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. Author manuscript; available in PMC: 2017 Aug 15.
Published in final edited form as: Cancer Res. 2016 Jun 14;76(16):4741–4751. doi: 10.1158/0008-5472.CAN-15-3367

MiR-196b is epigenetically silenced during the pre-malignant stage of lung carcinogenesis

Carmen S Tellez 1, Daniel E Juri 1, Kieu Do 1, Maria A Picchi 1, Teresa Wang 2, Gang Liu 2, Avrum Spira 2, Steven A Belinsky 1
PMCID: PMC4987256  NIHMSID: NIHMS795269  PMID: 27302168

Abstract

MicroRNA silencing by promoter hypermethylation may represent a mechanism by which lung cancer develops and progresses, but the microRNAs involved during malignant transformation are unknown. We previously established a model of pre-malignant lung cancer wherein we treated human bronchial epithelial cells (HBEC) with low doses of tobacco carcinogens. Here, we demonstrate that next-generation sequencing of carcinogen-transformed HBECs treated with the demethylating agent 5-aza-2′deoxycytidine revealed miR-196b and miR-34c-5p to be epigenetic targets. Bisulfite sequencing confirmed dense promoter hypermethylation indicative of silencing in multiple malignant cell lines and primary tumors. Chromatin immunoprecipitation studies further demonstrated an enrichment in repressive histone marks on the miR-196b promoter during HBEC transformation. Restoration of miR-196b expression by transfecting transformed HBECs with specific mimics led to cell cycle arrest mediated in part through transcriptional regulation of the FOS oncogene, and miR-196b re-expression also significantly reduced the growth of tumor xenografts. Luciferase assays demonstrated that forced expression of miR-196b inhibited the FOS promoter and AP-1 reporter activity. Finally, a case-control study revealed that methylation of miR-196b in sputum was strongly associated with lung cancer (OR = 4.7, p<0.001). Collectively, these studies highlight miR-196b as a tumor suppressor whose silencing early in lung carcinogenesis may provide a selective growth advantage to pre-malignant cells. Targeted delivery of miR-196b could therefore serve as a preventive or therapeutic strategy for the management of lung cancer.

Keywords: promoter hypermethylation, FOS, tumor suppressor, miR-196b

Introduction

Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases and is the leading cause of cancer-related deaths in the United States with a 5-year survival of approximately 15% (1). Drug resistance is a barrier for curative lung cancer therapies due in part to the molecular heterogeneity of tumors. A major contributor to intratumor heterogeneity is DNA methylation and repressive chromatin states that are also recognized to play major roles in lung cancer etiology (2, 3). Candidate, genome-wide profiling, and targeted sequencing approaches have identified hundreds of genes that are epigenetically silenced in NSCLC (4–8). Importantly, gene-specific hypermethylation is an early and likely an initiating event in pre-malignancy (5, 9, 10). Furthermore, the detection of gene promoter hypermethylation in sputum can serve as a molecular marker for lung cancer risk assessment (11). We recently validated a panel of seven hypermethylated genes that have a classification accuracy of up to 77% and is associated with a 22-fold increase in lung cancer risk when five of seven genes are hypermethylated in sputum (12).

Field cancerization and tumor heterogeneity are major barriers to early lung cancer detection and effective treatment, respectively. A better understanding of how tumors evolve will aid in diagnostic and mechanistic-based personalized medicine to improve clinical outcomes. To address this need, we have developed an in vitro model to identify key molecular changes driving cell transformation and clonal outgrowth of preneoplastic lung epithelial cells that occur in the chronic smoker (13, 14). This model uses normal human bronchial epithelial cells (HBECs; designated, HBEC2 and HBEC14 [are from smokers with lung cancer] and HBEC1 [from a cancer-free smoker]) that have been immortalized with hTERT/cdk4 that efficiently extends the life span without altering the characteristic phenotypic properties of the cells. Immortalization by hTERT/cdk4 is advantageous to SV40 large T antigen immortalization of normal cells because the cells do not go through cellular crisis that induces genomic instability prior to immortalization allowing the HBECs to retain normal p53 and Rb functions and G1S restriction checkpoint for responding to DNA damage. Our studies use low-dose weekly treatment (12 weeks) of HBEC lines with genotoxic, but not cytotoxic doses of the tobacco carcinogens methylnitrosourea (MNU) and benzo(a)pyrene-diolepoxide 1 (BPDE) that induce morphological transformation based on growth in soft agar. The MNU and BPDE (MB) transformed cells recovered from the soft agar and cells treated with these carcinogens for 4, 8, or 12 weeks are used to identify the timing for altered expression and methylation of genes and microRNAs (miRs) and their contribution to transformation. Key findings from these studies included the critical role for DNA methyltransferase 1 (DNMT1) in transformation and the induction of epithelial-to-mesenchymal transition (EMT) that occurred early during carcinogen exposure and persisted in MB transformed clones. The EMT was epigenetically regulated by the initial silencing of miR-200b, miR-200c, and miR-205 through enrichment of the chromatin repressive mark H3K27me3 and later by ensuing DNA methylation to sustain the silencing of these tumor suppressive miRs (14). Our findings extended prior concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can contribute to cancer initiation by promoting the clonal expansion of premalignant lung epithelial cells.

