Amanda M. Bernauer

EMT and stem cell-like properties associated with miR-205 and miR-200 epigenetic silencing are early manifestations during carcinogen-induced transformation of human lung epithelial cells

Epithelial-to-mesenchymal transition (EMT) is strongly associated with cancer progression, but its potential role during premalignant development has not been studied. Here, we show that a 4-week exposure of immortalized human bronchial epithelial cells (HBEC) to tobacco carcinogens can induce a persistent, irreversible, and multifaceted dedifferentiation program marked by EMT and the emergence of stem cell-like properties. EMT induction was epigenetically driven, initially by chromatin remodeling through H3K27me3 enrichment and later by ensuing DNA methylation to sustain silencing of tumor-suppressive microRNAs (miRNA), miR-200b, miR-200c, and miR-205, which were implicated in the dedifferentiation program in HBECs and also in primary lung tumors. Carcinogen-treated HBECs acquired stem cell-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44(high)/CD24(low), CD133, and ALDH1 stem cell-like markers. miRNA overexpression studies indicated that regulation of the EMT, stem-like, and transformed phenotypes in HBECs were distinct events. Our findings extend present concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can participate in cancer initiation to promote the clonal expansion of premalignant lung epithelial cells.

Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation.

Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could affect strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break (DSB) repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of DSB repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as three or more of eight genes methylated in sputum, compared with smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-PKc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with seven or more risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ataxia-telangiectasia mutated was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify DSB DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, which is, in turn, associated with elevated risk for lung cancer.

The A/G Allele of Rs16906252 Predicts for MGMT Methylation and Is Selectively Silenced in Premalignant Lesions from Smokers and in Lung Adenocarcinomas

Purpose: To address the association between sequence variants within the MGMT (O6-methylguanine-DNA methyltransferase) promoter–enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy. Experimental Design: Single nucleotide polymorphisms (SNP) identified through sequencing a 1.9 kb fragment 5′ of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1,731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide (TMZ) was assessed. Results: The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20 to 41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to TMZ was strongly correlated with levels of MGMT methylation and expression. Conclusions: These studies provide strong evidence that the A allele of a MGMT promoter–enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, TMZ treatment may benefit a subset of lung cancer patients methylated for MGMT. Clin Cancer Res; 17(7); 2014–23. ©2011 AACR.

HIF1α regulated expression of XPA contributes to cisplatin resistance in lung cancer

Factors regulating nucleotide excision repair probably contribute to the heterogenous response of advanced stage lung cancer patients to drugs such as cisplatin. Studies to identify the genes in the nucleotide excision repair pathway most closely associated with resistance to cisplatin have not been conclusive. We hypothesized that Xeroderma pigmentosum complementation group A (XPA), because of its dual role in sensing and recruiting other DNA repair proteins to the damaged template, would be critical in defining sensitivity to cisplatin. Studies were conducted to identify factors regulating transcription of XPA, to assess its role in modulating sensitivity to cisplatin and its expression in primary lung tumors. Hypoxia-inducible factor 1 alpha (HIF1α) subunit was found to bind with strong affinity to a hypoxia response element sequence in the promoter of XPA. Modulating expression of HIF1α by small interfering RNA or cobalt chloride markedly reduced or increased transcription of XPA in lung cancer cell lines, respectively. Protein levels of XPA were strongly correlated with sensitivity to cisplatin (r = 0.88; P < 0.001) in cell lines and sensitivity could be increased by small interfering RNA depletion of XPA. Expression of XPA determined in 54 primary lung tumors was elevated on average 5.2-fold when compared with normal bronchial epithelial cells and correlated with levels of HIF1α (r = 0.58; P < 0.01). Together, these studies identify XPA as a novel target for regulation by HIF1α whose modulation could impact lung cancer therapy.

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.

Haplotypes of DNMT1 and DNMT3B are associated with mutagen sensitivity induced by benzo[a]pyrene diol epoxide among smokers

The mutagen sensitivity assay is an in vitro measure of DNA repair capacity used to evaluate intrinsic susceptibility for cancer. The high heritability of mutagen sensitivity to different mutagens validates the use of this phenotype to predict cancer susceptibility. However, genetic determinants of mutagen sensitivity have not been fully characterized. Recently, several studies found that three major cytosine DNA methyltransferases (DNMTs), especially DNMT1, have a direct role in the DNA damage response, independent of their methyltransferase activity. This study evaluated the hypothesis that sequence variants in DNMT1, DNMT3A and DNMT3B are associated with mutagen sensitivity induced by the tobacco carcinogen benzo[a]pyrene diol epoxide (BPDE) in 278 cancer-free smokers. Single-nucleotide polymorphisms (n = 134) dispersed over the entire gene and regulatory regions of these DNMTs were genotyped by the Illumina Golden Gate Assay. DNA sequence variation in the DNMT1 and DNMT3B loci was globally associated with breaks per cell (P < 0.04 for both). No global association between DNMT3A and breaks per cell was seen (P = 0.09). Two haplotypes in block1 of DNMT1 (H284) and 3B (H70) were associated with 16 and 24% increase in breaks per cell, respectively. Subjects with three or four adverse haplotypes of both DNMT1 and 3B had a 50% elevation in mean level of breaks per cell compared with persons without adverse alleles (P = 0.004). The association between sequence variants of DNMT1 and 3B and mutagen sensitivity induced by BPDE supports the involvement of these DNMTs in protecting the cell from DNA damage.

