CpG Island Methylator Phenotype Involving Chromosome 3p Confers an Increased Risk of Non-Small Cell Lung Cancer Hong-Tao Zhang, Ph.D Soochow University Laboratory of Cancer Molecular Genetics Medical College of Soochow University May 21~23, 2010

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

In China, lung cancer is the fourth leading cause of cancer death in males and the fifth in females in However, it became the first leading cause of cancer death both in males and females in Authorized Reports from the 10th Annual Meeting of Chinese Society of Clinical Oncology (CSCO) Sep Harbin, China.

Yang L, et al. Estimation and projection of the national profile of cancer mortality in China: Br J Cancer 2004, 90:

Adenocarcinoma Squamous cell carcinoma Squamous cell carcinoma Large cell carcinoma Lung Cancer Small cell lung cancer Non-small cell lung cancer Pathology

Expression model Identical genotypes Different phenotypes Epigenetics: Changes in gene expression that are mitotically and/or meiotically heritable and do not involve a change in the DNA sequence.

DNA methylation Histone modification Chromatin remodeling Inactivation of X chromosome MicroRNA EpigeneticsEpigenetics

DNA methylation alterations are now widely recognized as a contributing factor in human tumorigenesis.

Baylin SB. Nat Clin Pract Oncol (2005) 2: S4-11. Review

Loss of heterozygosity involving several chromosome 3p regions accompanied by chromosome 3p deletions are detected in more than 90% NSCLCs. Zabarovsky ER, et al. Oncogene, 2002, 21:

However, CpG island methylator phenotype (CIMP) involving methylation abnormalities of TSGs on chromosome 3p has not been so far epigenetically elucidated. in NSCLCs. Liu Z, et al. Lung Cancer, 2008, 62:

FHIT: 表达产物具有调控细胞周期，诱导细胞凋亡、降 解等功能。 BLU: 参与调控细胞增生周期。 RASFF1A: Ras 信号转导通路上的一个效应器，涉及细 胞生长调节和凋亡通路。 SEMA3B: 在神经系统形成、胚胎发生、血管生成、免疫 反应和肿瘤发生、发展中发挥着重要作用。 hMLH1: 主要作用在于参与 DNA 复制后错配基因的修复。 RAR-b: 通过与视黄酸结合调控细胞生长。 VHL: 负性调节低氧诱导的如血管内皮生长因子 (VEGF) mRNA 表达，以此调控新生血管的生成。 hOGG1: 表达产物具有 DNA 糖苷酶和 AP 裂解酶活性，可 特异切除修复 8-oxoG 等。在 DNA 氧化损伤中， 8-oxoG 形成频率最高，致突变能力最强，是肿瘤发生和发展的 重要因素。 3p 3q

Primer sequences for methylation-specific PCR(MSP) analysis

Representative patterns of methylated (M) and unmethylated (U) alleles in MSP analysis for five NSCLC cell lines

CIMP in four NSCLC cell lines before and after treatment of demethylation agent 5'-aza-CdR Symbols – and + below 5-aza-CdR represent without and with treatment, respectively. Black boxes, presence of unmethylation and methylation; white boxes, presence of unmethylation. Symbols – and + below CIMP represent CIMP negative and positive, respectively.

MTT analysis Cell proliferation analysis for NSCLC cell lines without and with treatment of demethylation agent 5- aza-CdR. The cells were grown to 80% confluency when they were harvested on day 7. The Y-axis represents the absorbance of 550 nm wavelength of MTT assay. Symbols – and + below 5-aza-CdR represent without and with treatment, respectively. On day 7, significant difference in cell proliferation was observed between NSCLC cell lines (95-D and LTEP- a-2) without and with 5- aza-CdR treatment. *, P<0.05.

CIMP in 60 NSCLC tissues and paired paracancerous lung tissues AB

Frequency distributions of methylation per gene in 60 NSCLC and paired paracancerous lung tissues. Significantly methylated difference of hOGG1,RAR-B, RASSF1A, BLU and FHIT was observed between NSCLCs and paired normal tissues. *, P<0.05.

AB

Kaplan-Meier survival curves for 30 NSCLC patients who were classified into CIMP+ and CIMP-. A significant difference for survival rate was observed between CIMP+ and CIMP- using Log rank test (P=0.0166).

Representative patterns of methylated (M) and unmethylated (U) alleles in MSP analysis for six genes in peripheral blood mononuclear cell (PBMC) from patients with NSCLC. Two samples (2CB and 48CB) with CIMP+ have methylation of at least three genes. Two samples (4CB and 28CB) with CIMP- present methylation of less than three genes. MSP analysis of SPC-A-1 and A549 NSCLC cell lines served as a positive control (Pos) for hOGG1, RAR-B, SEMA3B, BLU and FHIT; and for RASSF1A, respectively. Distilled water and unbisulfited DNA were used as negative controls (Neg 1 and Neg 2, respectively).

Summary of methylation for hOGG1, RAR-B, SEMA3B, RASSF1A, BLU and FHIT in PBMC from 80 patients with NSCLC (A) and 80 controls (B). Green boxes, presence of unmethylation; yellow boxes, presence of unmethylation and methylation. CB, cancer blood sample; NB, normal blood sample; +, CIMP positive; -, CIMP negative.

Frequency of methylation for six genes in PBMC from 80 patients with NSCLC and 80 controls. Significantly methylated difference of hOGG1, SEMA3B and RASSF1A was observed between NSCLCs and controls (*, P=0.02, P<0.001 and P=0.001, respectively).

Liu Z, et al. Journal of Thoracic Oncology, 2010, in press.

 The present study shed light on the presence of chromosome 3p- specific CIMP, which might play an important role in tumorigenesis of NSCLC.  Insufficient evidence for an association of CIMP status with survival prognosis of NSCLC warranted us to perform a further confirmation.  Plus, this study suggests that PBMC DNA hypermethylation of TSGs, including hOGG1 and RASSF1A, and 3pCIMP could provide useful predictor for NSCLC. CONCLUSIONS

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