MOST104 – WWW Domain

2014/12/7: Arecoline-induced epigenomic changes in human oral keratinocytes

檳榔鹼誘導人類口腔角質細胞中表觀遺傳體改變之研究

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計畫內容(C12-1, C12-2 no more than 23 pages; references no more than 5 pages)

（一）近五年之研究計畫內容與主要研究成果說明。(C12-1)

台灣口腔癌細胞株中八十三個酪氨酸激酶表現剖繪

Expression profiling of 83 protein tyrosine kinases in Taiwanese oral cancer cell lines.

Figure 1 (DDR1 and collective cancer cell migration) (Eur J Cancer. 2012 48 (Suppl. 6): 167)

以次世代定序方法RNA-Seq分析台灣口腔癌細胞株與不朽化口腔角質細胞株

RNA-Seq analysis of Taiwanese oral cancer cell lines and immortalized keratinocyte lines.

Figure 2 (FGFR3-TACC3 gene fusion) (Cancer Discovery 2013 3: 636 pdf 3670)

參與建立台灣口腔癌與潛在惡性病症生物資料庫

Taiwan OPMD and Oral Cancer bioinformatic Database, TOP-OCD

– RNA-Seq FPKM values for each Taiwanese oral cancer cell lines under the following link (http://molas.iis.sinica.edu.tw/OralCancer)

– 40 Tumor/Normal paired oral cancer tissues (http://molas.iis.sinica.edu.tw/fold1)

– DOK projects (http://molas.iis.sinica.edu.tw/DOK2)

– New data from TOP-OCC (Taiwan OPMD and Oral Cancer Consortium, starts from Jan. 2015)

（二）研究計畫之背景及目的。請詳述本研究計畫之背景、目的、重要性及國內外有關本計畫之研究情況、重要參考文獻之評述等。本計畫如為整合型研究計畫之子計畫，請就以上各點分別述明與其他子計畫之相關性。(C12-2)

口腔潛在惡性病症及口腔癌簡述
Oral potentially malignant disorders (OPMD) and oral cancer.

Figure 3: OPMD to OSCC

葉酸攝取量與癌症間的關聯

Folate and cancer

–Celluar folate concentration represents a bell shape to maintain a homeostasis status of one-carbon metabolism, in which too little or too much causes troubles (Ulrich pdf 4100).

–While high folate level is required for a hyperactive metabolsim of cancer cells, which is the basis of anti-folate chemotherapy and has been used in clinic for decades (Locosale, pdf 4255), folate deficiency is emerging as a causal cause for cancer development (Crider in pdf 4146)

At precacner stage aduquate folate level is important for (A) Genome stability (B) SAM-mediated promoter methylations at genomic level.

–At molecular level, folate deficiency increased uracil misincorporation, reduced global DNA methylation and impaird the ability of host cell to repair oxidative and alkylation damage in immortalized normal huan colonocytes (2000, Nutr Cancer Duthie pdf 4259).

In vivo, folate insufficiency induced de novo site-specific methylation of p16 gene promoter was observed in preneoplastic liver tumors of rat modles (pdf 4260, 2002, Pogribny)

Figure 4: preliminary results of DOK projects (AC-induced MTHFR et expression changes)

Figure 5: preliminary results in MTHFR polymorphism in Taiwan OSCC

表觀遺傳體轉形作用: 專一位點的高度甲基化暨大規模的DNA低度甲基化

Epigenomic transformation: site-specific focal hypermethylation accompanied by genome-wide global hypomethylation

Observation/evidence of epigenomic transformation

(1) pdf 4102 2011/10 Increased methylation variation in epigenetic domains across cancer types (Hansen et al, Feinberg group, Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA)Tumor heterogeneity is a major barrier to effective cancer diagnosis and treatment. We recently identified cancer-specific differentially DNA-methylated regions (cDMRs) in colon cancer, which also distinguish normal tissue types from each other, suggesting that these cDMRs might be generalized across cancer types. Here we show stochastic methylation variation of the same cDMRs, distinguishing cancer from normal tissue, in colon, lung, breast, thyroid and Wilms’ tumors, with intermediate variation in adenomas. Whole-genome bisulfite sequencing shows these variable cDMRs are related to loss of sharply delimited methylation boundaries at CpG islands. Furthermore, we find hypomethylation of discrete blocks encompassing half the genome, with extreme gene expression variability. Genes associated with the cDMRs and large blocks are involved in mitosis and matrix remodeling, respectively. We suggest a model for cancer involving loss of epigenetic stability of well-defined genomic domains that underlies increased methylation variability in cancer that may contribute to tumor heterogeneity.