MiRs regulate gene expression by hybridizing to complementary sequences in the 3′UTR of the target mRNA to affect translation and degradation and are predicted to regulate 20–30% of all human genes with an average of 200 predicted targets per miR (15). Recent studies also indicate that miRs can affect gene transcription through binding within the gene promoter or 5′UTR sequences (16, 17). Thus, the silencing of miRs by methylation could play a significant role in lung cancer initiation and progression and could also be useful as biomarkers for lung cancer risk assessment. Recent studies by Xi et al. (18) support this supposition by demonstrating that miR-487b is epigenetically silenced and involved in the pathogenesis of lung cancer. We hypothesize that miRs silenced during transformation in the HBEC in vitro pre-malignancy model will be commonly silenced in primary tumors and serve as potential biomarkers for early lung cancer detection. Thus, the goal of this study was to identify miRs whose epigenetic silencing occurs during exposure of HBECs to tobacco carcinogens, elucidate the contribution of novel identified miR(s) to transformation and malignancy, and evaluate their potential as biomarkers for detection of lung cancer.

Materials and Methods

Patient samples and cell lines

The Institute’s Ethics Committees approved this study and all samples were obtained with written informed consent from participating patients. Lung tumor-normal pairs from 50 NSCLC patients were obtained from frozen tumor banks at The University of New Mexico (UNM) and the Mayo Clinic. NBEC collected through diagnostic bronchoscopy and PBMC were obtained from cancer-free smokers (19). NSCLC cell lines were obtained from and authenticated by the American Type Culture Collection (Manassas, VA). Three immortalized HBEC lines (HBEC1, HBEC2, and HBEC14) were obtained from Drs. Shay and Minna, Southwestern Medical Center, Dallas, TX (20). Cell lines were maintained for a maximum of 6 months (24 passages). HBECs exposed to the tobacco carcinogens MNU and BPDE for 4–12 weeks and MB transformed HBECs are selected for growth in soft-agar following 12 weeks of MB exposure are (summarized in Supplemental Table 1) were previously derived (13).

Study population

Sputum samples of cases and controls were obtained from the New Mexico Lung Cancer Cohort (NMLCC) and Lovelace Smokers Cohort (LSC), respectively. NMLCC was established in 2005 and lung cancer patients were recruited through the Multidisciplinary Chest Clinic at UNM. The lung cancer cases were current or former smokers with Stage I (60%), II (13%), or III (27%) NSCLC. As previously described, spontaneous sputum was collected approximately 1 – 2 weeks prior to surgery in the morning at home and 82 lung cancer cases were identified that produced cytological adequate sputum (12). LSC participants were current or former smokers with a minimum of 15 pack-years of smoking. Controls (n = 164) were frequency matched to cases (n = 82) by age (5 year intervals) and sex.

Small RNA sequencing and data analysis

Cell lines were treated daily with vehicle or 5-aza-2′deoxycytidine (500 nM) for four days, harvested, and RNA isolated with TRIzol reagent (Life Technologies, Grand Island, NY). Small molecular weight fraction (<200 bp) RNA was size fractioned using the Ambion (Life Technologies) flashPAGE fractionator to obtain small RNAs of 10–40nts. Small RNA sequencing libraries were created using SOLiD Small RNA Expression Kit (Ambion) starting with 200 ng of the size-fractionated material following the manufacturer’s instructions. Libraries were sequenced on an Applied Biosystems (Life Technologies) SOLiD System to obtain 36bp reads. Each read was trimmed and aligned to hg19 reference genome using Bowtie and an average of 9.8 million reads mapped to a known miR precursor from miRBase version 16.

MiR and gene expression

Total RNA was isolated from cell lines and tumor-normal samples with TRIzol reagent. RNA (1 μg) was reverse transcribed using the miScript Reverse Transcription Kit (Qiagen, Valencia, CA). RT-qPCR was performed using the miScript PCR Kit and miScript primers (Qiagen) for miR expression. For gene expression, RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). RT-qPCR was carried out with inventoried TaqMan assays (Life Technologies). Experiments were performed in triplicate and normalized to RNU44 or GAPDH using the 2(−ΔΔCt) method for miR and gene expression analysis, respectively.

Gene expression arrays

Total RNA isolated from cell lines was hybridized to Illumina Whole-genome HumanHT12v4 expression BeadChip using a standardized protocol to identify changes in gene expression. Arrays were normalized using robust spline normalization and transformed using the variance stabilization method. Significance analysis of microarrays (SAM) method was applied to identify genes significantly (P < 0.01) differentially expressed compared to control transfected cell lines. Array data analyses were performed in R (version 2.14.2). Metacore (Thomson Reuters, New York, NY) was used to identify pathways and networks statistically over represented in the set of altered genes (expression increasing and decreasing).

