||Studying the molecular mechanism responsible for the down-regulation of TGFBR3 in oral cancer
||Institute of Molecular Medicine
口腔癌自從2007成為國人十大癌症死因的第四名之後，仍然有繼續攀升的趨勢。近年來在不同種類的癌症包含頭頸癌中發現TGFBR3有降低表現的趨勢，於是我們推測TGFBR3可能具有抑制腫瘤生長的功能。從之前的臨床實驗數據指出，口腔癌病患的腫瘤組織相較於週邊正常的組織有較低的TGFBR3表現量，而且低TGFBR3表現量有較差的癒後結果。首先使用低TGFBR3表現量的兩株口腔癌細胞株，分別處理甲基轉移酶抑制劑或組織蛋白去乙醯酶抑制劑，結果發現TGFBR3的表現量明顯的上升，所以我們猜測在口腔癌中的TGFBR3表現量降低是藉由表觀基因調控的影響。將TGFBR3啟動子上的CpG island進行in vitro methylation會大幅降低啟動子的活性，更加證實DNA甲基化會對TGFBR3啟動子有負向調控功能。MeCP2是常見會與甲基化DNA結合的蛋白質，接著我們採用chromatin immunoprecipitation 去觀察MeCP2是否會附著在TGFBR3啟動子上，實驗結果顯示在TGFBR3啟動子-365到+406有偵測到MeCP2。除此之外，我們還偵測到微弱的抑制活性的組織蛋白標誌－H3K27me3，而後經過甲基轉移酶抑制劑的處理，我們偵測不到MeCP2訊號反而是偵測到增加活性的組織蛋白標誌－H3K4me3。DNA甲基轉移酶是進行DNA甲基化的重要酵素之一，進行DNA甲基化的同時也會吸引其他蛋白分子，例如MeCP2或是組織蛋白去乙醯酶。我們發現在處理了甲基轉移酶抑制劑或組織蛋白去乙醯酶抑制劑的口腔癌細胞株中，DNMT1，DNMT3B以及HDAC6都與TGFBR3表現量成反比。除了表觀基因調控之外，位於TGFBR3啟動子-1460到-1447上的retinoic acid response element也可以參與TGFBR3降低表現的機制。雖然需要更多實驗來證明，本實驗結果指出在口腔癌中的TGFBR3降低表現不僅是受到表觀基因調控，同時也可以透過TGFBR3啟動子上的RXR/RAR binding site所調控。
Oral cancer with alarmingly increase in the incidence rate has become the fourth cause of male cancer-related death in Taiwan since 2007.As a co-receptor for TGF-β, transforming growth factor β receptor III (TGFBR3, also known as betaglycan) is believed to exert tumor suppressor functions due to the frequent decrease of TGFBR3 expression in several cancer types including head and neck cancer. We also detected the down-regulation of TGFBR3 in oral cancer patients and the down-regulation was associated with poor clinical outcomes among these patients. Following the treatment of low-TGFBR3 expressing CAL-27 or SCC-15 cells with a DNA methyltransferase inhibitor, 5’azaC, or histone deacetylase (HDAC) inhibitors, 4-phenylbutyrate (PBA) or suberoylanilide hydroxamic acid (SAHA), there was a significant induction of TGFBR3 mRNA expression in both the single and co-treated cells, suggesting the involvement of epigenetics in regulating TGFBR3 expression. In vitro methylation drastically reduced the luciferase activity driven by the proximal promoter harboring a putative CpG island, indicating a negative role of DNA methylation in TGFBR3 expression. Methyl CpG binding protein 2 (MeCP2) is a protein frequently bound to the methylated DNA to regulate gene expression. We then used chromatin immunoprecipitation (ChIP) to examine if MeCP2 also bound to the methylated CpG sites in the TGFBR3 promoter. We did detect the in vivo binding of MeCP2 to the promoter region between -365 and +406bp. A repressive histone marker, histone3 lysine 27 tri-methylation (H3K27me3), was weakly detected in the promoter region. Following 5’azaC treatment, there was a decreased binding of MeCP2 but an increased binding of active histone markers, acetyl histone 3 (AcH3) and histone 3 lysine 4 tri-methylation (H3K4me3), on the same promoter. DNA methyltransferases (DNMTs) are the key enzymes participating in DNA methylation. Following binding to the methylated CpG sites, DNMTs often recruit repressive complex consisting of MeCP2 or HDACs. Consistent with the notion, we detected an inverse mRNA expression of TGFBR3 with DNMT1, DNMT3B and HDAC6 mRNA in CAL-27cells treated with 5’azaC or HDAC inhibitors. In addition to the epigenetic control, a predicted RXR/RAR binding site spanning -1460 to -1447 on the TGFBR3 promoter also participates in the TGFBR3 deregulation. Although more studies are needed to delineate the detailed action mechanism, epigenetic control together with RA metabolism dysfunction participates in the deregulation of TGFBR3 expression in oral cancer cells.
Abstract in Chinese I
Abstract in English II
List of Figures and tables IX
I. Introduction 1
1-1 Oral cancer 1
1-2 TGF-β and TGFBR3 1
1-3 TGFBR3 deregulation in cancer 2
1-4 Potential mechanisms involved in TGFBR3 deregulation 2
1-5 Impaired retinoic acid pathway in cancer 3
1-6 Retinoic acid cross talk with TGF-β/TGFBR3 3
1-7 Previous lab studies on TGFBR3 deregulation in oral cancer 3
II. Hypothesis 5
III. Specific aims 6
IV. Materials and methods 7
4-1 Cell culture and patient samples 7
4-2 Drug treatment 7
4-3 Plasmid constructs 8
4-4 Luciferase promoter assay 8
4-5 in vitro methylation treatment 8
4-6 RNA isolation and semi-quantitative and quantitative RT-PCR 9
4-7 Genomic DNA extraction and bisulfite sequencing PCR 9
4-8 Methylation specific PCR 10
4-9 Chromatin immunoprecipitation (ChIP) PCR 10
4-10 Site-directed mutagenesis 11
V. Results 12
5-1 Epigenetics involved in the control of TGFBR3 deregulation in oral cancer cells 12
5-2 The proximal TGFBR3 region spanning from -366 to +174 12
5-3 The impact of CpG island and its methylation on the promoter activity of the proximal TGFBR3 region spanning from -366 to +174 13
5-4 The inverse relation of TGFBR3 mRNA expression with promoter methylation in oral cancer lines but not clinical specimens 14
5-5 Bisulfite sequencing of the region spanning putative CpGisland in the TGFBR3 promoter 15
5-6 MeCP2 and histone modification markers bind to the TGFBR3 promoter region between -365 and +406 15
5-7 Inverse expression of TGFBR3 with DNMT1 and DNMT3B in oral cancer cells following 5’azaC and/or HDAC inhibitors treatment 16
5-8 The expression of TGFBR3 and DNMT1 and DNMT3B in clinical specimens 17
5-9 HDAC6 was not involved in the TGFBR3 deregulation despite its frequent increase of expression in oral cancer cells 17
5-10 RA metabolites decreased TGFBR3 promoter activity 18
5-11 The presence of RAR/RXR binding sites in the promoter region from -1460 to -1447 19
VI. Discussion 20
VII. References 23
VIII. Appendix 55
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