Mathewos Tessema

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.

Promoter Methylation of Genes in and around the Candidate Lung Cancer Susceptibility Locus 6q23-25

Chromosomal aberrations associated with lung cancer are frequently observed in the long arm of chromosome 6. A candidate susceptibility locus at 6q23-25 for lung cancer was recently identified; however, no tumor suppressor genes inactivated by mutation have been identified in this locus. Genetic, epigenetic, gene expression, and in silico screening approaches were used to select 43 genes located in 6q12-27 for characterization of methylation status. Twelve (28%) genes were methylated in at least one lung cancer cell line, and methylation of 8 genes was specific to lung cancer cell lines. Five of the 8 genes with the highest prevalence for methylation in cell lines (TCF21, SYNE1, AKAP12, IL20RA, and ACAT2) were examined in primary lung adenocarcinoma samples from smokers (n = 100) and never smokers (n = 75). The prevalence for methylation of these genes was 81%, 50%, 39%, 26%, and 14%, respectively, and did not differ by smoking status or age at diagnosis. Transcription of SYNE1, AKAP12, and IL20RA was completely silenced by hypermethylation and could be restored after treatment with 5-aza-2-deoxycytidine. Significant associations were found between methylation of SYNE1 and TCF21, SYNE1 and AKAP12, and AKAP12 and IL20RA, indicating a coordinated inactivation of these genes in tumors. A higher prevalence for methylation of these genes was not associated with early-onset lung cancer cases, most likely precluding their involvement in familial susceptibility to this disease. Together, our results indicate that frequent inactivation of multiple candidate tumor suppressor genes within chromosome 6q likely contributes to development of sporadic lung cancer.

Re-expression of CXCL14 , a common target for epigenetic silencing in lung cancer, induces tumor necrosis

Chemokines are important regulators of directional cell migration and tumor metastasis. A genome-wide transcriptome array designed to uncover novel genes silenced by methylation in lung cancer identified the CXC-subfamily of chemokines. Expression of 11 of the 16 known human CXC-chemokines was increased in lung adenocarcinoma cell lines after treatment with 5-aza-2′-deoxycytidine (DAC). Tumor-specific methylation leading to silencing of CXCL5, 12 and 14 was found in over 75% of primary lung adenocarcinomas and DAC treatment restored the expression of each of the silenced gene. Forced expression of CXCL14 in H23 cells, where this gene is silenced by methylation, increased cell death in vitro and dramatically reduced the in vivo growth of lung tumor xenografts through necrosis of up to 90% of the tumor mass. CXCL14 re-expression had a profound effect on the genome altering the transcription of over 1000 genes, including increased expression of 30 cell-cycle inhibitor and pro-apoptosis genes. In addition, CXCL14 methylation in sputum from asymptomatic early-stage lung cancer cases was associated with a 2.9-fold elevated risk for this disease compared with controls, substantiating its potential as a biomarker for early detection of lung cancer. Together, these findings identify CXCL14 as an important tumor suppressor gene epigenetically silenced during lung carcinogenesis.

Concomitant promoter methylation of multiple genes in lung adenocarcinomas from current, former and never smokers

Aberrant promoter hypermethylation is one of the major mechanisms in carcinogenesis and some critical growth regulatory genes have shown commonality in methylation across solid tumors. Twenty-six genes, 14 identified through methylation in colon and breast cancers, were evaluated using primary lung adenocarcinomas (n = 175) from current, former and never smokers. Tumor specificity of methylation was validated through comparison of 14 lung cancer cell lines to normal human bronchial epithelial cells derived from bronchoscopy of 20 cancer-free smokers. Twenty-five genes were methylated in 11-81% of primary tumors. Prevalence for methylation of TNFRSF10C, BHLHB5 and BOLL was significantly higher in adenocarcinomas from never smokers than smokers. The relation between methylation of individual genes was examined using pairwise comparisons. A significant association was seen between 138 (42%) of the possible 325 pairwise comparisons. Most notably, methylation of MMP2, BHLHB4 or p16 was significantly associated with methylation of 16-19 other genes, thus predicting for a widespread methylation phenotype. Kaplan-Meier log-rank test and proportional hazard models identified a significant association between methylation of SULF2 (a pro-growth, -angiogenesis and -migration gene) and better patient survival (hazard ratio = 0.23). These results demonstrate a high degree of commonality for targeted silencing of genes between lung and other solid tumors and suggest that promoter hypermethylation in cancer is a highly co-ordinated event.

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.

