EB病毒促進人類乳癌細胞癌化之研究

EBV enhances tumorigenic activity in human breast cancer cells

林君翰(Jiun-Han Lin)

指導教授 ： 許金玉

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.10188

摘要

EB病毒（Epstein-Barr virus）是一種普遍存在的人類γ泡疹病毒，全世界超過95%的成人均受過EB病毒的感染。EB病毒與一些惡性腫瘤有關，包括了伯奇氏淋巴瘤、霍奇金氏淋巴癌、鼻咽癌等。乳癌（Breast cancer）是女性最常被診斷出來惡性腫瘤之一，近年來許多實驗室研究結果顯示EB病毒可能也與乳癌的病變有所關係。由我們實驗室之前的研究顯示，在140個乳癌檢體中有66個（47.14%）可偵測到EB病毒基因的存在，而在12個乳癌組織旁的正常組織中只有1個（8.3%）偵測到EB病毒基因的存在，表示EB病毒基因主要在癌組織中被偵測到。經由生統分析之後，顯示EBV的存在與乳癌的預後較差有關。所以我們推斷EB病毒可能會促進乳癌的癌化情形。
為了研究EB病毒在乳癌中所扮演的角色，我們建立了EB病毒所感染的兩株乳癌細胞株，MCF7-A與BT474-A。在EB病毒所感染的乳癌細胞株中，EB病毒表現了三個典型第一期潛伏期所存在的基因產物(EBNA1, EBER1與 BARF0)。EB病毒感染的MCF7-A與BT474-A細胞擁有更能夠在軟洋膠中細胞獨立生長的能力。HER2 (亦稱Neu, ErbB2) 是酪胺酸激酶(RTKs)中表皮細胞生長因子接受器(EGFR; HER1/ErbB1)家族中的一員，另包括了HER3(ErbB3)與HER4(ErbB4)。在20-30%的人類乳癌中，可發現HER2這個原致癌基因的基因放大與過度表現，而這樣的現象又會與乳癌病人的預後不好與存活率低有所關係。此外，EB病毒的感染可增強HER2與HER3基因的表現並進而形成異質二元體（Heterodimer）。HER2與HER3形成的異質二元體可以刺激磷酯肌醇激酶(PI3K)作用器Akt與絲裂原活化蛋白激酶(MAPK)-ERK的磷酸化。為了了解HER2/HER3在細胞獨立生長中所扮演的角色，細胞種在含有HER2抗體（Herceptin）、PI3K抑制劑（LY294002）或MEK抑制劑（U0126）的軟洋膠中。結果MCF7-A細胞在軟洋膠中細胞獨立生長的能力在有HER2抗體（Herceptin）、PI3K抑制劑（LY294002）或MEK抑制劑（U0126）存在下都被減弱。另一方面，Wnt蛋白在老鼠中被證明具有促使乳腺癌化的能力，並且很清楚的知道Wnt/β-catenin訊息傳導在大部份的乳癌中是被活化的。這些證據包括了在細胞質中增加的β-catenin蛋白與常在細胞核中可觀察到β-catenin蛋白，以及β-catenin下游的轉錄基因的表現，如cyclin D1。在我們的研究中顯示EB病毒感染乳癌細胞之後，可以增加細胞中的β-catenin表現與累積，並且活化
β-catenin的下游標的基因，例如c-MYC 與 cyclin D1。
接著我們想要了解在受EB病毒感染的乳癌細胞中是哪一個EB病毒的基因產物(EBNA1, EBER1與 BARF0)促進了HER2/HER3與β-catenin表現上升。結果我們利用大量表現BARF0基因與利用干擾性核醣核酸（siRNA）的方法來證明BARF0在MCF7與BT474細胞中能夠促進HER2/HER3與β-catenin的表現，並同時能促進細胞的癌化活性。由冷光報導基因的實驗（Luciferase reporter assay）中，我們發現在MCF7細胞中表現BARF0，可使帶有HER2和HER3起動子的在轉譯上活化9.1和8.3倍。且相較親代細胞，於穩定的BARF0轉殖的MCF7細胞能夠在軟洋膠中形成更多的群落。同時,我們發現BARF0能夠增強BT474細胞中MMP2的量。而且BARF0可以透過PI3K/AKT訊息傳遞來活化β-catenin途徑來促進細胞的侵犯與轉移能力。這些發現意味著BARF0可能藉由活化HER2/HER3以及β-catenin的訊息傳遞來達到增強乳癌細胞癌化的能力。