(2) pdf 3610, 2013 Dynamic DNA methylation across diverse human cell lines and tissues (Varley/ Mayers RM group, Hudson Alpha Institute for Biotechnology, Huntsville, Alabama 35806, USA)

As studies of DNA methylation increase in scope, it has become evident that methylation has a complex relationship with gene expression, plays an important role in defining cell types, and is disrupted in many diseases. We describe large-scale single-base resolution DNA methylation profiling on a diverse collection of 82 human cell lines and tissues using reduced representation bisulfite sequencing (RRBS). Analysis integrating RNA-seq and ChIP-seq data illuminates the functional role of this dynamic mark. Loci that are hypermethylated across cancer types are enriched for sites bound by NANOG in embryonic stem cells, which supports and expands the model of a stem/progenitor cell signature in cancer. CpGs that are hypomethylated across cancer types are concentrated in megabase-scale domains that occur near the telomeres and centromeres of chromosomes, are depleted of genes, and are enriched for cancer-specific EZH2 binding and H3K27me3 (repressive chromatin). In noncancer samples, there are cell-type specific methylation signatures preserved in primary cell lines and tissues as well as methylation differences induced by cell culture. The relationship between methylation and expression is context-dependent, and we find that CpG-rich enhancers bound by EP300 in the bodies of expressed genes are unmethylated despite the dense gene-body methylation surrounding them. Non-CpG cytosine methylation occurs in human somatic tissue, is particularly prevalent in brain tissue, and is reproducible across many individuals. This study provides an atlas of DNA methylation across diverse and well-characterized samples and enables new discoveries about DNA methylation and its role in gene regulation and disease.

(3) pdf 4081, 2014/09 Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization (Hansen et al, Feinberg group)

Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.

Possible mechanisms:

(1) pdf 4103 2012/01 Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains Laird PW ; University of Southern California Epigenome Center, University of Southern California, Keck School of Medicine, Los Angeles, California, USA.)

Extensive changes in DNA methylation are common in cancer and may contribute to oncogenesis through transcriptional silencing of tumor-suppressor genes. Genome-scale studies have yielded important insights into these changes but have focused on CpG islands or gene promoters. We used whole-genome bisulfite sequencing (bisulfite-seq) to comprehensively profile a primary human colorectal tumor and adjacent normal colon tissue at single-basepair resolution. Regions of focal hypermethylation in the tumor were located primarily at CpG islands and were concentrated within regions of long-range (>100 kb) hypomethylation. These hypomethylated domains covered nearly half of the genome and coincided with late replication and attachment to the nuclear lamina in human cell lines. We confirmed the confluence of hypermethylation and hypomethylation within these domains in 25 diverse colorectal tumors and matched adjacent tissue. We propose that widespread DNA methylation changes in cancer are linked to silencing programs orchestrated by the three-dimensional organization of chromatin within the nucleus.

pdf 4105 2012/02 Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer (Ren Bing; Ludwig Institute for Cancer Research, La Jolla, California 92093, USA.)

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

pdf 4254 2014/10 RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation (Brenner, C. University of Iowa ; Iowa City , IA USA.)

Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, the molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in a gain of DNMT1 function. However, a substantial body of data suggested that RFTS is required for DNMT1 activity. Here, we demonstrate that deletion of RFTS alters DNMT1-dependent DNA methylation during malignant transformation. Compared to full-length DNMT1, ectopic expression of hyperactive DNMT1-DeltaRFTS caused greater malignant transformation and enhanced promoter methylation with condensed chromatin structure that silenced DAPK and DUOX1 expression. Simultaneously, deletion of RFTS impaired DNMT1 chromatin association with pericentromeric Satellite 2 (SAT2) repeat sequences and produced DNA demethylation at SAT2 repeats and globally. To our knowledge, RFTS-deleted DNMT1 is the first single factor that can reprogram focal hypermethylation and global hypomethylation in parallel during malignant transformation. Our evidence suggests that the RFTS domain of DNMT1 is a target responsible for epigenetic changes in cancer.

葉酸缺乏、p16基因轉動子高度甲基化與頭頸癌間的關聯

Folate deficiency, p16 hypermethylation and head and neck cancer.

–Folate deficiency emerges to be the usual suspect in tumor initiation.

–Ericson 2007 Breast cancer

–2005 review paper: the roles of folate insufficiency in squamous cell carcinoma of the head and neck (pdf 4099

Figure 6: working model in 2005 Rev paper)

–Folate deficiency is closely related to p16 hypermethylation in HNSCC.