DNA methylation analysis in cell lines and tumor samples

DNA was extracted from cell pellets (cell lines, NBEC, and PBMC) and patient tumor samples by digestion with proteinase K, RNase A, and RNase T1 followed by phenol/chloroform extraction and ethanol precipitation. DNA was bisulfite modified using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA). For the initial methylation screening 50 ng of bisulfite modified DNA was used for combined bisulfite restriction analysis (COBRA) and bisulfite sequencing was used to determine methylation density of promoter regions in selected cell lines. Methylation prevalence was assessed by Methylation Specific PCR (MSP) in cell lines and primary tumors for each miR. Primer sequences are provided in Supplemental Table 2.

Methylation specific PCR in sputum specimens

Sputum specimens were labeled only with study-specific coded identifiers to blind investigators from case or control status and the samples were randomized for methylation analysis. Two stage MSP assays were used for increased sensitivity for detection of promoter methylation in sputum. MSP stage 1 and stage 2 primer sequences are provided in Supplemental Table 2. Methylation was scored as positive or negative due to cellular heterogeneity in sputum where the epithelial fraction is typically less than 3% of the specimen thus limiting the ability to quantify methylation levels.

Transfections

Cells were seeded at 70% confluence for transient and stable cell transfections. After 24 hours cells were transfected using Lipofectamine 2000 (Life Technologies, Grand Island, NY). For stable expression of miR-196b, cells were transfected with precursor miR-196b expression vector pEZX-MR08 (HmiR0103-MR08 from GeneCopoeia, Rockville, MD) and selected with puromycin.

Chromatin immunoprecipation (ChIP)

Methylation of histone 3 (H3) lysine residues were examined using ChIP as described (14). ChIP grade antibodies specific for total Histone H3 and H3 di-methyl-K9 (H3K9me2) from Abcam (Cambridge, MA) and H3 tri-methyl-K27 (H3K27me3) from Cell Signaling (Danvers, MA) were used to capture protein–DNA complexes. Rabbit and mouse IgGs were used for isotype control. Results were generated by real-time qPCR performed in triplicate using Power SYBR® Green PCR Master Mix (Applied Biosystems). Primer sequences are provided in Supplemental Table 2. Results were quantified using a 2(−ΔΔCt) method.

MTT

Cells (104 per well) were plated in 96-well plates with six replicate wells for each condition. A cell growth assay was performed using MTT (Sigma, St. Louis, MO). Cell viability was determined using an enzyme-linked immunosorbent assay plate reader. All data points represent the mean of a minimum of six wells.

Cell cycle

Cells (106) were fixed with buffered paraformaldehyde, permeablized with ethanol and stained with propidium iodide solution. Analysis was performed by a FACSCalibur flow cytometer (Becton Dickinson, Mansfield, MA, USA). Data were analyzed by ModFit (Verify Software House, Inc., Mansfield, MA, USA) software. All experiments were performed in triplicate.

Western

Cells were plated at 50% confluence and deprived of serum for 24 hours. One hour prior to harvest cells were stimulated with normal growth media containing 10% FBS. The cells were lysed in RIPA buffer and 100 ug of protein was used for the detection of FOS (Cell Signaling) and β-actin (Sigma-Aldrich, St. Louis, MO).

Reporter assays

miRWalk2.0 was used to identify candidate genes regulated by miR-196b and to localize putative binding sites within the 3′UTR of the FOS gene (21). PITA, RNA22, and RNAhybrid 2.2 algorithms were used to localize putative binding sites for miR-196b within the FOS promoter and 5′UTR (−1500bp to translation start site +250bp) (22–24). The FOS promoter regions and the 3′UTR of the FOS gene were amplified by PCR and inserted into the pGL3-enhancer (Promega, Madison, WI) vector or pMIR-REPORT firefly Luciferase vector system (Life Technologies), respectively. The Luciferase vectors were co-transfected with either the pMIR-REPORT β-galactosidase reporter control vector or pRL Renilla (Promega) for normalizing transfection efficiency and miR-196b mimic or control mimic (Life Technologies) using Lipofectamine 3000. Luciferase and β-galactosidase activities are determined with the Dual-Light assay system (Life Technologies) and Luciferase and Renilla activities were determined by Dual-Luciferase reporter assay (Promega). The 3xAP-1pGL3 (3xAP-1 in pGL3-basic) was a gift from Alexander Dent (Addgene plasmid # 40342) (25). The AP-1 reporter plasmid was co-transfected with pRL Renilla for normalization. Reporter assays were performed in three independent experiments.