Functional Identification of Cancer-Specific Methylation of CDO1, HOXA9, and TAC1 for the Diagnosis of Lung Cancer

Purpose: Non–small cell lung cancer (NSCLC) is the leading cause of cancer mortality in the world. Novel diagnostic biomarkers may augment both existing NSCLC screening methods as well as molecular diagnostic tests of surgical specimens to more accurately stratify and stage candidates for adjuvant chemotherapy. Hypermethylation of CpG islands is a common and important alteration in the transition from normal tissue to cancer. Experimental Design: Following previously validated methods for the discovery of cancer-specific hypermethylation changes, we treated eight NSCLC cell lines with the hypomethylating agent deoxyazacitidine or trichostatin A. We validated the findings using a large publicly available database and two independent cohorts of primary samples. Results: We identified >300 candidate genes. Using The Cancer Genome Atlas (TCGA) and extensive filtering to refine our candidate genes for the greatest ability to distinguish tumor from normal, we define a three-gene panel, CDO1, HOXA9, and TAC1, which we subsequently validate in two independent cohorts of primary NSCLC samples. This three-gene panel is 100% specific, showing no methylation in 75 TCGA normal and seven primary normal samples and is 83% to 99% sensitive for NSCLC depending on the cohort. Conclusion: This degree of sensitivity and specificity may be of high value to diagnose the earliest stages of NSCLC. Addition of this three-gene panel to other previously validated methylation biomarkers holds great promise in both early diagnosis and molecular staging of NSCLC. Clin Cancer Res; 20(7); 1856–64. ©2014 AACR.

Mining the Epigenome for Methylated Genes in Lung Cancer

Lung cancer has become a global public health burden, further substantiating the need for early diagnosis and more effective targeted therapies. The key to accomplishing both these goals is a better understanding of the genes and pathways disrupted during the initiation and progression of this disease. Gene promoter hypermethylation is an epigenetic modification of DNA at promoter CpG islands that together with changes in histone structure culminates in loss of transcription. The fact that gene promoter hypermethylation is a major mechanism for silencing genes in lung cancer has stimulated the development of screening approaches to identify additional genes and pathways that are disrupted within the epigenome. Some of these approaches include restriction landmark scanning, methylation CpG island amplification coupled with representational difference analysis, and transcriptome-wide screening. Genes identified by these approaches, their function, and prevalence in lung cancer are described. Recently, we used global screening approaches to interrogate 43 genes in and around the candidate lung cancer susceptibility locus, 6q23-25. Five genes, TCF21, SYNE1, AKAP12, IL20RA, and ACAT2, were methylated at 14 to 81% prevalence, but methylation was not associated with age at diagnosis or stage of lung cancer. These candidate tumor suppressor genes likely play key roles in contributing to sporadic lung cancer. The realization that methylation is a dominant mechanism in lung cancer etiology and its reversibility by pharmacologic agents has led to the initiation of translational studies to develop biomarkers in sputum for early detection and the testing of demethylating and histone deacetylation inhibitors for treatment of lung cancer.

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.

Interaction and functional analyses of human VPS34/p150 phosphatidylinositol 3-kinase complex with Rab7.

The Rab7 GTPase is a key regulator of late endocytic membrane transport and autophagy. Rab7 exerts temporal and spatial control over late endocytic membrane transport through interactions with various effector proteins. Among Rab7 effectors, the hVPS34/p150 phosphatidylinositol (PtdIns) 3-kinase complex serves to regulate late endosomal phosphatidylinositol signaling that is important for protein sorting and intraluminal vesicle sequestration. In this chapter, reagents and methods for the characterization of the interactions and regulation of the Rab7/hVPS34/p150 complex are described. Using these methods we demonstrate the requirement for activated Rab7 in the regulation of hVPS34/p150 PtdIns 3-kinase activity on late endosomes in vivo.

Differential Epigenetic Regulation of TOX Subfamily High Mobility Group Box Genes in Lung and Breast Cancers

Aberrant cytosine methylation affects regulation of hundreds of genes during cancer development. In this study, a novel aberrantly hypermethylated CpG island in cancer was discovered within the TOX2 promoter. TOX2 was unmethylated in normal cells but 28% lung (n = 190) and 23% breast (n = 80) tumors were methylated. Expression of two novel TOX2 transcripts identified was significantly reduced in primary lung tumors than distant normal lung (p<0.05). These transcripts were silenced in methylated lung and breast cancer cells and 5-Aza-2-deoxycytidine treatment re-expressed both. Extension of these assays to TOX, TOX3, and TOX4 genes that share similar genomic structure and protein homology with TOX2 revealed distinct methylation profiles by smoking status, histology, and cancer type. TOX was almost exclusively methylated in breast (43%) than lung (5%) cancer, whereas TOX3 was frequently methylated in lung (58%) than breast (30%) tumors. TOX4 was unmethylated in all samples and showed the highest expression in normal lung. Compared to TOX4, expression of TOX, TOX2 and TOX3 in normal lung was 25, 44, and 88% lower, respectively, supporting the premise that reduced promoter activity confers increased susceptibility to methylation during lung carcinogenesis. Genome-wide assays revealed that siRNA-mediated TOX2 knockdown modulated multiple pathways while TOX3 inactivation targeted neuronal development and function. Although these knockdowns did not result in further phenotypic changes of lung cancer cells in vitro, the impact on tissue remodeling, inflammatory response, and cell differentiation pathways suggest a potential role for TOX2 in modulating tumor microenvironment.