Indeed, folate deficiency/p16 hypermethylation was proposed as an initial step of in thead and neck cancer pathogenesis by Kane MA U of Colorado in 2005 ((pdf 4099), followed by a series of experimental and seroepidemiologic data summarized as follows:

(Table 2 Summary of literatures regarding folate and p16 methylation)

–from pdf 4247

In a genetic progression model of head and neck squamous cell carcinoma (HNSCC) presented by Califano et al, (pdf 4262) p16 has been proposed as the earliest known tumor sup- pressor gene to be inactivated.

More than 80% of the head and neck tumors show early inactivation of p167,8 (Reed, AL and Papadimitrakopoulou)

(Rationale) Based on all the literature listed above and our preliminary result

In this 2-yr reserach grant, I propose to

Based on the aforementioned findings (ours and others)

Three main basisi of this grant

1. Arcoline, likely due to its alkylating function, in very short time, triggers the expressions of enzymes crucial to folate dependent one-carbon metabolism, including decreased MTHFR and CBS (Figure 4), which together lead to decread production of 5-methly THR and accumulation of homocysteine. The reduction of

Specific Aims of this study:

Aim 1: To identify acrecoline-induced epigenomic changes in primary human oral keratinocytes

Aim 2: To catalogue hypermethylated promoters contributed to malignant transformation of OPMD

Figure 7: working flowchart

（三）研究方法、進行步驟及執行進度。請分年列述：1.本計畫採用之研究方法與原因。2.預計可能遭遇之困難及解決途徑。3.重要儀器之配合使用情形。4.如為整合型研究計畫，請就以上各點分別說明與其他子計畫之相關性。

Aim 1: To identify arecoline (AC)-induced epigenomic changes in primary oral keratinocytes (HOK)
(Overview) To quickly validate that the synthesis of MTHFR mRNA will also be reduced by AC treatment in HOK cells, three independent experiments with similar condition used in the DOK project (detailed in p3) were conducted. Preliminary results showed that MTHFR was reduced by AC treatment in two out of three experiments, confirming that the general impacts of AC could be common in DOK and HOK cells.
Next, to gain an overall expression profile and epigenomic changes induced by AC treatment in HOK cells, RNA-Seq and whole-genome bisulfite sequencing (WGBS) will be performed. We have experience in RNA-Seq by which we decoded the transcriptome sequences of 7 Taiwanese OSCC and immortalized keratinocytes cell lines (detailed in p2). For WGBS, we have collected methods from Laird Lab [27] and Feinberg Lab [26] for bisulfite sequencing at genome-wide level. Next Gen Seq will be submitted to National Core Facility Program for Biotechnology (NCFPB) at National Yang-Ming University. Cost for the sequencing experiments will be budgeted accordingly. Complementary to WGBS, methylation specific PCR, bisulfite-pyrosequencing [24] and methylation enrichment pyrosequencing [39] will be applied to inspect the methylation state of gene promoters in small-scale experiments or in validation assays.
Aim 2: To catalogue hypermethylated promoters contributed to malignant transformation of OPMD
(Overview) This part of study involves the following principles. First,

Methods:
Cell culture: normal human oral keratinocytes (HOK) and keratinocytes immortalized by hTERT purchased from Dr. Rheinwald (Harvard Medical School) will be maintained in keratinocyte serum-free medium (KSFM) as described in their original paper [40]. Specifically, early passage cells will be grew at low to moderate density plating in a medium composed of KSFM supplemented with 25 μg/ml bovine pituitary extract, 0.2 ng/ml epidermal growth factor and 0.3 mM CaCl₂. The HOK cells will be maintained in low density, and will be subcultured before they have grown beyond 1/3 confluence.

Subculture of HOK

Aspirate the medium from the culture vessel, rinse with a small amount of trypsin/EDTA. Incubate cultures in trypsin/E at 37^oC for 5-12 minutes, until the cells have rounded up and have detached spontaneously. An equal volume of DMEM or DMEM/F12 medium containing 10% serum will be added onto the dish to neutralize the trypsin and EDTA, cells will be dispersed by pipette the cells up and down several times. Pellet the cells by low speed centrifugation (~400g) for 5 minutes, aspirate off the fluid, resuspend the cells in KSFM medium for cell count.

Grow HOK to high density for arecoline treatment

Normal keratinocytes grow at an exponential rate as long as they have adequate nutrients and have room on the culture dish to expand. Yet, KSFM medium was not designed to grow cells to high density with retention of viability. To grow keratinocytes to high density for arecoline treatment, HOK cells that have been grown up to ~1/3 confluence in KSFM will be switched to a 1:1 mixture of KSFM and DFK (DMEM/F12 (1:1 v/v), 2 mM L-glutamine, 25 µg/ml BPE, 0.4 mM CaCl2, 0.2 ng/ml EGF).