In vivo tumor growth

A one-to-one mixture of Matrigel Basement Membrane Matrix (Corning, Tewksbury, MA) and cell suspension was subcutaneously injected (2.5 × 106 cells/site) on each side of the shoulder blade in six female NCr-nu/nu athymic mice (Frederick, MD) per cell line. Tumor size and animal weight were measured weekly until the mice were sacrificed. Tumor volume was calculated as V = (a × b2)/2, with “a” and “b” representing the longer and shorter diameters. Tumors were collected, formalin fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin for pathology review.

Statistics

Demographic and methylation variables were summarized by case-control status with frequency and percent for categorical variables and mean and standard deviation for continuous variables. Differences in demographic variables between cases and controls were assessed with Chi square test for categorical variables and the Wilcoxon rank sum test for continuous variables. The association between the miR methylation and case-control status was expressed as odds ratios (OR) and their corresponding 95% confidence intervals (CI) obtained from logistic regression with adjustment for the design variables (age and sex) and other important covariates including race, smoking status, and pack years of smoking. Age and pack years were entered as continuous variables. Statistical significance was expressed by p values. All analyses were carried out using Statistical Analysis Software (SAS, version 9.2, SAS Institute, Inc., Cary, NC).

Study approval

Experiments using mice were approved and conducted in accordance with Institutional Animal Care and Use Committee guidelines.

Results

Next-generation sequencing identifies miRs frequently silenced by promoter hypermethylation

The goal for these studies was to identify miRs epigenetically silenced during lung pre-malignancy, thus our in vitro tobacco carcinogen transformation model was used. To unmask epigenetically silenced miRs, MB transformed HBECs (designated HBEC1MBT and HBEC2MBT) were treated with the cytosine demethylating agent 5-aza-2′deoxycytidine followed by next-generation sequencing (NGS) to display the mature miRNA transcriptome in comparison to untreated MB transformed HBECs and to the related parental lines. MiRs whose expression increases with 5-aza-2′deoxycytidine treatment is supportive of potential epigenetic regulation by cytosine promoter hypermethylation. Nine tumor-derived cell lines (H1568, H1975, H2023, H2170, H23, CALU6, SW900, H1993, and H2085) were also treated with vehicle or 5-aza-2′deoxycytidine and subjected to miRNA NGS to facilitate prioritization of epigenetically regulated miRs for further study. Three miRs (miR-196b, miR-34c-5p, and miR-497/195 cluster) whose expression was significantly reduced 2-fold (~80%) in HBEC2MBT cells were selected. Re-expression of miR-196b, miR-34c-5p, and miR-497/195 in HBEC1MBT and/or HBEC2MBT after treatment with 5-aza-2′deoxycytidine was 1.6 – 13-fold, and re-expressed 2–15-fold in ≥ 3 tumor-derived cell lines after treatment with 5-aza-2′deoxycytidine. Bisulfite sequencing of miR-196b, miR-34c-5p, and miR-497/195 promoter regions showed low basal levels of methylation in normal bronchial epithelial cells (NBEC), HBEC1, and HBEC2 that increased after 4 weeks of MB carcinogen (designated HBEC1MBW4 and HBEC2MBW4) treatment followed by a further significant increase in HBEC1MBT and HBEC2MBT and dense methylation in a malignant tumor cell line (H358 or CALU6) (Figure 1A–C).

Figure 1.

Figure 1

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Increased promoter methylation of miR-196b, miR-34c-5p, miR-497/195 is associated with reduced expression. Bisulfite sequencing of the promoter regions of miR-196b (A), miR-34c-5p (B), miR-497/195 (C) in NBEC, HBECs, carcinogen exposed (MB) HBECs, and lung cancer cell lines. Mean ± SEM of ten clones. DNA methylation of miR-196b, miR-34c-5p, miR-497/195 promoters increases during carcinogen induced transformation. Dense methylation is present at these promoters in lung cancer cell lines H358 and Calu6. MiR-196b expression was quantified (D) after 4 weeks and in 12 weeks-transformed MB carcinogen treated HBEC1, HBEC2, and HBEC14. The expression of miR-196b is reduced during transformation. MiR-196b expression is reduced in (E) lung cancer cell lines and (F) primary lung tumors with promoter methylation. All RT-qPCR expression analysis is shown as the mean ± SEM of three independent RT-qPCR experiments; *P < 0.05, **P < 0.01, ***P < 0.001.

