Functional Analysis of IGFBP-3 Gene Expression in Ovarian Endometrioid Carcinoma

確認哪些訊息傳遞路徑會與腫瘤轉移有關對於了解癌症病理的發生以及對腫瘤轉移機制的研究是非常重要的。腫瘤轉移發生的過程通常牽涉到數個步驟，其中包括增加癌細胞的移動能力、分解細胞外基質以及基底層膜、還有癌細胞的增生。在本篇實驗當中，利用卵巢癌侵襲模型(Ovarian cancer invasion model)來建立卵巢類子宮內膜腺癌細胞株-OVTW59的分株(P0~P4)。它們具有漸進式增強侵襲能力的特性可以用來研究與腫瘤侵襲、轉移相關的基因。藉著互補去氧核糖核酸微矩陣(cDNA microarray)和及時定量聚合酶連鎖反應(Quantitative Real-Time PCR)的幫助，我們可以做整個細胞內基因表現的掃描。並且已找出39個與腫瘤侵襲、轉移相關的基因。隨著卵巢癌細胞侵襲能力增加的時候有13個基因表現被抑制，另外有26個基因表現則是被促進。而在39個與腫瘤侵襲相關的基因之中，從P0進行到P4時，胰島素生長因子結合蛋白-3(IGFBP-3)的基因表現具有極大的差異，這也引起我們的好奇，想要去探討IGFBP-3在卵巢類子宮內膜細胞腺癌中所扮演的角色。為此目的我們首先建立一個能持續表現IGFBP-3的P0和P4的細胞株，把含有人類IGFBP-3基因的載體以及空的載體分別轉植到P0與P4之中，接著再進行下列一連串的實驗。實驗結果發現，IGFBP-3在細胞刮痕的實驗(Scratch Analysis)中能夠抑制癌細胞的移動；在侵襲室能力分析(Invasion Chamber Assay)中，能夠抑制癌細胞侵襲的能力。同時在動物實驗中發現IGFBP-3也能夠抑制腫瘤細胞的生長以及轉移，並且引起腫瘤細胞的凋亡(Apoptosis)以及腫瘤壞死(Tumor Necrosis)。對這些研究的結果，我們試著去探討IGFBP-3在卵巢類子宮內膜細胞腺癌細胞中是如何導致這些現象，以及有哪些訊息傳遞路徑會受到IGFBP-3的影響。研究結果發現IGFBP-3有可能透過抑制ERK-MAPK路徑的活化來達到抑制細胞活動能力(cell mobility)。同時我們也利用ERK路徑的抑制劑-PD98059來驗證，證明IGFBP-3抑制ERK的活化與IGFBP-3使得癌細胞的活動能力降低之間有高度相關。總括來說，本篇實驗發現IGFBP-3的確能夠抑制卵巢類子宮內膜腺癌細胞的移動、侵襲以及轉移的能力，並可誘發癌細胞的凋亡。To identify the signaling pathway responsible for the establishment of a metastatic phenotype in carcinoma cells is of crucial importance for the understanding of the pathogenesis of cancer metastasis. The process of metastasis usually involves several steps, including increase the cell motility to invade, degradation of extracellular matrix and basement membranes and cell proliferation. In our study, we used an ovarian cancer invasion model to define five OVTW59 sublines P0 to P4, with progressive increase in invasion capabilities for investigation of genes involved in invasion and metastasis. By cDNA microarray and quantitative real-time PCR analysis, we performed global gene screening and found 39 genes related with invasion and metastasis. Thirteen genes of them were down-regulated and twenty-six genes were up-regulated with the increase of ovarian cancer invasion. One of the down-regulated invasion-related genes, insulin-like growth factor-binding protein 3 (IGFBP-3), showed surprisingly differential expression in the ovarian cancer invasion model. For investigation of the role of the differentially expressed IGFBP-3 gene in the tumor progression, especially in the invasion and metastasis of ovarian cancer, we transfected a plasmid containing IGFBP-3 cDNA into the P0 and P4 cells. It was found that IGFBP-3 could inhibit cell migration in the scratch analysis and cell invasion in the invasion chamber assay. In addition, it could also inhibit tumor growth and metastasis and induce apoptosis in the animal model. These functions could be reversed when shRNA was transfected. We further studied the inhibitory mechanism of IGFBP-3 in cancer migration, invasion and metastasis, especially focusing in the signaling pathways and the interacting genes related to IGFBP-3. Our result revealed that IGFBP-3 could inhibit the activation of extracellular signal-regulated kinase (ERK)1/2 - mitogen-activated protein kinase (MAPK) signaling pathways. It is concluded that the IGFBP-3 gene, selected from the progressive increase in invasion capability sublines of ovarian cancer cell line, plays a role as a tumor suppressor to inhibit tumor cell migration, invasion and metastasis and to induce apoptosis in ovarian cancer pathogenesis.中文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1 Abstract ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 2 Introduction‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥3 Materials and Methods ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 9 Results ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 16 Discussion‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 22 Figures ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 26 Table‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 47 References‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥50 Appendix ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 5