Other cell cultures

All other cell lines will be grown under standard, MSDS-specified conditions. Cell proliferation of each cell line will be routinely determined by trypan blue exclusion method or WST-1 assay, to ensure appropriate propagation is performed.

RNA-Seq and data analysis
Paired-end (PE) transcriptome sequencing using Illumina HiSeq2000

cDNA libraries will be prepared in accordance with Illumina’s sample preparation protocol for PE mRNA sequencing with some modifications. Briefly, mRNA will be fragmented at 85 °C and converted to cDNA. The double-stranded cDNAs will be end-repaired and ligated with adaptor oligo mix. The adaptor-ligated cDNA library will be fractioned on a 3% agarose gel, and fragments corresponding to 300-350 nt will be excised, purified, and PCR amplified. The resulting PCR product will be purified free of primers using the Ampure XP solid phase reversible immobilization protocol. The libraries will be quantified with Agilent DNA 1000 kit on an Agilent 2100 Bioanalyzer following the manufacturer’s instructions. Libraries (10 nM) will be to prepare flowcells using an Illumina cBot and then sequenced in one flowcell lane on an Illumina HiSeq 2000, generating 80 to 120 million paired end reads per sample.

Gene expression analysis

Cufflinks is a program that calculates gene and transcript abundances in Fragments Per Kilobase of exon per Million fragments mapped (FPKM) [41]. Each gene or transcript has FPKM value as its expression level. MOLAS, a Multi-Omics onLine Analysis System built to identify differentially expressed genes (DEGs) and their downstream data analysis in two or more conditions and unravel their biological relationship [14]. MOLAS provides pairwise comparisons between libraries, clustering analysis, KEGG pathway, and GO terms enrichment analysis, to identify significantly altered networks and find the biological implication.

Whole-genome bisulfite sequencing (WGBS) and data analysis (WGBS)

Adapted from Feinberg Lab [26]

Library construction

Bisulfite sequencing libraries will be prepared using the Illumina TruSeq DNA Library Preparation Kit protocol. Specifically, thirty nanograms of unmethylated lambda DNA will be added to 3 mg of genomic DNA prior to shearing in order to monitor the efficiency of the bisulfite conversion. The sheared DNA ends will be repaired using 1 ´ NEB Buffer 2, 400 nm each of dATP, dGTP, and dTTP, 15 units of T4 DNA polymeras 5 units of Klenow DNA polymerase and 50 units of T4 Polynucleotide kinase. In the bisulfite conversion step, 24 mL of formamide will be added to an equal volume of DNA and incubated at 95°C for 5 min. Subsequently, 100 mL of Zymo Gold bisulfite conversion reagent (Zymo) will be added, and the mixture will be incubated for 8 h in 50°C. Samples will be desulphonated and purified using spin columns following the Zymo EZ DNA Methylation-Gold Kit protocol. The bisulfite converted library will be amplified in 1 ´ PCR buffer, 0.2 mM dNTP, 5 mL of the TruSeq PCR Primer Cocktail, 5 units of Taq, and 0.25 units of PfuTurbo DNA polymerase (Stratagene). The DNA will be subjected to 10 cycles of PCR reaction.

Mapping and quality control of WGBS reads

The 100-by-100 bp HiSeq 2000 paired end sequencing reads obtained for each sample will be ran on the BSmooth bisulfite alignment pipeline using Bowtie 2 [42] and the GRCh37 build of the human genome including sex chromosomes, nonchromosomal sequences, and mitochondrial sequence. BSmooth is capable of extracting read-level measurements, and filtering out unreliable read-level measurements.

Adapted from Laird Lab [27]

Library construction and sequencing

Sequencing adapters with fully methylated cytosines (Integrated DNA Technologies) will be used to create Illumina Genome Analyzer IIx sequencing libraries followed by bisulfite conversion (Zymo EZ DNA Methylation Kit) and PCR. Two libraries will be made for each sample, and each library will be PCR amplified by dividing it into nine independent PCR reactions and then pooling the PCR products. Attachment of the library DNA to the Genome Analyzer flow cell will be performed on an Illumina Cluster Station fluidics device. Single-end DNA sequencing (76-bp reads) will be performed using the Illumina Genome Analyzer II. Only reads passing the Illumina chastity quality filter will be retained.

