Donna M. Klinge

Gene Promoter Methylation in Plasma and Sputum Increases with Lung Cancer Risk

Purpose: Lung cancer is the leading cause of cancer mortality in the United States, due in part to the lack of a validated and effective screening approach for early detection. The prevalence for methylation of seven and three genes was examined in DNA from sputum and plasma, respectively, from women at different risk for lung cancer. Experimental Design: Lung cancer survivors (n = 56), clinically cancer-free smokers (n = 121), and never smokers (n = 74) comprised the study population. Plasma was collected from all three groups, whereas sputum was collected from lung cancer survivors and smokers. Results: Methylation was detected in plasma DNA from 10 of 74 women who never smoked. Prevalence for methylation of the p16 gene in plasma was highest in lung cancer survivors. Lung cancer survivors showed a significant increase in the odds of having at least one or more genes methylated in plasma (odds ratio, 3.6; 95% confidence interval, 1.9-9.1) than never smokers. The prevalence for methylation of the O6-methylguanine-DNA methyltransferase, ras effector homologue 1, death associated protein kinase, and PAX5α genes in sputum was significantly higher in lung cancer survivors compared with smokers. Lung cancer survivors had 6.2-fold greater odds (95% confidence interval, 2.1-18.5) for methylation of three or more genes in sputum compared with smokers. Methylation was more commonly detected in sputum than plasma for O6-methylguanine-DNA methyltransferase and ras effector homologue 1, but not p16, in lung cancer survivors. Conclusion: Concomitant methylation of multiple gene promoters in sputum is strongly associated with lung cancer risk.

SULF2 methylation is prognostic for lung cancer survival and increases sensitivity to topoisomerase-I inhibitors via induction of ISG15

The heparan sulfate 6-O-endosulfatase (SULF2) promotes growth and metastasis of solid tumors. We recently identified that cytosine methylation of the SULF2 promoter is associated with better survival of resected lung adenocarcinoma patients, and now also demonstrates a marginal improvement in survival of advanced non-small cell lung cancer (NSCLC) patients receiving standard chemotherapy (hazard ratio=0.63, P=0.07). Subsequent studies focused on investigating the effect of methylation on SULF2 expression and its genome-wide impact. The genes and pathways modulated by epigenetic inactivation of SULF2 and the effects on sensitivity to chemotherapy were characterized in vitro and in vivo. Silencing SULF2 through small interfering RNA or methylation primarily increased expression of interferon-inducible genes including ISG15, a marker for increased sensitivity to topoisomerase-1 inhibitors such as camptothecin (CPT). NSCLC cell lines with methylated SULF2 (SULF2M) express 60-fold higher ISG15 compared with SULF2 unmethylated (SULF2U) NSCLC cell lines and normal human bronchial epithelial cells. In vitro, SULF2M and high ISG15 (ISG15H)-expressing NSCLC cell lines were 134-fold more sensitive to CPT than SULF2U and low ISG15 (ISG15L)-expressing cell lines. Topotecan, a soluble analog of CPT and FDA-approved anticancer drug, dramatically arrested the growth of SULF2M-ISG15H, but not SULF2U-ISG15L lung tumors in nude mice (P<0.002). Similarly, high ISG15 expression that is comparable to the topotecan (TPT)-sensitive NSCLC cell lines was found in tumors from 25% of NSCLC patients compared with normal lung, indicating a potential to identify and target the most sensitive NSCLC subpopulation for personalized TPT therapy.

Rosiglitazone prevents the progression of preinvasive lung cancer in a murine model

There is a critical need to identify efficacious chemopreventive agents for lung cancer that can be taken chronically with no side effects and whose mechanisms of action do not involve genotoxicity that could drive, rather than impede, cancer progression. We evaluated the ability of a chemopreventive cocktail that included selenium (antioxidant), rosiglitazone (peroxisome proliferator-activated receptor gamma agonist), sodium phenylbutyrate or valproic acid (histone deacetylase inhibitors) and hydralazine (cytosine-demethylating agent) to prevent the progression of lung cancer in A/J mice treated with NNK. Agents were administered alone or in various combinations. Effects of the chemopreventive agents were quantified based on the proportion of hyperplasias and adenomas within the mouse lung. Significant effects on tumor progression were seen in all treatment groups that included rosiglitazone as reflected by a 47-57% increase in number of hyperplasias and a 10-30% decrease in adenomas. Cell proliferation was also reduced in these treatment groups by approximately 40%. Interestingly, while treatment with rosiglitazone alone did not significantly affect lesion size, striking effects were seen in the combination therapy group that included sodium phenylbutyrate, with the volume of hyperplasias and adenomas decreasing by 40 and 77%, respectively. These studies demonstrate for the first time that chronic in vivo administration of rosiglitazone, used in the management of diabetes mellitus, can significantly block the progression of premalignant lung cancer in the A/J mouse model.

