Promoter Methylation of Genes in and around the candidate Lung Cancer Susceptibility

Locus 6q23-25

Cancer research 2008; MarchSpeaker: 徐君瑋Advisor: 徐明達

Copyright ©2007 American Cancer Society

From Jemal, A. et al. CA Cancer J Clin 2007;57:43-66.

Lung Cancer, the leading cause of cancer death among human

Some earliest changes detected in the bronchial epithelium of smokers include loss of heterozygosity (LOH ) at chromosome 3p21, 9p21, and 17p13. (J Natl Cancer Inst 1997)

SmokeTobaccoSmokeTobacco

Bronchi-al lesionBronchi-al lesion

Pneumonia

Pneumonia

Inflammatory reaction

Inflammatory reaction

Lung carcinoma

Lung carcinoma

Early genetic alteration

late genetic alteration

Allelic loss, inactivation of the remaining allele by promoter hypermethylation of RASSF1A and p16 and mutation of p53 gene is commonly seen in non-small cell lung cancer (NSCLC)

( Cancer Lett 2005)

Allelic loss, inactivation of the remaining allele by promoter hypermethylation of RASSF1A and p16 and mutation of p53 gene is commonly seen in non-small cell lung cancer (NSCLC)

( Cancer Lett 2005)

Lung tumors develop through mutation or epigenetic silencing via promoter hypermethylation of many genes

Lung tumor

Cell cycle control & Growth regulation

Adhesion

(Cancer J. 2002)

Methylation of the p16 gene is one of the earliest changes in lung cancer development, especially induced by carcinogens like tobacco and increasing in prevalence in adenocarcinoma and squamous cell carcinoma . (Nat Rev Cancer 2004 ; PNAS 1998; Cancer Res 2001 )

More identified LOH region frequently observed in adenocarcinomas from both smoker and never smoker is 6q

Genome alteration

LOH in 6q22-27 occurs in 30% to 55% of tumor. ( Cancer research ,2008 )

Deletions between 6q14 and 6q24 have been detected in 60% of primary lung

tumor. ( J. Pathology 2002)

Genome-wide linkage analysis of three family member with aerodigestive

cancer identified a lung cancer susceptibility locus at chromosome

6q23-25. (Am J Hum Genet 2004)

More identified LOH region frequently observed in adenocarcinomas from both smoker and never smoker is 6q

• Chromosome 6 is one of the gene-rich and CpG island –rich chromosomes that contains ~ 1557 genes and 1070 CpG islands. (Nat Genet 2006 ; Nature 2003)

• Estrogen receptor α in 6q25 have been shown by promoter hypermethylation in 20% and 36% of lung tumors repectively from smoker and never smokers. (Cancer research 1999)

• TCF21, within the 6q23-24 locus that normally expressed in lung airway epithelial cells but silenced in aerodigestive tumors. (PNAS 2006)

Purpose

To identify novel genes in and around the candidate lung cancer susceptibility locus 6q23-25 that are inactivated by

promoter hypermethylation and to compare their prevalence in adenocarcinomas from smokers and never smokers

array CGH analysis of genome copy number change

Lung cancer cell lines (H1435, H1568, H2009, Calu-6)

Genomic DNA isolation then fragmentation

Random prime labeled, and hybridize to oligonucleotide

microarray. (22.5K )

Fluorescence ratios of scanned images of the array were calculated as the average of two

paired arrays

Data analysis

Transcriptome array

6 adenocarcinoma cell lines

H23, H1568, and H1993

SmokersH2023, H2085, and H228

Never smokers

Treated with DAC and TSA alone

TSA : Tricostatin A, HDAC inhibitor DAC: 5-aza-2-deoxycytidine

RNA extraction and labeled with dye

Hybridized to oligoarray

TSA : Tricostatin A, HDAC inhibitor DAC: 5-aza-2-deoxycytidine

PLoS Genet, 2007

Green : Complete promoter methylation in WtYellow : 2-fold change or above in DKO cell

Gene sensitive to DAC but not TSA as new informatics filter to identify the majority of DNA hypermethylated genes in cancer

In silico analysis

Analysis performed using computers in conjunction with informatics capabilities

Chromosome 6

Single-copy deletion between 6p12.1 and 6q22.33

H1435

~ 70Mbp

array CGH analysis of genome copy number change

In H1568 and H2009 cell line showed no significant copy number alteration.

Calu-6 cell line show ~ 15.41 Mbp single deletion region between 6q22.1 and 6q12.2 and 6q24-27 . (data not shown)

To exam genes in these chromosome alter site and whether these genes harbor CpG island in their promoter regions

1. CpG island criteria of Takai and Jones. (PNAS, 2002)

2. Obs/Exp ≥ 0.61. CpG island criteria of Takai and Jones. (PNAS, 2002)

2. Obs/Exp ≥ 0.6

Calu-6 and H1435 cell lines

1. CpG island criteria of Takai and Jones. (PNAS, 2002)

2. ObsCpG/ExpCpG ≥ 0.651. CpG island criteria of Takai and Jones. (PNAS, 2002)

2. ObsCpG/ExpCpG ≥ 0.65

CpG island criteria formula

• GC content of 50% or greater

• length greater than 200 bp

• ratio greater than 0.6 of observed number of CG dinucleotides to the expected number on the basis of the number of Gs and Cs in the segment

• Obs/Exp CpG = Number of CpG * N / (Number of C * Number of G) where N = length of sequence.

References Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J. Mol. Biol. 1987 Jul 20;196(2):261-282.