Alignment and extraction of 5mC levels

Human genome build hg19 (NCBI v.37) will be used for all analyses. MAQ (http://maq.sourceforge.net) will be used for sequence alignment, using the ‘-c’ option to match any C or T in the sequencing read to a C in the reference genome. SAMtools [43] will be used to perform processing and merging of BAM files, and duplicate reads starting at the same genomic position will be removed for each of the two sequencing libraries per sample. Reads will be filtered out if they had a MAQ mapping quality score of less than 30, which removed alignments with many mismatches as well as those reads aligning equally well to multiple locations in the genome. A Java library (http://ngsgenomelibs.sourceforge.net/) will be used to transform BAM alignments to percent methylation for each cytosine in the genome. Each cytosine in the reference genome will be included for analysis if it were covered by three or more C or T reads on the bisulfite-converted strand. Cytosines will be considered high-confidence CpGs if greater than 90% of reads at the following position were G or high-confidence CpHs if greater than 90% of reads at the next position were H (A, C, T).

Identification of focal methylation changes

Unmethylated regions will be identified by scanning all windows of at least ten individual CpG cytosines within five CpG dinucleotides (each CpG dinucleotide contains two cytosines, one on each strand). Only those cytosines covered by at least three cytosine or thymine reads will be counted, and each CpG dinucleotide will be assigned a weighting factor defined as the span (in bp) between the next CpG dinucleotide upstream and the next CpG dinucleotide downstream. A weighted average will be calculated for each window, and those windows with an average DNA methylation of less than 5% in both tumor and adjacent normal tissue will be categorized as methylation resistant. Those windows with methylation of less than 5% in the control cells and greater than 35% in the AC-treated cells will be methylation prone, and those windows with methylation of greater than 35% in the control cells and less than 5% in the AC-treated cells will be characterized as methylation loss. Two or more overlapping regions from a single methylation class will be merged into one.

Methylation-specific polymerase chain reaction (MS-PCR)
MS-PCR distinguishes unmethylated from methylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated cytosines to uracil, and subsequent PCR using primers designed for either methylated or unmethylated DNA. The primers for each desired promoter genes will be designed using NCBI Primer 3. PCR will be performed using an amplification kit (AmpliTag Gold, Applied Biosystems) and a thermal cycler (PTC-100, MJ Research). Each PCR product will loaded directly onto non-denaturing 2% agar gels. As a positive control for the methylation, CpGenomeTM Universal Methylation DNA (Serologicals Co., CA) will be used.
Bisulfite-pyrosequencing
Four hundred nanograms of genomic DNA will be bisulfite converted using the EZ DNA Methylation-Gold Kit (Zymo). Nested PCR will be performed using the appropriate primer sets of desired genes. The annealing temperature used for all PCR reactions will be 50°C. The resulting PCR reactions will be used directly for pyrosequencing.
Methylation enrichment-pyrosequencing
This is a newly developed method by Shaw et al that combines the above two techniques, MS-PCR and bisulfite-pyrosequencing, to utilize methylation-specific primers and followe by a confirmatory pyrosequencing step that allows elimination of false positives [39]. Thus, Hot-start PCR will be carried out with HotStar Taq Master Mix kit (Qiagen Ltd.) using 120 ng bisulfite-treated DNA. MEP primers will be designed accordingly for each desired promoters by using NCBI Primer 3. All PCR annealing temperature will be carried out at 50°C. The presence or absence of PCR products and freedom from PCR contamination will be inspected on 2% agarose gels. Gel-positive PCR products will be subjected to confirmatory pyrosequencing to ensure that methylation was >95% at all CpG dinucleotides interrogated.
DNA Extraction
For all promoter methylation experiments, DNA will be extracted using a phenol-chloroform–based extraction procedure that gives superior yield from small samples (DNeasy Blood & Tissue Kit, Qiagen). Bisulfite treatment of each sample will be undertaken using the EZ DNA methylation kit (Zymo Research). A quality check of converted DNA will be done using pyrosequencing methylation assay primers specific for bisulfite-treated DNA {Shaw, 2006 #3504}. Any samples showing <100 any="" are="" as="" assay.="" be="" conversion="" false="" in="" likely="" methylation-dependent="" p="" positives="" produce="" rejected="" these="" to="" will="">Other general methods
(遺珠) Reduced representation bisulfite sequencing experimental procedure (in pdf 3610)

（四）預期完成之工作項目、成果及績效。

1.預期完成之工作項目。

(A) To understand the molecular process of arecoline-induced epigenomic changes in primary human oral keratinocytes. (B) To provide a panel of highly specific and sensitive methylated gene promoters that can be used to screen high-risk OPMD patients.