COBRA was used to assess the promoter methylation status of miR-196b, miR-34c-5p, and miR-497/195 in peripheral blood mononuclear cells (PBMC) (n = 10), NBEC (n = 10), and tumor-derived cell lines (n = 23). MiR-196b and miR-34c-5p were unmethylated in nine PBMC and ten NBEC samples, while methylation was seen in 71% and 42% of tumor lines, respectively (Supplemental Figure 1). In contrast, miR-497/195 was methylated in 100% of PBMC, 90% NBEC samples, and 91% of tumor-derived cell lines. Due to the high frequency of methylation in PBMC and NBEC, miR-497/195 was not evaluated further in this study (Supplemental Figure 1). MiR-196b and miR-34c-5p were frequently methylated in adenocarcinoma (ADC) (88% [n = 25]) and squamous cell carcinoma (SCC) (76% and 96% [n = 25], Supplemental Figure 2). Methylation of miR-34c-5p in carcinomas including colorectal, breast and lung, as well as a progressive loss of expression during lung squamous carcinogenesis has been shown by others (26, 27). In addition, miR-34c-5p is functionally regulated by p53, participates in the control of cell proliferation and adhesion-independent growth and its tumor suppressor role is well-defined (28). Due to sequence homology the miR-34 family members control a similar set of target genes and appear to be functionally redundant. The broad anti-oncogenic functions of the miR-34 mimic is also being evaluated as a therapeutic in a clinical study in solid tumors and hematologic malignancies (29). In contrast, the timing for loss of miR-196b expression and its role in lung carcinogenesis has not been elucidated, thus studies described below focused on these questions.

MiR-196b is silenced through chromatin remodeling that is acquired during pre-malignancy

Our in vitro pre-malignancy model developed previously was used to characterize the timing for reduced expression of miR-196b during carcinogen-induced transformation (13, 14). In HBEC-MBW4 the expression of miR-196b was reduced 26–60% and by 40–82% in HBEC-MBT (Figure 1D). A similar magnitude for loss of expression was observed in malignant lung cancer cell lines and primary lung tumors in which miR-196b was methylated (Figure 1E and 1F). Epigenetic mediated gene silencing is accompanied by changes in chromatin structure and DNA methylation (30), thus enrichment of two common transcriptional repressive chromatin marks (H3K27me3 and H3K9me2) were evaluated. ChIP-qPCR analysis for H3K27me3 and H3K9me2 enrichment at the miR-196b promoter region (268bp to 454bp upstream of the precursor miR-196b) was performed in HBEC2, HBEC2MBW4, HBEC2MBT, H1975, and CALU6. H3K27me3 enrichment was increased 3-fold in HBEC2MBW4, 7-fold in HBEC2MBT, and 3-fold in H1975 compared to HBEC2. Similarly, H3K9me2 enrichment increased 5-fold to 7-fold in HBEC2MBW4 and HBEC2MBT and 2-fold in H1975 compared to HBEC2 (Figure 2A and 2B). Neither repressive chromatin mark was enriched in CALU6 that has an unmethylated miR-196b promoter region (Figure 3).

Figure 2.

Figure 2

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Increasing levels of repressive chromatin at miR-196b promoter in HBEC2 during carcinogen exposure and effects the expression of cell cycle signaling pathways. Enrichment of repressive marks H3K27me3 (A) and H3K9me2 (B) are quantified at the miR-196b promoter by ChIP-qPCR in triplicate experiments; mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001. (C) Pathways significantly altered in response to miR-196b mimic are ranked by −1 × log10 P value. Genes within the core cell cycle pathways are the major pathways affected by miR-196b re-expression. (D) MiR-196b was stably re-expressed in HBEC2MBT, H1975, and H358. Expression was compared to the control cell line. (E) Re-expression of miR-196b significantly reduced the mRNA expression of FOS in miR-196b stable cell lines compared to control cell lines. All RT-qPCR expression analysis is shown as the mean ± SEM of three independent RT-qPCR experiments; *P < 0.05, **P < 0.01, ***P < 0.001. (F) Re-expression of miR-196b significantly reduced the protein levels of FOS in miR-196b stable cell lines compared to control cell lines.

Figure 3.

Figure 3

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Stable re-expression of miR-196b reduces cell cycle and cell proliferation. (A) Cell cycle analysis of miR-196b stable cell lines and control cell lines. Data shows flow cytometric profiles of HBEC2MBT, HBEC2MBT+mir-196b, H358, H358+mir-196b, H1975, and H1975+mir-196b. (B) Re-expression of miR-196b in HBEC2MBT, H1975, and H358 reduced cell growth rates. Cell proliferation activity was measured every 24 hrs with MTT assay and the data are presented as mean ± SEM of triplicate experiments; *P < 0.05, **P < 0.01.

Re-expression of miR-196b reduces expression of FOS and cell proliferation

The initial studies to identify whether miR-196b functions as a tumor suppressor in the pathogenesis of lung cancer focused on identifying genes with significantly changed expression and the pathways in which they reside in response to re-expression of miR-196b. HBEC2MBT and H1975 were transiently transfected with a miR-196b mimic and subjected to array-based genome-wide gene expression profiling 48 hours post transfection. Re-expression of miR-196b resulted in decreased expression of 100 genes (20 miR-196b predicted target genes obtained from miRWalk) in H1975 and 261 genes (86 miR-196b predicted target genes obtained from miRWalk) were identified in HBEC2MBT. There was a small overlap of 9 predicted target genes (CHMP1B, CXCL2, ECHDC2, EGR1, FOS, IDH1, NFKBIZ, NR4A2, ZFP36) whose expression was reduced in both cell lines. Moreover, of the 9 genes that showed reduced expression and were predicted targets of miR-196b, the FOS gene was the most significantly altered (~6-fold) by re-expression of miR-196b. Metacore pathway analysis showed that all major aspects of cell cycle regulation (G2-M, G1-S, and mitosis) were significantly influenced by restored expression of miR-196b (Supplemental Figure 2C). In addition, pathways regulating the cytoskeleton, proteolysis-ubiquitination, cell adhesion, and mRNA processing were also affected by re-expression of miR-196b.