Radiation-Induced Lung Adenocarcinoma is Associated with Increased Frequency of Genes Inactivated by Promoter Hypermethylation

Abstract Lyon, C. M., Klinge, D. M., Liechty, K. C., Gentry, F. D., March, T. H., Kang, T., Gilliland, F. D., Adamova, G., Rusinova, G., Telnov, V. and Belinsky, S. A. Radiation-Induced Lung Adenocarcinoma is Associated with Increased Frequency of Genes Inactivated by Promoter Hypermethylation. Radiat. Res. 168, 409–414 (2007). Epigenetic inactivation of genes by promoter hypermethylation, a major mechanism in the initiation and progression of tobacco-induced cancer, has also been associated with lung cancer induced through environmental and occupational exposures. Our previous study of gene methylation in workers from the MAYAK nuclear enterprise identified a significantly higher prevalence for methylation of the p16 gene (CDKN2A) in adenocarcinomas from workers compared to tumors from non-worker controls. The purpose of this investigation was to determine whether genes in addition to p16 are “targeted” for silencing and whether overall gene methylation was more common in radiation-induced adenocarcinoma. A significant increase in the prevalence of methylation of GATA5 was seen in tumors from workers compared to tumors from controls. The prevalence for methylation of PAX5 β and H-cadherin did not differ in tumors from workers and controls. Evaluating the frequency for methylation of a five-gene panel revealed that 93% of adenocarcinomas from workers compared to 66% of tumors from controls were methylated for at least one gene. Moreover, a twofold increase was seen in the number of tumors methylated for three or more genes for tumors from workers compared to controls. Increased frequency for inactivation of genes by promoter hypermethylation and targeting of tumor suppressor genes such as GATA5 may be factors that contribute to the increased risk for lung cancer associated with radiation exposure.

Global identification of genes targeted by DNMT3b for epigenetic silencing in lung cancer

The maintenance cytosine DNA methyltransferase DNMT1 and de novo methyltransferase DNMT3b cooperate to establish aberrant DNA methylation and chromatin complexes to repress gene transcription during cancer development. The expression of DNMT3b was constitutively increased 5–20-fold in hTERT/CDK4-immortalized human bronchial epithelial cells (HBECs) before treatment with low doses of tobacco carcinogens. Overexpression of DNMT3b increased and accelerated carcinogen-induced transformation. Genome-wide profiling of transformed HBECs identified 143 DNMT3b-target genes, many of which were transcriptionally regulated by the polycomb repressive complex 2 (PRC2) complex and silenced through aberrant methylation in non-small-cell lung cancer cell lines. Two genes studied in detail, MAL and OLIG2, were silenced during transformation, initially through enrichment for H3K27me3 and H3K9me2, commonly methylated in lung cancer, and exert tumor suppressor effects in vivo through modulating cancer-related pathways. Re-expression of MAL and OLIG2 to physiological levels dramatically reduced the growth of lung tumor xenografts. Our results identify a key role for DNMT3b in the earliest stages of initiation and provide a comprehensive catalog of genes targeted for silencing by this methyltransferase in non-small-cell lung cancer.