關鍵字

Epstein-Barr 病毒 ； BARF0 ； HER2 ； HER3 ； β-catenin ； 乳癌

並列摘要

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus estimated to have infected about 95% of the adult population worldwide. EBV is associated with certain malignancies, such as Burkitt lymphoma, Hodgkin lymphoma and nasopharyngeal carcinoma. Several laboratories have reported recently that EBV may also be involved in the pathogenesis of breast carcinoma, the most frequently diagnosed malignancies in women. In our laboratory, the presence of the EBV genome in breast tumors (66/140, 47.14%) and in normal tissues adjacent to breasr tumors (1/12, 8.3%) indicated that EBV was mainly detected in tumors. After statistical data analysis, EBV was correlated with poor prognosis in breast cancer. It suggested that EBV might be related to the development of some breast carcinomas.
To investigate the potential role of EBV in breast cancer, we established EBV-infected breast cancer cell lines, MCF7-A and BT474-A. Three EBV latency genes (EBNA1, EBER1, and BARF0) were expressed in EBV-infected breast cancer cells and this pattern was typical of EBV latency I. EBV-infected MCF7-A and BT474-A cells exhibited increased anchorage-independent growth in soft agar. HER2 (also known as Neu, ErbB2) is a member of the epidermial growth factor receptor (EGFR; HER1/ErbB1) family of receptor tyrosine kinases (RTKs), which also includes HER3(ErbB3) and HER4(ErbB4). Amplification and overexpression of the HER2 proto-oncogene is observed in 20–30% of human breast cancers and is correlated with poor prognosis and decreased patient survival. Moreover, EBV infection enhances expression of HER2 and HER3 gene, and formation of HER2/HER3 heterodimer. HER2/HER3 heterodimer also stimulated phosphorylation of the phosphatidylinositol 3'-kinase (PI3K)-dependent effector kinase Akt, as well as that of the MEK-dependent mitogen-activated kinase (MAPK), extracellular signal-regulated kinase (ERK). To realize the role of HER2/HER3 in anchorage-independent growth, cells were seeded in the soft agar in the presence of an anti-HER2 antibody, herceptin, PI3K inhibitor LY294002 or the MEK inhibitor U0126. The ability of colony formation in soft agar was substantially reduced by either herceptin or the PI3K inhibitor LY294002 or the MEK inhibitor U0126 in MCF7-A cells. In the other hand, the oncogenic potential of Wnt proteins in the mammary gland has been amply demonstrated in mouse models, and it is clear that at least distal components of the Wnt/β-catenin pathway are activated in majority of breast carcinomas. The evidence for this includes elevated levels of cytoplasmic β-catenin, the frequent presence of β-catenin in the nucleus, and expression of transcriptional targets of β-catenin such as cyclin D1. In our study, EBV infection enhances expression and accumulation of β-catenin, and activates expression of β-catenin target genes, c-MYC and cyclin D1. Next, it was examined which of the three EBV genes expressed in these cells (EBNA1, EBER1 and BARF0) was responsible for HER2/HER3 and β-catenin expression. Consequently, we showed that BARF0 alone was sufficient to efficiently up-regulate HER2/HER3 and β-catenin expression and promoted tumorigenic activity in MCF7 and BT474 cells by both overexpression and small interfering RNA knock-down. According to the luciferase reporter assay, expression of BARF0 resulted in 9.1- and 8.3-fold transcriptional activation of the HER2 and HER3 promoter construct in MCF7 cells, respectively. Stably BARF0-tranfected MCF7 cells formed more colonies in soft agar than parental cells. Meanwhile, we found that BARF0 enhances the expression of MMP2 in BT474 cells. And then BARF0 promotes BT474 cells migration and invasion ability through activating β-catenin signaling by PI3K/AKT pathway. These findings imply that BARF0 is likely to enhance the breast cancer tumorigenic activity through activating HER2/HER3 and β-catenin signaling pathway.

並列關鍵字

Epstein-Barr Virus ； BARF0 ； HER2 ； HER3 ； β-catenin ； breast cancer

參考文獻

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