2.對於學術研究、國家發展及其他應用方面預期之貢獻。

口腔癌的發生是漸進性的，多數的病人在口腔癌發生前，常會先被診斷出患有口腔潛在惡性病症 (oral potentially malignant disorders, OPMD) 。最新的分析資料顯示約有5–10%的口腔黏膜下纖維化及白斑患者在五到十年內將惡性轉化為口腔癌，若能於此黃金時段定期追蹤或施予有效之化學預防療程，將可直接降低國人罹患口腔癌的機率。此外、儘管國外有許多進行口腔潛在惡性病症化學預防的研究，但其病因多與抽煙與喝酒有關，缺少本土性口腔癌「檳榔」因子。本計畫乃基於這兩個迫切的議題，深入探討檳榔鹼對人類口腔角質細胞的癌變初始化(initiation of cancer)機制、並輔以最新之臨床分子診斷技術，希望研究產出能轉譯於臨床上、直接加惠於口腔潛在惡性病症之患者。

3.對於參與之工作人員，預期可獲之訓練。

計畫執行過程中至少培養出一位博士班畢業生，嫻熟於癌症表觀遺傳體學、生物資訊分析、以及次世代定序技術。此生將融合這些知識與技術，進而將其應用於篩檢口腔潛在惡性病症之高危險群。本計畫中擬定的生物檢體為相對穩定的甲基化DNA，多可由口腔黏膜抹片或是血液中萃取而得，屬於非襲性的篩檢方式。

4.預期完成之研究成果及績效（如期刊論文、研討會論文、專書、技術報告、專利或技術移轉等質與量之預期績效）

研究成果預期產生至少兩篇研討會論文及一篇臨床醫學相關期刊論文。

_____________

審查委員意見

委員1
[Significance & Novelty:]
Arecoline might impair cell growth and proliferation via interfering with folate and one-carbon metabolism in human oral keratinocytes. In this proposed study, Dr. Lin will use the RNA-Seq and whole-genome bisulfite sequencing (WGBS) to interrogate the arecoline induced gene expression alterations and the associated DNA methylation changes.
[Weakness:]
This is an ambitious project to link the WGBS and RNA-Seq data together in systematical interrogation on methylation alterations and transcriptome modifications. From the previous experience, Dr. Lin should have the expertise in carry out the needed experimental procedures; however, the more challenging part is the integrated bioinformatic analysis. Dr. Lin failed to provide the details on this critical issue. It is suggested to have a bioinformatic co-PI from NHRI to join the study. The depth of NGS is also a critical issue should be take into consideration in such a systems biology study.

委員2
(1)Significance & Novelty:
本研究計畫部分preliminary results support本計畫之進行，研究成果可對arecoline影響正常口腔角質細胞的基因甲基化的情形有全盤性理解，亦可更清楚葉酸以及單碳代謝路徑如何參與癌化形成。搭配分析transcriptome生物資料庫，未來可作為高危險群口腔癌前病變患者的臨床檢測之參考。
(2)Weakness:
腫瘤組織或口腔癌前病變過程參與微環境的各式細胞，而促進cancer progression，計畫以primary human oral keratinocytes經arecoline處理所得的基因甲基化改變可能與實際癌變過程的表觀修飾之改變有很大出入。
(3) Specific Comment:
1. 該計畫中對於RNA-seq及Genome-wide bisulfite seq 的分析，應有bioinformatics背景co-PI共同協助分析為宜。
2. 計畫中僅分析arecoline對口腔細胞基因甲基化改變，並進一步與台灣口腔癌與潛在惡性疾病資料庫連結分析，應審慎評估其中之落差，係因台灣口腔癌有菸、酒、檳榔同時使用之病患比例相當高，基因表現與單純arecoline處理之細胞應有相當差異。主持人應於資料分析時加入菸、酒、檳榔之考量。

委員3
(1)Significance & Novelty:
檳榔鹼影響正常口腔角質細胞的基因甲基化的情形有全盤性理解，此結果將可作為高危險群口腔癌前病變患者的臨床檢測之參考。
(2)Weakness:
1.此計畫應用很多高階檢測技術, WGBS and RNA-Seq data分析檳榔鹼影響正常口腔角質細胞的基因甲基化的情形，龐大的資料分析，應該與生物資訊的PI合作，此計畫沒看到共同主持人有此專長。
2.口腔癌前病變相當複雜是否能只用簡單檳榔鹼改變HOK epigenome configuration來回答？而不考慮微環境的各式細胞之影響？

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40TN為either Illumina HumanCNV370-Duo or HumanCNV370-quad

Wishing list:

Loss

1p36.3: MTHFR (ACshort 0.53x folate pathway related) *
1q12-q23: DPT (40TN 0.36x, 4NQO mouse 0.5x, DOK project array上沒有此基因)
3p21.1: BAP1 (ACshort 0.92x)
3p21.3: RASSF1(ACshort 1.48x)
3q26.3-q27: LAMP3 (ACshort 0.05x) *
4q23: ADH1A, ADH1B, ADH1C (DOK project array上沒有此些基因 40TN 0.33x, 0.08x, 0.14x)
4q28: MAML3
4q31.1: FBXW7 (40TN 0.9x)
9p21.3: CDKN2A (40TN 5.33x)
9q21.13: ALDH1A1 (ACshort 0.8x)
9q34.3: NOTCH1 (ACshort 0.84x, 40TN 0.93x, qRT-PCR primer 小丸有~)
9q34.3: NOXA1 (ACshort 0.15x) *
12q23.3: ALDH1L2 (ACshort 0.38x folate pathway related) *
17p13.1: TP53

