析後，獲得22個顯著上升表現的基因與K-ras基因之致癌機轉最為相關，因此著手以這22個基因為研發標的，製備K-ras突變基因之診斷晶片，並收集所有受K-ras致癌基因調控之胰臟癌、大腸直腸癌、肺癌、胃癌等癌症血液檢體進行臨床試驗，經生物統計軟體統整後證實此診斷晶片的敏感度為83.7%,特異性為90.9%而準確度為86.8%。根據以上實驗結果，得証此K-ras oncogene診斷晶片極具開發潛力的新方法! 只要收集病人血液即可偵測其體內之K-ras致癌基因，因此只要將此診斷晶片應用於所有相關癌症，不但將我們的研究成果之應用性由腎上腺相關領域推廣至胰臟癌、大腸直腸癌、肺癌、胃癌等所有相關癌症，想必一定能為這些癌症的診治打開另一扇窗，做到早期診斷早期治療，同時將癌症之診斷帶入另一個全新紀元。

關鍵字

腎上腺皮質細胞；診斷晶片； K-ras致癌基因；功能性分析

並列摘要

Abstract K-ras is the oncogene with the most frequent occurrence of mutation in cancers. The mutation rate of K-ras gene is 75% in pancreas cancer, 50% in colorectal cancer, and 40% in lung cancer. Besides, K-ras mutation is also significantly found in breast cancer, gastric cancer, endocervical adenocarcinoma, and so on. In our laboratory, a series of studies focusing on K-ras mutation in human adrenocortical tumor have determined that the frequency of K-ras mutation in adrenocortical tumor is 44%, and that K-ras mutation commonly occurs at codons 15, 16, 18, and 31. Biochemical analysis has confirmed that K-ras proteins mutated at amino acid residues at position 15, 16, and 31 in human adrenocortical tumor display altered sensitivity to GAP. In addition, a human adrenocortical cell line stably transfected with mutant K-ras gene has been established successfully, serving as material for exploring the functions of K-ras mutants. Later, a study of the stably transfected cell line demonstrated that K-ras gene affected both proliferation and steroid hormone synthesis of normal adrenocortical cells. Based on the brilliant achievement described above, the author intended to conduct further exploration on the molecular mechanisms of mutant K-ras mediated regulation over tumorigenesis and steroid hormone synthesis. The study results can hopefully provide valuable contribution to clinical interventions against adrenocortical carcinoma or even other kinds of cancers. This thesis is consisted of two parts. Part I: This part of study mainly focuses on the expression levels and signal pathways of genes regulated by K-ras in steroid hormone synthesis and tumorigenesis in adrenocortical cells. The techniques applied in Part I for determining involved factors and mechanism of mutant K-ras mediated regulation in adrenocortical cells included conventional molecular biological approaches and innovative techniques in genomics, e.g.. microarray assay and bioinformatics tools. At first, in the “exploration of molecular mechanism(s) by which K-ras oncogene regulates steroid hormone synthesis in adrenocortical cells,” the analysis of expression levels of genes involving in steroid hormone synthesis as well as relevant protein analysis were carried out. The result determined that K-ras oncogene regulated steroid hormone synthesis through Ras-MAPK signaling pathway in the stably transfected human adrenocortical cell line. Then, in the “exploration of expression and mechanism(s) of genes regulated by K-ras oncogene in adrenocortical cells,” the author endeavored to elucidate the downstream genes regulated by K-ras oncogene and their roles in adrenocortical cells. For this purpose, microarray assays were carried out to screen for all genes differentially expressed in response to the activation of mutant K-ras gene, and subsequently obtained 94 differentially expressed genes associated with K-ras activity. Further analysis revealed a pRB activation mechanism present in adrenocortical cells. Namely, pRB was significantly phosphorylated after IPTG-induced activation of K-ras gene. Finally, for the purpose of clarifying the details in tumorigenesis of adrenocortical cells in the “identification of factors and mechanisms underlying cell proliferation and transformation regulated by K-ras oncogene,” the above 94 differentially expressed genes were analyzed by bioinformatics tools to identify genes associated with cell proliferation or tumorigenesis, with which the underlying mechanisms were conjectured subsequently. After that, experiments were performed to confirm the results of bioinformatical analysis. The experimental results included the identification of a panel of K-ras oncogene-regulated genes associated with tumorigenesis and an activation pathway for Ets1 in adrenocortial cells, a gene significantly associated with cell transformation. In addition, the feasibility and value of applying bioinformatics tools in cancer research were also established in this part of study. Part II: “development of innovative diagnostic tool specific to K-ras oncogene.” The study results obtained in Part I were then applied to the development of innovative diagnostic tool, urgently needed in clinical practice. K-ras is by far the most prevalent oncogene among cancers. K-ras mutation is widely found in colon cancer, lung cancer, breast cancer, and so on. Although efforts have been made to develop therapeutic agents targeting K-ras mutants, a well-establish diagnostic system monitoring K-ras mutation is not yet available. In order to resolve the deficiency as well as to enhance early diagnosis of cancer, the author managed to develop an innovative K-ras oncogene diagnostic system on the basis of results described in Part I. Plus, biochip assay is rather precise, and does not suffer the drawbacks as conventional diagnostic methods do such as being invasive or difficult in specimen acquisition. Therefore, the author dedicated this part of study to development of a K-ras oncogene diagnosis chip. At first, program packages Go miner and DAVID were used to analyze the 94 differentially expressed genes screened by microarray assay in Part I, and subsequently determined 22 up-regulated genes most associated with K-ras oncogenesis. These 22 genes were adopted as target genes for the preparation of K-ras diagnosis chip. Then, blood specimens were collected from patients with K-ras-related cancers such as pancreas cancer, colorectal cancer, lung cancer, and gastric cancer in a clinical trial for the diagnosis chip. Analyzed by a biological statistics package, the sensitivity, specificity, and accuracy of K-ras oncogene diagnosis chip were shown to reach 83.7%, 90.9%, and 86.8%, respectively. This result verified a great potential of this diagnosis chip in clinical application. That is, the innovative diagnosis chip cannot only monitor K-ras oncogene in patients through collecting a small amount of blood specimen, but also be applied in the diagnosis of pancreas, colorectal, lung, and stomach cancers in addition to adrenocortical carcinoma. Therefore, this study certainly provides promising results contributing to remarkable advance in both early diagnosis and therapy for various cancers in the medical practice of the future.

並列關鍵字

adrenocortical cells ； Functional analysis ； K-ras oncogene ； diagnostic chip

參考文獻

第七章參考文獻

Google Scholar

Alcorta D, Preston G, Munger W, Sullivan P, Yang JJ, Waga I, Jennette JC and Falk R. Microarray studies of gene expression in circulating leukocytes in kidneydiseases. Exp Nephrol(2002) 101:39-49.

Google Scholar

Augenlicht LH, Velcich A, Klampfer L, Huang J, Corner G, Aranes M, Laboisse C, Rigas B, Lipkin M, Yang K, Shi Q, Lesser M, Heerdt B, Arango D, Yang W, Wilson A, Mariadason JM. Application of gene expression profiling to colon cell maturation, transformation and chemoprevention. J Nutr. (2003) 133 : 2410S-2416S.

Google Scholar

Ballschmieter P, Braig M, Lindemann RK, Nordheim A, Dittmer J. Splicing variant DeltaVII-Ets1 is downregulated in invasive Ets1-expressing breast cancer cells. Int J Oncol. (2003)22(4):849-53.