Genetic variation in SIRT1 affects susceptibility of lung squamous cell carcinomas in former uranium miners from the Colorado plateau

Epidemiological studies of underground miners suggested that occupational exposure to radon causes lung cancer with squamous cell carcinoma (SCC) as the predominant histological type. However, the genetic determinants for susceptibility of radon-induced SCC in miners are unclear. Double-strand breaks induced by radioactive radon daughters are repaired primarily by non-homologous end joining (NHEJ) that is accompanied by the dynamic changes in surrounding chromatin, including nucleosome repositioning and histone modifications. Thus, a molecular epidemiological study was conducted to assess whether genetic variation in 16 genes involved in NHEJ and related histone modification affected susceptibility for SCC in radon-exposed former miners (267 SCC cases and 383 controls) from the Colorado plateau. A global association between genetic variation in the haplotype block where SIRT1 resides and the risk for SCC in miners (P = 0.003) was identified. Haplotype alleles tagged by the A allele of SIRT1 rs7097008 were associated with increased risk for SCC (odds ratio = 1.69, P = 8.2 × 10(-5)) and greater survival in SCC cases (hazard ratio = 0.79, P = 0.03) in miners. Functional validation of rs7097008 demonstrated that the A allele was associated with reduced gene expression in bronchial epithelial cells and compromised DNA repair capacity in peripheral lymphocytes. Together, these findings substantiate genetic variation in SIRT1 as a risk modifier for developing SCC in miners and suggest that SIRT1 may also play a tumor suppressor role in radon-induced cancer in miners.

Association between common genetic variation in Cockayne syndrome A and B genes and nucleotide excision repair capacity among smokers.

Mutagen sensitivity in in vitro cultured lymphocytes challenged by benzo[a]pyrene diolepoxide (BPDE) has been validated as an intrinsic susceptibility factor for several cancers. Bulky BPDE-DNA adducts are repaired via either transcription-coupled repair or global genome nucleotide excision repair depending on the location of lesions. Cockayne syndrome A (CSA) and B (CSB) play essential roles in integrating the recognition of damage, chromatin remodeling, and the core nucleotide excision repair proteins. This study evaluated the hypothesis that common genetic variation in CSA and CSB is associated with mutagen sensitivity induced by BPDE in 276 cancer-free smokers. Tag single nucleotide polymorphisms (SNP; n = 37) selected across the entire coding and putative regulatory regions of CSA and CSB based on a high-density SNP database were genotyped by the Illumina Golden Gate assay. Major principal components of CSA and CSB that captured the linkage disequilibrium from multiple SNPs were globally associated with the number of breaks per cell at the threshold of 80% (P ≤ 0.02 for both genes). Haplotype H125 in CSA and H97 in CSB as well as SNPs in high linkage disequilibrium with these two haplotypes were significantly associated with a 13% to 15% reduction in the mean number of chromatid breaks per cell (P < 0.05). A resampling-based omnibus test supported the significant association between SNPs and haplotypes in CSA and mutagen sensitivity induced by BPDE (P = 0.035). This study implicates transcription-coupled repair in protecting the cell from BPDE-induced DNA damage. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2062–9)

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

Abstract LB-358: Carcinogen-induced epigenetic silencing of miR-205 and miR-200 family promotes EMT and transformation of lung epithelial cells

An in vitro carcinogen-induced transformation model was developed using immortalized bronchial epithelial cells (HBECs) to identify key molecular changes in preneoplastic lung epithelial cells that occur during the development of lung cancer in smokers. HBECs were treated for twelve weeks (one exposure per week) with low-doses of tobacco carcinogens (MNU and BPDE) that resulted in transformation from genotoxic stress (DNA damage). The HBECs displayed a morphological change consistent with an epithelial-to-mesenchymal transition (EMT) after 4 weeks of carcinogen treatment that persisted in colonies that were recovered and expanded from soft agar (transformed cell line). Expression of mesenchymal markers vimentin, fibronectin, and N-cadherin increased significantly, while the epithelial marker E-cadherin was significantly decreased. The most notable changes associated with EMT included 4 - 55 fold reduced expression of miR-200b, -200c, and -205 that correlated with 2-1500 fold increased expression of E-box binding transcription factors (SNAI1, ZEB1, ZEB2) and basic helix-loop-helix transcription factor (TWIST1). Restoring the expression of miR-200b, -200c or -205 in carcinogen transformed cells prevented growth in soft agar; meanwhile the cells maintained a fibroblast-like mesenchymal appearance. Levels of DNA methyltransferase 1 (DNMT1) protein also increased significantly during carcinogen treatment and stable knockdown of DNMT1, but not DNMT3A and DNMT3B prior to carcinogen prevented EMT and transformation. ChIP for H3K4me2 (indicative of active/open chromatin) revealed a progressive decrease of this mark at the promoters of miR-200b, -200c, and -205 over 12 weeks of carcinogen treatment and during transformation. A modest enrichment for H3K9me2 (inactive/closed chromatin mark) was observed at all time points. In contrast, H3K27me3 (indicative of inactive/closed chromatin) was enriched at the miR promoters after 4 weeks of carcinogen treatment and enrichment declined over time. Bisulfite sequencing revealed CpG DNA methylation increased from 3% to 42% in the miR-200 family and miR-205 promoters during transformation. To examine the clinical relevance of miR-200b, -200c, and -205 their expression was analyzed in lung tumors relative to distant normal lung tissue from 24 patients. Reduced expression of at least one miR was observed in 50% of lung adenocarcinomas. These findings implicate a major role for epigenetic regulation of EMT that extends beyond that of cancer metastasis to causality for development of premalignant lung cancer. Supported by ES008801. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-358.