探討組蛋白甲基轉移酶 EZH2 於卵巢癌成癌過程中針對TGF-β標的基因/抑癌基因 LTBP2 所造成之表觀基因調控/默化之過程

The Role of EZH2 in the Epigenetic Silencing of the TGF-β Target and Putative Tumor Suppressor, LTBP2, in Ovarian Cancer

謝昀臻(Jacqueline Shay)

指導教授 ： 陳永恩

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2014年)

摘要

根據前人研究得知TGF-訊息傳遞對於抑制卵巢上皮細胞的生長扮演重要的角色。然而TGF-在卵巢癌細胞中不但無法發揮抑制生長的功能，反而會促進癌細胞的轉移及入侵。因此TGF-訊息傳遞在卵巢癌成癌過程以及其對癌細胞轉移過程的影響一直是許多研究團隊所關注的主題之一。而探究過去相關文獻可觀察到受到此訊息傳導路徑所調控的下游基因在卵巢癌成癌過程中往往受到表基因靜默機制調控而導致許多重要的抑癌基因失去正常作用。 然而造成此類表基因靜默的DNA甲基化或組蛋白修飾等分子機制仍待探索。
透過實驗室過去所進行過expression microarray及ChIP-chip實驗，我們挑選出一個可能的抑癌基因，LTBP2，作為此論文的主要研究對象。另外，我們同時利用了ENCODE project之中進行在胚胎幹細的ChIP-seq結果發現此基因除了受到TGF-訊息傳導路徑所調控之外，更受到一種著名的組蛋白甲基轉移酶，Enhancer of Zeste Homolog 2 (EZH2)，所調控。由於過去研究指出此蛋白不但能透過加入抑制性的組蛋白修飾來抑制基因轉錄，更可能帶來DNA甲基化修飾，使得這樣的抑制作用能夠持續進行。因此在這份研究中我們首先探測LTBP2基因分別在正常的卵巢上皮細胞 (IOSE) 與六株卵巢癌細胞株中表現量並觀察到其表現量除了在IOSE與其中一株卵巢癌細胞HeyC2較高之外，在其餘五株卵巢癌細胞表現量都頗低。爾後我們分別進行組合重亞硫酸鹽限制分析法（COBRA) 、焦磷酸定序法 (Pyro-Sequencing) 與染色體免疫沈澱搭配即時聚合酶鏈鎖反應(Chromatin Immunoprecipitation combined qRT-PCR) 等方式觀察到EZH2確實結合到LTBP2並導致抑制性的組蛋白修飾與DNA甲基化等情形。而透過加以DNMT, HDAC, EZH2抑制劑能夠還原LTBP2表現則更進一步的驗證了我們的觀察。除此之外，當TGF-訊息傳導路徑中的Smad4被抑制時，我們也觀察到此基因的表現受到了更高程度的DNA甲基化抑制而導致其表現更為低落，而更使得卵巢癌細胞的爬行/轉移能力下降。加入EZH2抑制劑也發現到相同的結果。因此兩者顯然對於抑制卵巢癌細胞轉移的能力有所助益。最後我們也發現當LTBP2被大量表現時，CP70這株細胞的轉移能力以及非貼附性生長能力都受到抑制，由此我們更能確認其為抑癌基因的假說。
總而言之，透過觀察LTBP2，我們發現異常的TGF- 訊息傳遞與EZH2含量會導致下游抑癌基因受到表基因靜默；另一方面，在惡性的卵巢癌中，擾亂TGF-訊息傳遞與較高的EZH2呈現能夠促使卵巢癌進一步惡化。

關鍵字

組蛋白甲基轉移酶 EZH2 ； 抑癌基因靜默 ； 表基因 ； 卵巢癌 ； DNA甲基化

並列摘要

The TGF-β signaling regulates numerous cellular processes, including cell proliferation, cell differentiation, apoptosis, migration and cell fate. It was also demonstrated that TGF-β functions as a tumor suppressor in normal ovarian surface epithelium (OSE) cells but promotes tumor proliferation and epithelial–mesenchymal transition (EMT) during ovarian cancer progression. Nevertheless, the molecular mechanisms leading to this divert role of TGF-β signaling in ovarian cancer remains to be elucidated. Our previous studies have demonstrated a population of TGF-/SMAD4 regulated targets are epigenetically silenced by DNA methylation and histone modification, including H3K27me3. We hypothesized that the histone-lysine N-methyltransferase, enhancer of zeste homolog 2 (EZH2), may act as an epigenetic switch to facilitate the TGF-β mediated EMT in ovarian cancer. In this study, we utilized our previously identified TGF-β responsive targets obtained by a combination of ChIP-chip and expression arrays with an immortalized ovarian surface epithelial (IOSE) cell. Bioinformatics analysis using ENCODE ChiP-Seq data identified that that several of these TGF-β targets are also marked by EZH2. Deep sequencing data from MBDcap-Seq in CP70 cells confirmed that promoter CpG island of several of these targets are differentially methylated. Interestingly, knockdown of SMAD4 demonstrated a further increase in DNA methylation in some of these targets. One of the targets, LTBP2, which was previously found to be a tumor suppressor, was expressed in IOSE but down regulated in a panel of ovarian cancer cells showing overexpression of EZH2. Except for IOSE, promoter hypermethylation was found in ovarian cancer cells as revealed by COBRA assay and pyro sequencing. Treatment of demethylation agent, 5azaDC, partially restored its expression in these cancer cells. Importantly, synergistic treatment of 5azaDC and EZH2 inhibitor, GSK343 resulted in a dramatic increase of LTBP2 expression in MCP3 and CP70 cells. Interestingly, knockdown of SMAD4 in CP70 cells resulted in increase of promoter methylation in LTBP2 suggesting that SMAD4 and EZH2 plays a contradictory role in the transcriptional control of LTBP2. Finally, overexpression of LTBP2 inhibited invasion in CP70 ovarian cancer cells. Taken together, our result suggested that EZH2 might be involved in the epigenetic silencing of TGF-β/SMAD4-regulated tumor suppressors in ovarian cancer. The differential occupancy of EZH2 into these SMAD4 loci may act as an epigenetic switch to turn the function of TGF-β from a tumor suppressor into an EMT regulator. The therapeutic potential of targeting EZH2 in the inhibition of EMT in ovarian cancer deserves further investigation.

並列關鍵字

histone methyltransferase, EZH2 ； tumor suppressor silencing ； epigenetics ； ovarian cancer ； DNA methylation

參考文獻

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