Gains

3q26.3: SOX2 (ACshort 0.4x)/PI3KCA(ACshort 0.82x)
4p16.3: FGFR3
7q35-q36: EZH2 (ACshort 2x) *
8p12: FGFR1
8q24.21: MYC
9p24: JAK2
10q26: FGFR2
11q13.3: CCND1 (ACshort 0.76x; 40TN 0.9x)
11q22.3: MMP3 (ACshort 4x) *
11q23.2: TAGLN (ACshort 9.8x) *
12p12.1: KRAS
19p13.2: DNMT1 (ACshort 1.82x) *
19q13.1: AKT2
19q13.12: WDR62 (ACshort 6.11x)

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另外兩篇原載於LOH post的paper

2. Loss of Heterozygosity (LOH) Profiles–Validated Risk Predictors for Progression to Oral Cancer, 2012 Cancer Prev Res (pdf 3498)

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Hypermethylation of the p16 gene in normal oral mucosa of smoker

Int J Mol Med. 2004 Nov;14(5):807-11.
Hypermethylation of the p16 gene in normal oral mucosa of smokers. (PubMed) (pdf requires $$)
von Zeidler SV1, Miracca EC, Nagai MA, Birman EG.
Author information
Abstract

The oral cavity is the sixth most common anatomical localization of head and neck carcinoma in men. Detection of oral carcinomas in the early asymptomatic stages improves cure rates and the quality of life. Tobacco smoking and alcohol drinking are the most important known risk factors for the development of head and neck tumors, suggesting that the exposure to these risk factors may increase the predisposition for genetic and epigenetic alterations, such as DNA methylation. The presence of methylated CpG islands in the promoter region of human genes can suppress their expression due to the presence of 5-methylcytosine that interferes with the binding of transcription factors or other DNA-binding proteins repressing transcription activity. Hypermethylation leading to the inactivation of some tumor suppressor genes, such as p16, has been pointed out as an initial event in head and neck cancer. Our aim was to evaluate an early diagnostic method of oral pre-cancerous lesions through the analysis of methylation of the p16 gene. DNA samples from normal oral mucosa and posterior tongue border from 258 smokers, without oral cancer, were investigated for the occurrence of p16 promoter hypermethylation. The methylation status of the p16 gene was analyzed using MS-PCR (methylation-sensitive restriction enzymes and PCR amplification), MSP (Methylation-specific PCR) or direct DNA sequence of bisulfite modified DNA. Hyper-methylation was detected in 9.7% (25/258) of the cases analyzed. These findings provide further evidence that epigenetic alteration, leading to the inactivation of the p16 tumor suppressor gene is an early event that might confer cell growth advantages contributing to the tumorigenic process. Thus, the detection of abnormal p16 methylation pattern may be a valuable tool for early oral cancer detection.

PMID: 15492849

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# 7 Use of allelic loss to predict malignant risk for low-grade oral

Clin Cancer Res. 2000 Feb;6(2):357-62.
Use of allelic loss to predict malignant risk for low-grade oral epithelial dysplasia. (in OPMD db, referred by YWC no pdf yet)
Rosin MP1, Cheng X, Poh C, Lam WL, Huang Y, Lovas J, Berean K, Epstein JB, Priddy R, Le ND, Zhang L.
Author information
Abstract

One of the best approaches to identifying genetic changes critical to oral cancer progression is to compare progressing and nonprogressing oral premalignant lesions. However, such samples are rare, and they require long-term follow-up. The current study used the large archive network and clinical database in British Columbia to study loss of heterozygosity (LOH) in cases of early oral premalignancies, comparing those with a history of progression to carcinoma in situ or invasive cancer and those without a history of progression (referred to as nonprogressing cases). Each of 116 cases was analyzed for LOH at 19 microsatellite loci on seven chromosome arms (3p, 4q, 8p, 9p, 11q, 13q, and 17p). The progressing and nonprogressing cases showed dramatically different LOH patterns of multiple allelic losses. An essential step for progression seems to involve LOH at 3p and/or 9p because virtually all progressing cases showed such loss. However, LOH at 3p and/or 9p also occurred in nonprogressing cases. Individuals with LOH at 3p and/or 9p but at no other arms exhibit only a slight increase of 3.8-fold in relative risk for developing cancer. In contrast, individuals with additional losses (on 4q, 8p, 11q, or 17p), which appeared uncommon in nonprogressing cases, showed 33-fold increases in relative cancer risk. In conclusion, analysis of LOH at 3p and 9p could serve as an initial screening for cancer risk of early premalignancies. Follow-up investigation for additional losses would be essential for predicting cancer progression. PMID: 10690511
Comment in Can molecular assessment improve classification of head and neck premalignancy? [Clin Cancer Res. 2000]