These studies were expanded to investigate the potential tumor suppressor function of miR-196b in vitro and in vivo by stable integration and expression of the pre-miR-196b regulated by the CMV promoter into HBEC2MBT, H1975, and H358 (malignant cell lines with silenced miR-196b). The stable miR-196b cell lines (designated HBEC2MBT+miR-196b, H1975+miR-196b, and H358+miR-196b) exhibited increased miR-196b expression levels of 3–47-fold and also showed a 3–10-fold reduced expression of FOS (Figure 2D and 2E). Serum stimulated H358, H358+miR-196b, H1975, and H1975+miR-196b cells showed that FOS protein levels diminished in a pattern similar to that of FOS mRNA expression (Figure 2F). The effect of miR-196b re-expression on the cell cycle of HBEC2MBT, H1975, and H358 was analyzed by propidium iodide staining using flow cytometry. The stable miR-196b expressing cell lines also showed a dramatic increase in percentage of cells within G1 and reduction in S phase consistent with G0/G1 cell cycle arrest compared with the control cell lines that coincided with a reduction in cell growth (Figure 3A and 3B).

MiR-196b regulates FOS promoter and activity

Epigenetic silencing of miR-196b was associated with increased transcription and protein levels of FOS suggesting regulation through binding to the promoter/5′UTR and/or 3′UTR region of this gene. Software algorithms predicted two and nine binding sites within the FOS 3′UTR and promoter/5′UTR region, respectively. Thus, to investigate these potential interactions, the human FOS 3′UTR was subcloned into a firefly Luciferase reporter vector and transfected into H358 and H1975. There was no decrease in relative Luciferase activity with a miR-196b mimic compared with the control with the FOS 3′UTR (Figure 4A). The FOS promoter region was amplified and cloned in three fragments spanning −1169/−619, −304/+1, +1/+205 bases from the transcription start site (+1). Each fragment contained three miR-196b predicted binding sites. Transfection with the miR-196b mimic in H358 cells decreased luciferase activity by 2-fold in the −304/+1 and +1/+205 fragments (Figure 4B). We investigated FOS transcriptional activity in the context of AP-1 reporter activity. The inducible transcriptional complex AP-1 composed of FOS and JUN proteins can act positively on transcription of a variety of genes. H358 cells were transfected with 3xAP-1 Luciferase reporter construct and co-transfected with miR-196b or negative control mimic. In the presence of miR-196b the activity of 3xAP-1 Luciferase was significantly reduced by 50% (Figure 4C).

Figure 4.

Figure 4

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Effects of mir-196b on luciferase reporter gene bearing FOS 3′UTR, FOS Promoter/5′UTR, or 3xAP-1 binding elements. (A) H358 cells transfected with pMIR, FOS 3′UTR-pMIR with control mimic or miR-196b mimic. (B) Segments of the FOS Promoter/5′UTR were transfected into H358 with control mimic or miR-196b mimic. The data are presented as a ratio indicating the fold change in luciferase activity. (C) H358 cells were transfected with the AP-1 reporter plasmid containing three binding sites for the FOS/JUN complex and with control mimic or miR-196b mimic. The data are presented as a ratio indicating the fold change in luciferase activity. Luciferase assays are results from three independent experiments; mean ± SEM, *P < 0.05, **P < 0.01.

Re-expression of miR-196b reduces growth of tumor xenografts

The effect of miR-196b on in vivo tumor growth was evaluated by xenograft studies of H358, H358+miR-196b, H1975, and H1975+miR-196b cells in nude mice. HBECMBT was not studied because these cells do not grow as a xenograft (13). The time required to form tumors of approximately >100 mm3 in diameter was 7 days for mice in the four groups. Tumor growth from cells expressing miR-196b was significantly slower than parent H358 and H1975 cell lines. By day 28 the mean tumor size of H358+miR-196b and H1975+miR-196b xenograft tumors was less than 73% of H358 or H1975 tumors (mean volumes of 162 mm3 and 196 mm3 for H358+miR-196b and H1975+miR-196b compared to 595 mm3 and 767 mm3 H358 and H1975 tumors at day 28) and the difference in tumor size was statistically significant P <0.001 (Figure 5A). Histologic evaluation revealed a higher mitotic index (arrows) and more apoptosis/single cell necrosis (arrowheads) in parent compared to miR-196b expressing tumors (Figure 5B).

