RALYL Hypermethylation: A Potential Diagnostic Marker of Esophageal Squamous Cell Carcinoma (ESCC) Junwei Liu, MD

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1 RALYL Hypermethylation: A Potential Diagnostic Marker of Esophageal Squamous Cell Carcinoma (ESCC) Junwei Liu, MD Aurora Healthcare, Milwaukee, WI

2 INTRODUCTION v Epigenetic aberration and genetic alteration are equally responsible for cancer development. v DNA methylation is one of the most frequent types of epigenetic changes. v Methylation are most frequently found in promoter regions of the genes. v CpG islands are targets of methylation.

3 v Aberrant methylation of DNA promoters includes hypermethylation of TSGs and hypomethylation of oncogene. v DNA hypermethylation is a common mechanism in suppressing TSGs and is a common event in carcinogenesis. v DNA-methylation based technologies could be useful in cancer detection, risk assessment, chemoprediction and prognosis.

4 v Esophageal cancer is the 8 th most common malignancy. The highest rate occurs in Asia and Africa. v The most common types of esophageal cancer are squamous cell carcinoma and adenocarcinoma. Squamous cell carcinoma (ESCC) is the predominant type worldwide. v Most esophageal cancer is diagnosed at advance stages stage (III/IV), the ratio of mortality to incidence is high (0.83). It is the 6 th most frequent cause of death from malignancy worldwide. v Therefore, it is very critical to understand molecular events in diagnosis and management of esophageal cancer.

5 METHODS AND RESULTS

6 Screening of methylation gene in esophageal cancer Methods * RNA re-expression microarray * Expression profile analysis through gene database search * RT-PCR * Methylation specific PCR * Direct sequencing

7 Screening of methylation gene in esophageal cancer Microarray genes 500 genes Upregulated > 3 folds with demethylation treatment 120 genes Expression profile: expressed in normal esophagus Background genes TSGs which evaded the previous method Methylated genes

8 Selected 19 genes from ESCC database Analyze CG density of the promoter

9 Results of promoter analysis gene No. Accession No. Gene Name CG content 1 AK hypothetical protein LOC U22376 c-myb NM_ aspartyl beta-hydroxylase W28545 chromosome 6 open reading frame 166, mrna ++ 5 X54936 placenta growth factor NM_ Histone (HISTIH2BJ) ++ 7 M60299 alpha 1 collagen type II ++ 8 J03133 SP U03270 centrin, EF-hand protein, AB ubiquitous tetratricopeptide containing protein RoXaN U66078 DAZLA L40385 integrin alpha 6 subunit AF connective tissue growth factor related protein WISP X81420 hhkb X16663 hematopoitic cell-specific Lyn substrate X74764 dicoidin domain receptor family, member AF nm23-h X72755 chemokine (C-X-C) ligand 9-22 M12625 lecithin-cholesterol acyltransferase + -, no methylation site; +, methylaton sites; ++, dense methylation; +++, very densemethylation.

10 19 genes from ESCC database Analysis of the CG density of the promoter 12 genes are CG dense Check the methylation level in ESCC cell lines, primary normal ES tissues and ESCC tissues

11 Methylation status of genes in ESCC cell lines and primary ES tissues Gene No. Accession E105N E106N E107N E108N KY30 KY E100T E101T E102T E103T E104T E108T E109T E110T 1 AK M U U J NM_ U W X NM_ AB L40385 Methylation Partial methylation Unmethylation

12 19 genes from ESCC database Analysis of the CG density of the promoter 12 genes are CG dense Check the methylation level in ESCC cell lines primary normal ES tissues and ESCC tissues 1 potential cancer-specific methylation gene Quantitative methylation (Taqman) analysis

13 Taqman analysis by using MSP primer and probe 5 primer probe 3 primer

14 Quantitative analysis (Taqman) in primary ESCC tissue ESCC PN TaqMeth /56 (61%) 0/17 (0%)

15 Clinicopathologic correlation with RALYL methylation in ESCC RALYL Taqman Methylation analysis Methylation Methylation P (+), n = 19 (-), n = 22 Age (median) 59 (40-76) 65 (50-70) Sex Male 15 (43%) 20 (57%) Female 4 (67%) 2 (33%) Location Upper 0 (0%) 2 (100%) Middle 12 (52%) 11 (48%) Lower 7 (44%) 9 (56%) NS NS Differentiation P = Well 7 (88%) 1 (12%) Moderately or poor 12 (36%) 21 (64%) Depth of invasion Submucosa 2 (100%) 0 (0%) Muscularis propria 2 (40%) 3 (60%) Adventitia 15 (44%) 19 (56%) Lymph node metastasis Absent 3 (60%) 2 (40%) Present 16 (44%) 20 (56%) Vascular penetration Absent 5 (56%) 4 (44%) Present 14 (44%) 18 (56%) TNM staging Stage I and II 3 (50%) 3 (50%) Stage III and IV 16 (46%) 19 (54%) NS NS NS NS NS: no significance.

16 mrna expression of RALYL in ESCC NC PC N T N T N T N T N T N T N T N T N T RALYL beta- ac8n

17 DISCUSSION v RALYL: RNA-binding protein Raly-like. v Its ORF encodes 291 a.a. which contains RNA binding domain. v RALYL gene located on chromosome 8q v Its mrna are ubiquitously expressed in human tissue with various degrees. v Its function may be related to RNA binding, but largely remains unknown.

18 Our study showed: v RALYL promoter was hypermethylated in 61% (34 out of 56) of ESCC tissues, whereas none was found in normal esophageal tissues (0 out of 17), suggesting RALYL promoter hypermethylation is a common and specific event in primary esophageal cancer. v RALYL mrna expression is downregulated or absent in ESCC and is closely associated with its promoter methylation status.

19 CONCLUSION v RALYL promoter methylation may serve as a potential molecular diagnostic marker in ESCC. v Downregulation of RALYL expression in ESCC through hypermthylation indicating it may play an important role on ESCC development.

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