Gene methylation biomarkers in sputum as a classifier for lung cancer risk

CT screening for lung cancer reduces mortality, but will cost Medicare ~2 billion dollars due in part to high false positive rates. Molecular biomarkers could augment current risk stratification used to select smokers for screening. Gene methylation in sputum reflects lung field cancerization that remains in lung cancer patients post-resection. This population was used in conjunction with cancer-free smokers to evaluate classification accuracy of a validated eight-gene methylation panel in sputum for cancer risk. Sputum from resected lung cancer patients (n=487) and smokers from Lovelace (n=1380) and PLuSS (n=718) cohorts was studied for methylation of an 8-gene panel. Area under a receiver operating characteristic curve was calculated to assess the prediction performance in logistic regressions with different sets of variables. The prevalence for methylation of all genes was significantly increased in the ECOG-ACRIN patients compared to cancer-free smokers as evident by elevated odds ratios that ranged from 1.6 to 8.9. The gene methylation panel showed lung cancer prediction accuracy of 82-86% and with addition of clinical variables improved to 87-90%. With sensitivity at 95%, specificity increased from 25% to 54% comparing clinical variables alone to their inclusion with methylation. The addition of methylation biomarkers to clinical variables would reduce false positive screens by ruling out one-third of smokers eligible for CT screening and could increase cancer detection rates through expanding risk assessment criteria.CT screening for lung cancer reduces mortality, but will cost Medicare ∼2 billion dollars due in part to high false positive rates. Molecular biomarkers could augment current risk stratification used to select smokers for screening. Gene methylation in sputum reflects lung field cancerization that remains in lung cancer patients post-resection. This population was used in conjunction with cancer-free smokers to evaluate classification accuracy of a validated eight-gene methylation panel in sputum for cancer risk. Sputum from resected lung cancer patients (n=487) and smokers from Lovelace (n=1380) and PLuSS (n=718) cohorts was studied for methylation of an 8-gene panel. Area under a receiver operating characteristic curve was calculated to assess the prediction performance in logistic regressions with different sets of variables. The prevalence for methylation of all genes was significantly increased in the ECOG-ACRIN patients compared to cancer-free smokers as evident by elevated odds ratios that ranged from 1.6 to 8.9. The gene methylation panel showed lung cancer prediction accuracy of 82–86% and with addition of clinical variables improved to 87–90%. With sensitivity at 95%, specificity increased from 25% to 54% comparing clinical variables alone to their inclusion with methylation. The addition of methylation biomarkers to clinical variables would reduce false positive screens by ruling out one-third of smokers eligible for CT screening and could increase cancer detection rates through expanding risk assessment criteria.

Abstract 87: SULF2 methylation is a prognostic biomarker for lung cancer survival and increases sensitivity to camptothecin analogues via expression of ISG15

Heparan sulfate proteoglycans (HSPGs) serve as co-receptors for numerous signaling molecules including growth factors and cytokines. The sulfation pattern on the heparan sulfate (HS) motif is central to the interaction between HSPGs and the myriads of molecules including interferon (INF). SULF2 encodes for HS 6-O-endosulfatase enzyme that removes the 6-O-sulfate moieties from HS and regulates the specificity of this interaction. Increased expression of SULF2 promotes angiogenic and oncogenic signaling. In hepatocellular carcinoma higher expression of SULF2 is associated with worse prognosis after resection. Conversely, we recently reported that methylation-mediated SULF2 silencing is associated better survival of resected lung cancer patients. Here we show that SULF2 methylation is also a favorable survival marker in unresectable lung cancer. Furthermore, epigenetic silencing of SULF2 suppresses cell migration and induces the expression of multiple INF-inducible genes. Knocking down 80% of SULF2 expression in Calu-3 cells using siRNA resulted in 7-fold increase in the expression of INF-inducible regulator of ubiquitination, ISG15. Similarly, lung cancer cell lines with methylated SULF2 (SULF2M) on average express 60-fold higher ISG15 than cell lines with unmethylated SULF2 (SULF2U). ISG15 is a key marker for increased sensitivity of cancer cells to topoisomerase-1 inhibitors such as camptothecin and topotecan. In vitro treatment of lung cancer cell lines with camptothecin revealed a 65-fold higher sensitivity in cell lines with SULF2M and ISG15 high (ISG15H) phenotype compared to SULF2U and ISG15 low (ISG15L) cell lines. In nude mice, 10mg/kg/week topotecan treatment dramatically arrested the growth of SULF2M and ISG15H lung cancer xenografts but had no effect on SULF2U and ISG15L xenografts. Heterogeneity for ISG15 expression was also seen in primary tumors. One third of primary lung tumors have ISG15H phenotype with an average of 5-fold higher expression than the remaining 2/3 rd tumors in which the ISG15 level is comparable to normal lung. Taken together these results indicate SULF2 methylation is not only a favorable prognostic marker for lung cancer, but through its association with increased expression of ISG15 could indicate a higher sensitivity to topoisomerase-1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 87. doi:10.1158/1538-7445.AM2011-87