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# 8 The precancer risk of betel quid chewing, tobacco use and alc

Br J Cancer. 2003 Feb 10;88(3):366-72.
The precancer risk of betel quid chewing, tobacco use and alcohol consumption in oral leukoplakia and oral submucous fibrosis in southern Taiwan. (in OPMD db, referred by YWC no pdf yet)
Lee CH1, Ko YC, Huang HL, Chao YY, Tsai CC, Shieh TY, Lin LM.

Author information
Abstract

In areas where the practise of betel quid chewing is widespread and the chewers also often smoke and drink alcohol, the relation between oral precancerous lesion and condition to the three habits is probably complex. To explore such association and their attributable effect on oral leukoplakia (OL) and oral submucous fibrosis (OSF), a gender-age-matched case-control study was conducted at Kaohsiung, southern Taiwan. This study included 219 patients with newly diagnosed and histologically confirmed OL or OSF, and 876 randomly selected community controls. All information was collected by a structured questionnaire through in-person interviews. A preponderance of younger patients had OSF, while a predominance of older patients had OL. Betel quid chewing was strongly associated with both these oral diseases, the attributable fraction of OL being 73.2% and of OSF 85.4%. While the heterogeneity in risk for areca nut chewing across the two diseases was not apparent, betel quid chewing patients with OSF experienced a higher risk at each exposure level of chewing duration, quantity and cumulative measure than those who had OL. Alcohol intake did not appear to be a risk factor. However, cigarette smoking had a significant contribution to the risk of OL, and modified the effect of chewing based on an additive interaction model. For the two oral premalignant diseases combined, 86.5% was attributable to chewing and smoking. Our results suggested that, although betel quid chewing was a major cause for both OL and OSF, its effect might be difference between the two diseases. Cigarette smoking has a modifying effect in the development of oral leukoplakia. PMID: 12569378

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Molecular screening of oral precancer.

Oral Oncol. 2013 Dec;49(12):1129-35. doi: 10.1016/j.oraloncology.2013.09.005. Epub 2013 Oct 9. (in OPMD db, referred by YWC no pdf yet)

Molecular screening of oral precancer. (PubMed)

Graveland AP1, Bremmer JF, de Maaker M, Brink A, Cobussen P, Zwart M, Braakhuis BJ, Bloemena E, van der Waal I, Leemans CR, Brakenhoff RH.
Author information

Abstract
OBJECTIVES:

Early detection and treatment of high risk premalignant mucosal changes of the oral cavity, will expectedly improve survival and reduce treatment-related morbidity. Aims of this study were to evaluate a non-invasive screening approach and to assess the value of molecular markers to identify patients at risk for oral cancer.
MATERIALS AND METHODS:

Exfoliated cells and biopsies were obtained from oral leukoplakia lesions of 43 patients, of whom six developed oral cancer. All samples were investigated for loss of heterozygosity (LOH) at chromosomes 3p, 9p, 11q and 17p using microsatellite markers. On the biopsy specimen additional immunohistochemical staining for p53, TP53 mutation analysis and histopathological grading were performed.
RESULTS:

The analytical sensitivity of the non-invasive assay using exfoliated cells to detect genetic changes present in the lesions was 45% (9 of 20), the specificity was 100% (19 of 19), and the positive predictive value was also 100% (9 of 9). LOH was present in 20 of 39 (51%) of the biopsies with uniformly LOH at 9p. Mutated TP53 and LOH at 9p in the biopsy, as single markers and in combination, were significant risk factors for malignant progression of leukoplakia to oral cancer (Kaplan-Meier analysis, p<0 .05="" br="" style="margin: 0px; padding: 0px;">CONCLUSION:

A non-invasive genetic screening approach using LOH in exfoliated cells has limited value for monitoring patients with leukoplakia. However, LOH at 9p, but also mutated TP53 in biopsies of oral leukoplakia have a significant association with malignant transformation and are promising candidate biomarkers to predict the risk for malignant progression. PMID: 24120275

Leukoplakia; Loss of heterozygosity; Malignant transformation; Molecular diagnosis; Oral cancer; Premalignant oral lesions; Screening; p53