RECK轉移抑制基因之調控

Gene Regulation of a Metastasis Suppressor RECK

劉立德(Li-Teh Liu)

指導教授 ： 洪文俊

高雄醫學大學/醫學院/醫學研究所/博士(2004年)

摘要

癌細胞是由正常細胞經長期累積多種基因變異或基因表達異常而成的，造成癌症治療不易及致命的原因主要是腫瘤細胞經侵入循環系統（血管及淋巴）並轉移至其他器官而形成的腫瘤。基質金屬蛋白酶（matrix metalloproteinases；MMPs）的過度表現及活化在腫瘤細胞轉移的過程中扮演重要的角色，RECK基因是一個新發現的基因，它可以逆轉v-Ki-ras（oncogenic ras）所造成的小鼠NIH3T3細胞轉型（transformation），其蛋白質功能是抑制MMP-2及MMP-9的釋放及活化，所以被認為是抑制癌細胞轉移的基因。

在本論文中的研究開始進行前，尚無任何研究結果討論人類RECK基因表達受何種訊息傳遞路徑的影響。本論文包含4個關於RECK基因表達的研究，涵蓋1. 非類固醇抗發炎藥物對RECK基因表達的調節（第2章） 2. 組蛋白去乙醯酶抑制劑對RECK基因表達的調節（第3章） 3. Epstein-Barr virus的latent membrane protein 1在鼻咽癌細胞轉移過程中扮演的角色（第4章） 4. 人類RECK基因啟動子的選殖（第5章） 等4個章節。

在第一部份的研究，我們發現非類固醇抗發炎藥物抑制癌細胞轉移的機制可能是透過活化RECK基因表達而成，選擇性的cyclooxygenase-2 inhibitor NS398可以促進RECK基因轉錄，並提高CL-1肺癌細胞表面RECK蛋白的含量，從而抑制MMP-2的釋放及活化，這樣的現象會降低癌細胞侵犯循環系統及周邊組織的能力。第二部份的研究，我們發現組蛋白去乙醯酶抑制劑可以活化RECK啟動子的活性來促進基因的表達，也增加了CL-1細胞膜上RECK蛋白的含量，釋放到細胞培養液的MMP-2活性因而降低，最後減弱了CL-1細胞的侵犯能力。第三部份的研究，我們釐清了Epstein-Barr virus的latent membrane protein-1會經由活化MEK/ERK訊息傳遞路徑而抑制RECK基因啟動子的活性，而此抑制作用在鼻咽癌細胞MMP-9的釋放及活化扮演關鍵的角色，而MMP-9的活化加強了鼻咽癌細胞的侵犯能力，這或許是LMP1陽性的鼻咽癌病人預後差的原因。第四部份的研究，我們進行人類RECK基因啟動子的選殖，我們發現人類RECK基因啟動子和小鼠RECK基因啟動子的結構相似，但是其調節基因轉錄的方式可能不一樣，詳細的情況尚待進一步的研究。

關鍵字

組蛋白去乙醯?抑制劑 ； 非類固醇抗發炎藥物 ； 肺癌 ； 鼻咽癌

並列摘要

Cancer is a disease results from the accumulation of multiple gene mutations and/or deregulation of gene expression. The major cause of therapy failure and death of cancer patients is metastasis. Metastasis is a process involves extracellular matrix degradation, invaion of circulation system, settlement in target organ, and secondary tumor formation. Previous studies showed that overexpression and/or activation of matrix metalloproteinase genes (MMPs) is a common event in metastasis of cancer cells. RECK is a newly identified gene which was isolated by using a expression cloning strategy designed to find human cDNA that can induce flat reversion of oncogenic v-Ki-ras-transformed NIH3T3 cells. It is a 110 kDa membrane-anchored glycoprotein, which inhibits the release and activation of MMP-2 and MMP-9. Thus, it is considered a metastasis suppressor gene.

Before the research projects in this thesis began, there was no scientific report describing the regulation of human RECK gene expression. There are four major topics in this thesis including 1. Up-regulation of RECK gene expression by non-steroidal anti-inflammatory drugs (NSAIDs) 2. Histone deacetylase inhibitor (HDACi) upregulates RECK expression to inhibit MMP-2 activity and cancer cell invasion 3. RECK is a target of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) 4. Cloning and identification of transcriptional regulation site(s) of human RECK promoter.

In part I, we demonstrated that NSAIDs reduced metastasis of tumor cells via increase of RECK mRNA. NS398 induced RECK protein expression on the surface of CL-1 lung cancer cells and repressed MMP-2 activity detected in CL-1 culture medium. The mechanism of NS398-induced RECK expression was COX-2 independent. This phenomenon may be one of the reasons by which NSAIDs inhibit metastasis. In part II, we results showed that histone deacetylase inhibitor (HDACi) trichostatin A (TSA) could reduce the invasive ability of CL-1 cells via induction or derepression of RECK gene expression. Flow cytometric analysis showed that TSA-upregulated RECK protein was presented on the surface of CL-1 cells. Promoter activity analysis demonstrated that TSA upregulated RECK gene expression by transcriptional activation. The results suggested that HDACi repressed metastatic potential of cancer cells in part, if not all, by upregulation of RECK expression. In part III, we identified a molecular pathway by which latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) acts to induce metastasis. Ectopic expression of LMP1 in NPC-TW04 (a LMP1 negative nasopharyngeal carcinoma cell line) repressed RECK gene expression via inhibition of RECK promoter activity. Our data suggested that LMP1 repressed RECK gene transcription by activation of Raf/MEK/ERK signaling pathway. Downregulation of RECK protein is a critical step in LMP1 induced MMP-9 activity and results in the increase of invasive capacity of NPC-TW04 cells. Our results is consistent with the fact that prognosis of LMP1 positive NPC patients are worse than that of LMP1 negative patients. In part IV, we described the cloning of human RECK promoter. With alignment of human RECK mRNA sequence to human genomic DNA sequence, human RECK promoter was located to the nucleotide sequence AL138834 deposited in GeneBank. We amplified a 2163 base pairs DNA fragment by polymerase chain reaction. The result of comparison（sequence alignment）of the PCR-amplified DNA fragment to published mouse RECK promoter demonstrated that predicted human RECK promoter was homologous to mouse RECK promoter at the proximal end to transcription start site. This region contains two SP1 site. The transcription activity driven by this DNA fragment was upregulated by NSAIDs (aspirin) and TSA. The data was in agreement with our previous results that aspirin and TSA increased RECK mRNA in CL-1 cells. Mutation analysis of both SP1 sites of human RECK promoter showed that SP1A was a transcriptional activator while SP1B was a repressor. Further works are needed to address the molecular mechanism that controls human RECK gene expression.

並列關鍵字

NSAIDs ； RECK ； nasopharyngeal carcinoma ； lung cancer ； HDAC

參考文獻

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