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1.  Genetic variation in SIRT1 affects susceptibility of lung squamous cell carcinomas in former uranium miners from the Colorado plateau 
Carcinogenesis  2013;34(5):1044-1050.
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.
doi:10.1093/carcin/bgt024
PMCID: PMC3643420  PMID: 23354305
2.  HIF1α regulated expression of XPA contributes to cisplatin resistance in lung cancer 
Carcinogenesis  2012;33(6):1187-1192.
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.
doi:10.1093/carcin/bgs142
PMCID: PMC3388491  PMID: 22467238
3.  SULF2 Methylation is Prognostic for Lung Cancer Survival and Increases Sensitivity to Topoisomerase-I inhibitors via Induction of ISG15 
Oncogene  2011;31(37):4107-4116.
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 demonstrate a marginal improvement in survival of advanced non-small cell lung cancer (NSCLC) patients receiving standard chemotherapy (HR = 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 siRNA or methylation primarily increased expression of interferon-inducible genes including ISG15, a marker for increased sensitivity to topoisomerase-1 inhibitors such as camptothecin. NSCLC cell lines with methylated SULF2 (SULF2M) express 60-fold higher ISG15 compared to 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 camptothecin than SULF2U and low ISG15 (ISG15L) expressing cell lines. Topotecan, a soluble analogue of camptothecin and FDA approved anti-cancer 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 sensitive NSCLC cell lines was found in tumors from 25% of NSCLC patients compared to normal lung indicating a potential to identify and target the most sensitive NSCLC subpopulation for personalized topotecan therapy.
doi:10.1038/onc.2011.577
PMCID: PMC3307938  PMID: 22158045
NSCLC; Camptothecin; SULF-2; Oncogene; Topotecan
4.  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 
Cancer research  2011;71(8):3087-3097.
Epithelial 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 four-week exposure of immortalized human bronchial epithelial cells (HBECs) 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 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-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44high/CD24low, 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.
doi:10.1158/0008-5472.CAN-10-3035
PMCID: PMC3078195  PMID: 21363915
methylation; miRNA; EMT; stem cells; transformation
5.  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 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
SNPs identified through sequencing a 1.9kb fragment 5' of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1731 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 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 20–41% 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 temozolamide 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, temozolamide treatment may benefit a subset of lung cancer patients methylated for MGMT.
doi:10.1158/1078-0432.CCR-10-3026
PMCID: PMC3070839  PMID: 21355081
MGMT; allele specific methylation; single nucleotide polymorphism; sputum; lung cancer
6.  Discovery of common SNPs in the miR-205/200 family-regulated epithelial to mesenchymal transition pathway and their association with risk for non-small cell lung cancer 
The activation of the epithelial-to-mesenchymal transition (EMT) program is an important step for tumor initiation, invasion, and metastasis in solid tumors, including lung cancer. The purpose of this study was to identify the sequence variants in the miR-205/200 family-regulated EMT pathway and test their association with risk for lung cancer. Fifty samples were resequenced to identify sequence variants in the miR-205/200 family-regulated EMT pathway. The association between tagSNPs and risk for non-small cell lung cancer was discovered and validated in New Mexico (386 cases and 514 controls) and Massachusetts (2453 cases and 1555 controls) case-control studies, respectively. The function of SNPs on miR-200b-a-429 promoter activity was tested using luciferase reporter and expression assays. Forty-one sequence variants with minor allele frequency ≥ 0.03 were identified, and 16 variants were selected as tagSNPs. Genetic association analysis identified that the G allele of rs61768479 was associated with a 50% reduced risk for lung cancer (OR=0.50, 95%CI=0.30-0.85, uncorr-P=0.01); however, this association was not validated (OR=0.90, 95%CI=0.72-1.13, uncorr-P=0.35). The G allele of rs61768479 was associated with lower promoter activity and miR expression by disrupting the binding of NKX2.5. In summary, no association was identified between sequence variants in the miR-205/200 family-regulated EMT pathway and risk for lung cancer. However, this study identified a comprehensive panel of tagSNPs (n=16) in the miR-205/200 family-regulated EMT pathway that can be applied to other EMT-related phenotypes such as cancer chemoresistence and prognosis.
PMCID: PMC3110389  PMID: 21686129
miR-200 family; miR-205; sequence variant; risk; lung cancer
7.  Haplotypes of DNMT1 and DNMT3B are associated with mutagen sensitivity induced by benzo[a]pyrene diol epoxide among smokers 
Carcinogenesis  2008;29(7):1380-1385.
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.
doi:10.1093/carcin/bgn121
PMCID: PMC2899849  PMID: 18499700

Results 1-7 (7)