利用人類癌症細胞株分析荷爾蒙接受體α (ESR1基因)多個啟動子之DNA甲基化情形

Abstract

雌激素對於女性生殖器官的生長與發育扮演重要的角色，除了人體正常生理的調控外，也可能造成骨質疏鬆、乳癌與子宮內膜相關癌症等的病理變化。典型的荷爾蒙作用機轉是來自與細胞核的接受器結合後，再進一步向細胞內傳遞訊息，目前較為人知的接受器包括ERα和ERβ(負責的基因分別為ESR1和ESR2)，人體中以ERα的表現較為重要。根據研究發現不同器官會有不同的ER蛋白質表現量，而且就算是同一個器官亦會隨著不同階段之生理需求而不同表現。人體內ERα是否因為空間與時間上的不同表現而造成如此多樣性的生理反應，尚待釐清? 唯有透過了解荷爾蒙與其受體調控的分子機制後，才有助於探討荷爾蒙在人體器官的生理與病理變化。 人類ESR1 cDNA序列於1986年發現，位於染色體6q25.1的位置上，全長約為300kb其中含8個外顯子。在多種癌症的研究發現ESR1基因發生鹼基突變的機會很低，但是除了基因的突變外，近來超基因學(epigenetic)的蓬勃發展中又以基因體中發生甲基化的現象最為常見，由於帶有甲基化的基因表現是被抑制的，多數研究團隊發現腫瘤抑癌基因啟動子的甲基化(包括ESR1基因)與各種腫瘤的形成有關。根據先前的研究發現ERα存在有多個RNA的isoforms，推測應該存在多個基因啟動子可以調控ER蛋白質的表現。而目前已知ESR1至少有6個以上的基因啟動子，可以轉錄出不同mRNA isoforms，再轉譯成蛋白質。其中不同基因啟動子與mRNA isoforms在各個器官組織的表現能力不一樣，具有組織專一性，而這些差異性可能來自於不同基因啟動子的調控，過去利用螢光活性之檢測方式，證實了不同癌細胞株包括乳癌、卵巢與子宮內膜癌有不同的基因啟動子活性，於是我們假設不同組織中ERα的表現是來自於ESR1基因中不同啟動子的甲基化(methylation)現象。 本研究是利用癌症細胞株與部分正常人體組織，採用bisulfite direct sequencing技術，經由特殊引子對的設計來進行ESR1基因六個啟動子過度甲基化的分析。以人類不同癌症細胞株為研究對象，發現不同癌症細胞株在不同啟動子確實有不同過度甲基化的特點。以乳癌細胞株為例，由於荷爾蒙受體的表現與否影響臨床乳癌患者的預後與治療方式，代表ER(+)的MCF-7和T47D與代表ER(-)的MDA-MB-231和MDA-MB-435在不同啟動子(A、B、C、D和F)有不同過度甲基化的特點。我們認為不同啟動子的選擇使用可能來自於啟動子甲基化的發生與否，希望這個結果有助於未來進行臨床不同癌症組織的超基因學分析。 實驗結果同時發現，癌症細胞株中ESR1基因啟動子附近之CpG位置發生甲基化為全有全無的表現，有人指出這是因為癌症細胞株為癌化末期之病變組織所致。另外我們發現子宮頸癌細胞株(HeLa和CaSki)與正常子宮頸細胞在啟動子A、B與F有不同甲基化的特點，由於癌症的發生屬於heterogenous processes，未來我們希望能進一步運用於臨床癌症組織檢體以了解是否仍存在這樣的特異性，另外同時定量CpG甲基化程度與分析不同mRNA isoforms與蛋白質的表現量，以釐清甲基化的發生與荷爾蒙受體表現的相關性也是很重要的。

It is well-known that estrogens play an important role in sexual development, reproduction and pathological processes in gynecologic tissues such as the mammary and uterine carcinomas. Estrogens exert these changes through the superfamily of nuclear hormone receptors which act as ligand-activated transcriptional factors. Currently two ER receptors (αandβ) coded on different genes (ESR1 and ESR2 genes respectively) have been identified. Comparing to ERβ, ERα is expressed in a great variety of human tissues. Its expression level varies considerably among different cell types and tissues due to the specific stimulation or repression by estrogens in a tissue-dependent manner. The possibility for the tissue-specific effects of estrogens may be from the differential and spatio-temporal expression of ERα. To understand the molecular mechanisms that control the tissue specific pattern of ERα could be valuable in identifying the various effects of estrogens on different biological processes. The human ESR1 cDNA was cloned in 1986 and the gene consists of 8 coding exons spanning 140 kb of chromosome 6q25.1 locus. Until now, there have been very few reports about mutations of coding regions or other structural alterations of the ESR1 gene in various cancers. One possible mechanism for inactivating the transcriptional status is methylation of CpG islands in ESR1 gene due to the large distances between promoters. Previous studies on the structure and organization of the human ESR1 gene indicated that its transcriptional activity was probably controlled by up to six promoters. The differential expression of ERα protein in different tissues was demonstrated to be achieved by multiple promoter usage that aroused the possibility of responding to distinct combinations of factors present in different tissues. The discovery of six promoters at least in the ESR1 gene has led us to suggest that differential promoter usage through CGIs methylation pathway could be one mechanism regulating expression of the estrogen receptor α in various tissues. In this study we comprehensively analyzed methylation status of six promoter regions in ESR1 gene in cancer cell lines and normal tissues by using bisulfite direct sequencing assay. Our results indicate that methylation status in six ESR1 promoters varies in different cancer cell lines. Currently ERαis a well-established prognostic marker in breast cancer, predicting the likelihood of a patient’s response to adjuvant endocrine therapy. The major finding of this work is that methylation status in ESR1 promoter A, B, C, D and F regions is dramatically different in ER(+) and ER(-) breast cancer cell lines. Selective promoter usage in genes containing multiple promoters such as ESR1 gene may be possible clinical application of promoter-specific methylation. Therefore, the different methylation profiles of ESR1 promoters in different cancer cell lines may provide a basis for further epigenetic analysis to distinguish different cancers. To enrich the information obtained from bisulfite sequencing technique, we observed all or none methylated CpG sites within the amplified fragments in various cancer cell lines (0% or 100% methylation density, the percentage of methylated CpG sites within the amplified CpG sites). Smiraglia et al. reported that cancer cell lines exhibit a higher degree of methylation than normal tissues resulting from the end stage of carcinogenesis. In addition, it shows different methylation status in promoter A, B and F between cervical cancer cell lines and normal cervical tissues. However they may not truly represent each cancer in tissue and this result can be probably used for advanced analysis or as a molecular marker of cancer if we get the same result in primary cancer tissues. Further research in primary tumors is required in order to clarify the possible connection to cancer and quantification of methylation severity should also be emphasized due to the heterogeneous process of carcinogenesis.