The roles of imprinted SLC22A18 and SLC22A18AS gene overexpression caused by promoter CPG island hypomethylation as diagnostic and prognostic biomarkers for non-small cell lung cancer patients

cancers Article The Roles of Imprinted SLC22A18 and SLC22A18AS Gene Overexpression Caused by Promoter CpG Island Hypomethylation as Diagnostic and Prognostic Biomarkers for Non-Small Cell Lung Cancer Patients JoséFrancisco Noguera-Uclés1,†, Laura Boyero 1,†, Ana Salinas 1, Juan Antonio Cordero Varela 1, Johana Cristina Benedetti 1,2, Reyes Bernabé-Caro 1,2, Amparo Sánchez-Gastaldo 1,2, Miriam Alonso 1,2, Luis Paz-Ares 3,4,5 and Sonia Molina-Pinelo 1,2,3,* 1Institute of Biomedicine of Seville (IBiS) (HUVR, CSIC, Universidad de Sevilla), 41013 Seville, Spain; [email protected] (J.F.N.-U.); [email protected] (L.B.); [email protected] (A.S.); jacorder[email protected] (J.A.C.V.); [email protected] (J.C.B.); [email protected] (R.B.-C.); [email protected] (A.S.-G.); [email protected] (M.A.) 2Medical Oncology Department, Hospital Universitario Virgen del Rocío, 41013 Seville, Spain 3Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), 28029 Madrid, Spain; [email protected]g 4H12O-CNIO Lung Cancer Clinical Research Unit, Instituto de Investigación Hospital 12 de Octubre & Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, Spain 5Medical Oncology Department, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain *Correspondence: [email protected] †These authors contributed equally to this work. Received: 9 July 2020; Accepted: 23 July 2020; Published: 27 July 2020   Abstract: Genomic imprinting is a process that involves one gene copy turned-offin a parent-of-origin-dependent manner. The regulation of imprinted genes is broadly dependent on promoter methylation marks, which are frequently associated with both oncogenes and tumor suppressors. The purpose of this study was to assess the DNA methylation patterns of the imprinted solute-carrier family 22 member 18 (SLC22A18) and SLC22A18 antisense (SLC22A18AS) genes in non-small cell lung cancer (NSCLC) patients to study their relevance to the disease. We found that both genes were hypomethylated in adenocarcinoma and squamous cell carcinoma patients. Due to this imprinting loss, SLC22A18 and SLC22A18AS were found to be overexpressed in NSCLC tissues, which is significantly more evident in lung adenocarcinoma patients. These results were validated through analyses of public databases of NSCLC patients. The reversed gene profile of both genes was achieved in vitro by treatment with ademetionine. We then showed that high SLC22A18 and SLC22A18AS expression levels were significantly associated with worsening disease progression. In addition, low levels of SLC22A18AS were also correlated with better overall survival for lung adenocarcinoma patients. We found that SLC22A18 and SLC22A18AS knockdown inhibits cell proliferation in vitro . All these results suggest that both genes may be useful as diagnostic and prognostic biomarkers in NSCLC, revealing novel therapeutic opportunities. Keywords: NSCLC; SLC22A18;IMPT1;TSSC5;SLC22A18AS; genomic imprinting; prognosis; diagnostic; biomarkers Cancers 2020,12, 2075; doi:10.3390/cancers12082075 www.mdpi.com/journal/cancers Cancers 2020,12, 2075 2 of 18 1. Introduction Epigenetic studies have revealed inheritable and reversible changes in the genome, which, without modifying nucleic acid sequences, alter all key DNA-dependent processes, such as replication, repair, recombination and transcription [ 1 ]. Epigenetic mechanisms include DNA methylation, histone modifications, nucleosome remodeling, and alterations in microRNA expression [ 2 ]. Currently, one of the most widely studied epigenetic processes is DNA methylation, in which a methyl group is added at the fifth carbon position of cytosine residues in the context of a CpG dinucleotide [ 3 , 4 ]. The CpG dinucleotides are not distributed uniformly across the human genome; they tend to cluster into small dense sequences known as CpG islands, which make up 1% of the full genome sequence [ 2 ]. CpG islands are enriched in gene promoter sequences, playing key roles in the regulation of gene expression under physiological conditions. However, aberrations to DNA methylation contribute to the development and progression of diseases such as cancer [ 5 ]. For instance, hypermethylation in the promoter region of tumor suppressor genes reduces their expression, and in contrast, cancer-linked DNA hypomethylation is associated with the overexpression of oncogenes, whereas methylation in a transcribed region has various effects on gene expression in tumor cells [4]. Over the past decade, there has been increasing evidence showing the relevant role of epigenetic markers in non-small cell lung cancer (NSCLC); these tumors are the deadliest tumors worldwide and represent 80% of all types of lung cancer, with the other 20% being small cell lung cancer (SCLC). NSCLC can be histologically subclassified into squamous cell carcinoma (SCC) (40%), adenocarcinoma (30%) and large cell carcinoma (10%). The