ObsCpG/ExpCpG ≥ 0.65(PNAS 2005)

COBRA

• Combined bisulfite restriction analysis

TaqI :

BstUI :

methylated unmethylated

2. COBRA analysis revealed that 31 genes (72.1%) were devoid of promoter methylation in lung cancer cell lines

COBRA: Combined bisulfite modification and restriction analysis

PNLDC1 and PLAGL1 showed a high prevalence for methylation in the lung cancer cell lines also were methylated in 100% HBECs and PBMCs (Figure 3A)

PNLDC1 and PLAGL1 showed a high prevalence for methylation in the lung cancer cell lines also were methylated in 100% HBECs and PBMCs (Figure 3A)

12 lung tumor cell lines

12 genes remain: AIM1M, NR2E1, IL20RA, TCF21, SASH1, PLAGL1, LATS1,

SYNE1, AKAP12, PNLDC1, M ACAT2, DACT2.

HBECs: Human bronchial epithelia cells PBMCs: Perpheral blood mononuclear cells

BstUI :

PLAG1 DMR showed partial digestion

PNLDC1 promoter showed complete methylation.

Breast mammary gland carcinoma cell line

Breast mammary gland carcinoma cell line

COBRA

DACT2 and NR2E1 genes show 100% PBMCs but no in normal HBECs

Five of remaining eight genes methylated at prevalence of > 20% in cancer cell lines

3. Further analysis in primary adenocarcinoma

AIM1M, NR2E1, IL20RA, TCF21, SASH1, LATS1, SYNE1, AKAP12,

Hypermethylation of 6q genes in primary lung adenocarcinoma

Non of the genes showed significant difference in prevalence of methylation between tumors from smoker versus never smokers and no age variation

4. Promoter region of SYNE1, AKAP12, IL20RA was characterized further by bisulfite sequencing.

4. Promoter region of SYNE1, AKAP12, IL20RA was characterized further by bisulfite sequencing.

Bisulfite sequence

Promoter region in these three genes showed no methylation in normal PBMCs and HBECs whereas methylated in primary adenocarcinoma cell.

80%

68%

43%

ADC: primary adenocarcinoma cell

DAC treatment restores expression of silence genes

In Calu-6 cell, expression of five SYNE1-A, SYNE1-B, AKAP12-A, AKAP12-B, and IL20RA transcript was restored to normal level after DAC treatment.

Treatment with TSA did not affect the expression of these genes

ControlRT-PCR

Survival and the association of methylation between genes

• No significant association was seen between survival and promoter hypermethylation for any of the five genes individually or in combination.

• The association of methylation of the five genes was assessed in tumors independent of smoking status.

• A positive association was found in early-stage adenocarcinomas between methylation of SYNE1 & TCF21 (P= 0.02) 88%, SYNE1 & AKAP12 (P < 0.001) 74%

AKAP12 & IL20RA (P=0.045) SYNE1 & the other four genes (P< 0.001)

AKAP12 & the other four genes (P< 0.002)

Survival and the association of methylation between genes

Summary• Genetic, epigenetic, gene expression, and in silico screening approaches were used to

select 43 genes located in 6q12-27 for characterization of methylation status.

• 12 genes were methylated in at least one lung cancer cell lines, and methylation of 8 genes was specific to lung cancer cell lines.

• 5 of 8 genes with the highest prevalence for methylation in cell lines.(TCF21, SYNE1, AKAP12, ILRA20, and ACAT2)

• No significant association was seen between survival and promoter hypermethylation for any of the five genes individually or in combination.

• The association of methylation of the five genes was assessed in tumors independent of smoking status.

• These five genes in and around the 6q23-25 region show similar methylation in lung cancer adenocarcinoma.

SYNE1

• SYNE1 gene span 0.5 Mbs of genomic DNA, comprising 147 exons and encoding four different transcripts through alternative splicing. (26)

• It is a multifunctional gene involved in cytokinesis nuclear organization and the structural integrity, and function of the Golgi apparatus.(27, 28)

AKAP12/Gravin

• AKAP12 is one of the A-kinas anchoring proteins that regulates mitogenesis by anchoring key signaling protein (PKA and PKC). And modulating the expression of genes involved in cell cycle and apoptosis. (30)

• It suppresses tumor cell viability and growth by inducing apoptosis via caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 expression.(30)

• AKAP12 induces cell cycle arrest by increasing the expression of cell cycle checkpoint proteins p21 and p27 and reducing the cyclin D1 expression.

• AKAP12 shown methylated in many kinds of tumor support that this gene represents another common target for silencing in cancer.

Il20RA

• Il20RA encodes a receptor for interleukin IL-20 and IL-24(MDA-7). IL-24 induces G2-M cell cycle arrest and apoptotic cell death through up-regulation of proapoptotic proteins (Bax and Bak) and down-regulation of antiapoptotic proteins (Bcl-2 and Bcl-xL). (35,36)

• Silencing of IL20RA by promoter methylation represents another survival strategy of lung cancer cells by blocking cell cycle arrest and apoptotic signals from IL-23.

• IL20RA is significantly associated with methylation of AKAP12.

Nat Rev Cancer. 2004 Sep;4(9):707-17.

Comprehensive analysis of CpG islands in human chromosome 21 and 22 (PNAS 2005)

• ObsCpG/ExpCpG ≥ 0.65

• Regions of DNA of greater than 500 bp with a G+C equal to or greater than 55% and observed CpG / expected CpG of 0.65 were more likely to be associated with the 5’ regions of genes and this definition exclude most Alu-repetitive elements.