Figure 5.

Figure 5

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MiR-196b significantly reduces tumor growth in nude mice. (A) Forced expression of miR-196b in H358 and H1975 suppresses tumor growth rates. (B) Hematoxylin and eosin staining of tumors revealed that the controls had a higher mitotic index (arrows) and more single cell necrosis (arrowheads). Error bars indicate the mean ± SEM, ***P <0.001.

MiR-196b is a potential biomarker for lung cancer risk assessment

The specificity to epithelial cells and high prevalence for silencing of miR-196b and miR-34c-5p in primary tumors supported their interrogation as biomarkers for lung cancer in sputum samples in a case-control study. Clinical covariates and smoking history for cases and controls are described in Table 1. The prevalence for methylation of miR-196b in sputum from cases and controls was 58% and 23%, respectively. Methylation of miR-196b in sputum was strongly associated with lung cancer with adjustment for age, sex, race, smoking status and pack-years (OR = 4.7, CI: 2.6–8.3, p< 0.001) (Table 2). Methylation of miR-34c-5p was very common in sputum from cases (88%) and controls (70%). However, a significant association with lung cancer was also seen for miR-34c-5p (OR = 3.2, CI: 1.5–6.7, p<0.003). Neither lung cancer stage nor histology assessed as a categorical or dichotomized variable was associated with methylation of either miR.

Table 1.

Clinical Covariates for miR-196b and miR-34c-5p Methylation Analysis in Sputum

Covariates Cases (N=82) Controls (N=162) p-Value1
Sex N (%) N (%) 0.71
 Male 63 (77) 121 (75)
 Female 19 (23)   41 (25)
Age (Years) 0.91
 Mean ± SD     65 ± 7.6     65 ± 7.3
 Range 45, 80 46, 76
Race2 N (%) N (%) 0.66
 Non-Hispanic White 57 (70) 117 (72)
 Hispanic 20 (24)   40 (25)
 Other 5 (6) 5 (3)
Smoking Status N (%) N (%) 0.80
 Current 27 (33)   56 (35)
 Former 55 (67) 106 (65)
Pack Years 0.27
 Mean ± SD     56 ± 30.6     52 ± 26.5
Lung Cancer Stage N (%)
 I 49 (60)
 II 11 (13)
 III 22 (27)
Histology N (%)
 Squamous Cell 30 (36)
 Adenocarcinoma 40 (49)
 NSCLC 8 (10)
 Other 4 (5)
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1

Differences in covariates between cases and controls were assessed using Chi-square for categorical variables and Wilcoxon rank-sum test for continuous variables.

2

Race was dichotomized to non-Hispanic white and other.

Table 2.

Association Between miR Methylation and Lung Cancer Risk

miR Cases (N=82)
Methylation
Frequency (%)		 Controls (N=162)
Methylation
Frequency (%)		 OR (95% CI) p-Value
196b 48 (59) 37 (23) 4.7 (2.6 – 8.3) <0.0001
34c-5p 72 (88) 113 (70) 3.2 (1.5 – 6.7) 0.003
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In addition to adjustment for age, gender, race, smoking status and pack years, age and pack years were entered as continuous variables. Neither lung cancer stage nor histology (assessed as either a categorical or dichotomized variable) were associated with miR methylation and were not included in the logistic regression model.

Discussion

NGS identified that miR-196b and miR-34c-5p were first silenced via promoter hypermethylation in an in vitro lung pre-malignancy model that was replicated in lung tumor-derived malignant cell lines and primary tumors. While epigenetic regulation of miR-34c-5p has been observed in primary lung and other cancers, our results demonstrate a major role for miR-196b in the molecular pathogenesis during the progression of NSCLC. An integrated analysis of miR-196b silencing and genome-wide gene expression patterns demonstrated that miR-196b is epigenetically silenced by chromatin modification and DNA methylation during MB transformation of HBECs and that re-expression in MB transformed and tumor-derived cell lines was associated with expression changes in genes related to cell cycle regulation. Most important, stable re-expression of miR-196b to physiological levels led to a G0/G1 cell cycle arrest likely mediated in part through its regulation of the FOS oncogene. The in vitro reduction in cell growth was replicated in vivo by a profound reduction in growth of tumor xenografts in which function of miR-196b was restored. Finally, miR-196b promoter hypermethylation was a highly significant molecular marker in sputum for predicting lung cancer.