detection of lung cancer at an early stage is correlated with a better prognosis; however, unfortunately, this occurs only in 15–20% of diagnosed patients [6]. The relationship between tobacco and lung cancer is well known, with approximately 80% of cases directly related to smoking in Western countries [ 7 ]. Smoking produces chronic inflammation and an increase in reactive oxygen species, leading to alterations in DNA methylation in defined nuclear positions and chromosome domains [ 8 ]. It is notable that, although most lung cancer patients are smokers, only a minority of lifetime smokers develop the disease. This finding suggests that lung carcinogenesis is strongly related to genetic and/or epigenetic susceptibility [4]. In general, NSCLC shows global hypomethylation [ 9 , 10 ], which is associated with genomic instability. This global demethylation is generally an early event, occurring during tumor initiation and progression, and its significance depends the part of the genome and the genes affected [ 5 , 11 ]. In addition, hypermethylation also occurs, but specifically on tumor suppressor genes, such as P16. In lung cancer, hypermethylated genes are involved in functions such as DNA repair, apoptosis, cell cycle, cell adhesion and invasion, and transcription regulation [ 2 ]. In summary, gene methylation status has shown potential diagnostic, prognostic and treatment-response predictive value, which makes it a promising epigenetic tumor biomarker. The majority of the genes deregulated due to DNA methylation are inherited from two functionally equivalent parental copies. However, there is a small subset in which one allele is turned offthrough an epigenetic mechanism in a parent-of-origin-dependent manner, known as genomic imprinting [ 12 ]. Loss of genomic imprinting is often associated with oncogenes or tumor suppressor genes. For example, the imprinted P53 and IGF2 genes have been reported to be tumor suppressor genes and oncogenes, respectively [ 12 , 13 ]. Alternatively, overexpression of some imprinted miRNAs has been proposed as a regulator of tumor suppressor genes [ 14 ]. Thus, imprinted messenger RNA aberrations seem to play a key role in cancer. However, there are still many imprinted genes whose underlying role in NSCLC remains unknown. Therefore, we analyzed whether the methylation status of imprinted solute carrier (SLC) transporters, namely, SLC22A18 and its antisense gene (SLC22A18AS), have clinical significance in patients with NSCLC. The SLC22A18 and SLC22A18AS genes are a sense-antisense pair located on chromosomal segment 11p15.5, an imprinted region that is 1.25 Mb long with a total of 21 genes [ 15 ]. These genes partially overlap in divergent orientations such that the first exon of SLC22A18AS shares 31 bp with the second exon of SLC22A18 [ 16 ]. While the sense gene (SLC22A18) is a 10 transmembrane domain member of a family of polyspecific transporters and multidrug resistance genes and is expressed in the liver and Cancers 2020,12, 2075 3 of 18 kidney, little is known about its antisense partner (SLC22A18AS). There is still no consensus among the scientific community on the role of SLC22A18 in the tumor process, since, depending on the type of cancer, it will have either a tumor suppressor function or be a promoter of tumorigenesis [ 17 ]. Due to the importance of genomic imprinting in correct gene expression and the role that epigenetic imbalances play in tumorigenesis and its progression, the SLC22A18 and SLC22A18AS genes may be potential biomarkers of NSCLC. However, little is known about the function(s) of these genes in the development and prognosis of this disease. Therefore, the purpose of this study was to elucidate the roles of the SLC22A18 and SLC22A18AS genes in NSCLC. To this end, we analyzed the transcriptional regulation mechanisms of both imprinted genes and their potential uses as prognostic markers in patients with NSCLC. 2. Results 2.1. CpG Promoter Methylation Status of the Imprinted SLC22A18 and SLC22A18AS Genes in NSCLC Patients To evaluate the potential role of the SLC22A18 and SLC22A18AS genes in lung cancer, we analyzed the DNA methylation pattern of its promoter in human lung tissues (Figure S1). The DNA methylation profile of SLC22A18 and SLC22A18AS was studied in tumor samples and compared to nontumor tissue in the first patient cohort using an Illumina Infinium Human Methylation 450 BeadChip. Significant differences were identified for the SLC22A18 (p<0.0001) and SLC22A18AS (p<0.0001) genes in tumors in relation to the nontumor tissue. Both genes were hypomethylated in NSCLC patients. The hypomethylation status was consistently observed for patients subclassified into the two main histological subtypes of NSCLC. We found that adenocarcinoma patients showed significantly lower methylation levels of both the SLC22A18 and SLC22A18AS genes than the SCC patients. In addition, methylation levels for both genes were significantly lower in adenocarcinoma with respect to SCC subtype (p<0.01). To validate these findings, we analyzed the differential