Mir-196b is encoded within the HOXA10-A9 gene in an evolutionarily conserved locus on chromosome 7 and has been shown to regulate the expression of HOXB8 during vertebrate development (31, 32). Dysregulation of the HOX network has been implicated in cancer development and abnormal expression during proliferation, differentiation, apoptosis, and signal transduction (33). The mechanisms through which miR-196b impacts cancer development are likely influenced by cell function and the local microenvironment. With respect to cancers of the digestive track that includes oral, esophagus, and gastric, miR-196b expression is increased and postulated to promote an invasive phenotype through the NME4-JNK-TIMP1-MMP signaling pathway (34–36). MiR-196b over expression has also been implicated in the development of Mixed Lineage Leukemia through inhibiting cell differentiation and apoptosis, while promoting cell proliferation and survival (37, 38). In contrast, reduced expression of miR-196b associated with promoter hypermethylation is seen for chronic myeloid leukemia, glioblastoma, prostate cancer, hepatocellular carcinoma, and in cervical cancer miR-196b is down-regulated (39–43). Re-expression of miR-196b in cervical cancer resulted in reduced cell growth, clonogenicity, migration and invasion in vitro, as well as reduced tumor angiogenesis and tumor cell proliferation in vivo (43), findings corroborated by our studies in lung cancer. The tumor suppressor properties of miR-196b in NSCLC appear to be mediated through its direct regulation of FOS through binding the sites within the promoter/5′UTR thereby affecting transacting activity of the AP-1 complex. FOS proteins are required for cell cycle progression and for entering S phase during the induction of cell proliferation and exponential growth (44). The FOS gene is constitutively expressed in a number of tissues as well as in certain tumors cells, and is inducible by a variety of stimuli and rapidly degraded in virtually all cell types (45). The FOS gene belongs to a multi-gene family encoding several structurally and functionally related transcription factors (FOS, FOSB, FRA-1 and FRA-2) contributing to the formation of the AP-1 transcription complex. FOS is involved in important cellular events, including cell proliferation, angiogenesis, differentiation, and survival which makes its de-regulation an important factor for cell transformation and cancer progression (46, 47).

The silencing of miR-196b that appears early in lung cancer pre-malignancy and the strong tumor suppressive effect associated with its re-expression offer opportunities for translation with respect to risk assessment and therapeutics, respectively. The development of the next generation of risk prediction models for lung cancer to combine with CT screening to improve its predictive value requires a clear mechanistic understanding of lung carcinogenesis prior to invasive disease that enables development of novel molecular biomarkers. Our group showed that detecting gene methylation in exfoliated cells from the lungs of smokers provides an assessment of the extent of field cancerization and is a validated biomarker for predicting lung cancer risk (12). MiR-196b methylation in sputum is a promising novel molecular biomarker whose inclusion in a seven gene methylation biomarker panel increased lung cancer classification accuracy from 77% to 82%, which could improve risk stratification of smokers who would most likely benefit from CT screening (S. A. Belinsky, unpublished).

Prostate cancer cells expressing prostate-specific membrane antigen have been targeted effectively with RNA aptamer-small interfering RNA chimeras (48). This strategy, while in early stage development, should be amenable to delivering pre-miR-196b to treat field cancerization or lung cancer with constructs targeting aberrantly expressed tumor antigens to facilitate cell internalization and processing by dicer to yield a functional miR (49). Furthermore, the opportunity for aerosol delivery, that has proven effective for epigenetic therapy, obviates the need for higher therapeutic doses and reduces potential systemic toxicity (50). Collectively, these studies identified miR-196b as a lung tumor suppressor whose silencing early in cancer may contribute to the etiology of this disease by providing a selective growth advantage to premalignant cells, while also affording novel opportunities for early detection and therapeutic targeting to prevent and treat NSCLC.

Supplementary Material

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Acknowledgments

The authors thank Dr. Hitendra Chand for technical assistance with flow cytometry.

Grant Support

This work was supported by NIH/NCI 5R01CA183296-02 and The State of New Mexico as a direct appropriation from the Tobacco Settlement Fund through collaboration with the University of New Mexico to establish and support the LSC. Additional support was provided by NIH/NCI 5R01CA097356-10 and NIH/NCI P30CA118100.

Footnotes

Data Deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database (NGS data accession no. GSE79982 and expression array data accession no. GSE79987).

The following link has been created to allow review of record GSE79982: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=urmbmqqqttuxrmd&acc=GSE79982

The following link has been created to allow review of record GSE79987: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=qvapqwqatncplwp&acc=GSE79987

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors’ contributions

Concept and design: C.S. Tellez, S.A. Belinsky

Development of methodology: C.S. Tellez, M.A. Picchi, T.Wang, G. Liu, A. Spira, S.A. Belinsky

Acquisition of data (provided animals acquired and managed patients, provided facilities, etc.): K.Do M.A. Picchi, A. Spira, S.A. Belinsky

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.S. Tellez, M.A. Picchi, T.Wang, G. Liu, A. Spira, S.A. Belinsky

Writing, review, and/or revision of the manuscript: C.S. Tellez, S.A. Belinsky

Study supervision: C.S. Tellez, S.A. Belinsky

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NIHMS795269-supplement-1.tif (62.4KB, tif)
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NIHMS795269-supplement-3.doc (30.5KB, doc)
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