methylation levels of both genes from an independent cohort of NSCLC patients identified in The Cancer Genome Atlas (TCGA) database. Significant differences in the methylation levels between tumor and nontumor tissues were observed for both genes (p<0.001). Then, we analyzed the methylation levels according to the disease stage, comparing late stage (III–IV) and early stage (I–II) samples. However, we did not find significant differences in methylation levels in tissues of different tumor stages. To explore the CpG density in the 11p15.5 chromosomic region, which carries the imprinted SLC22A18 and SLC22A18AS genes, we generated an epigenome map using the WashU Epigenome Browser (Figure 1). A high CpG rate was observed throughout the genomic sequence of both genes (>50%). We found five CpG islands in the chromosome region, one of which was located in the promoter region of the SLC22A18 gene and its antisense gene. CpG shores and CpG shelves flanked all the CpG islands, whose CpG density was progressively lower away from the CpG islands. Cancers 2020, 12, 2075 3 of 18 the second exon of SLC22A18 [16]. While the sense gene (SLC22A18) is a 10 transmembrane domain member of a family of polyspecific transporters and multidrug resistance genes and is expressed in the liver and kidney, little is known about its antisense partner (SLC22A18AS). There is still no consensus among the scientific community on the role of SLC22A18 in the tumor process, since, depending on the type of cancer, it will have either a tumor suppressor function or be a promoter of tumorigenesis [17]. Due to the importance of genomic imprinting in correct gene expression and the role that epigenetic imbalances play in tumorigenesis and its progression, the SLC22A18 and SLC22A18AS genes may be potential biomarkers of NSCLC. However, little is known about the function(s) of these genes in the development and prognosis of this disease. Therefore, the purpose of this study was to elucidate the roles of the SLC22A18 and SLC22A18AS genes in NSCLC. To this end, we analyzed the transcriptional regulation mechanisms of both imprinted genes and their potential uses as prognostic markers in patients with NSCLC. 2. Results 2.1. CpG Promoter Methylation Status of the Imprinted SLC22A18 and SLC22A18AS Genes in NSCLC Patients To evaluate the potential role of the SLC22A18 and SLC22A18AS genes in lung cancer, we analyzed the DNA methylation pattern of its promoter in human lung tissues (Figure S1). The DNA methylation profile of SLC22A18 and SLC22A18AS was studied in tumor samples and compared to nontumor tissue in the first patient cohort using an Illumina Infinium Human Methylation 450 BeadChip. Significant differences were identified for the SLC22A18 (p < 0.0001) and SLC22A18AS (p < 0.0001) genes in tumors in relation to the nontumor tissue. Both genes were hypomethylated in NSCLC patients. The hypomethylation status was consistently observed for patients subclassified into the two main histological subtypes of NSCLC. We found that adenocarcinoma patients showed significantly lower methylation levels of both the SLC22A18 and SLC22A18AS genes than the SCC patients. In addition, methylation levels for both genes were significantly lower in adenocarcinoma with respect to SCC subtype (p < 0.01). To validate these findings, we analyzed the differential methylation levels of both genes from an independent cohort of NSCLC patients identified in The Cancer Genome Atlas (TCGA) database. Significant differences in the methylation levels between tumor and nontumor tissues were observed for both genes (p < 0.001). Then, we analyzed the methylation levels according to the disease stage, comparing late stage (III–IV) and early stage (I–II) samples. However, we did not find significant differences in methylation levels in tissues of different tumor stages. To explore the CpG density in the 11p15.5 chromosomic region, which carries the imprinted SLC22A18 and SLC22A18AS genes, we generated an epigenome map using the WashU Epigenome Browser (Figure 1). A high CpG rate was observed throughout the genomic sequence of both genes (>50%). We found five CpG islands in the chromosome region, one of which was located in the promoter region of the SLC22A18 gene and its antisense gene. CpG shores and CpG shelves flanked all the CpG islands, whose CpG density was progressively lower away from the CpG islands. Figure 1. CpG density in the SLC22A18 and SLC22A18AS genes. This image illustrates the CpG content level and CpG percentage in both genes. Purple arrows represent the exact position of both genes on Chr.11p15.5. CpG-rich regions (CpG islands, “I”) are highlighted in black; regions up to 2 kb from the CpG island (shores) are marked in dark gray; regions from 2 to 4 kb from the CpG island Figure 1. CpG density in the SLC22A18 and SLC22A18AS genes. This image illustrates the CpG content level and CpG percentage in both genes. Purple arrows represent the exact position of both genes on Chr.11p15.5. CpG-rich regions (CpG islands, “I”) are highlighted in black; regions up to 2 kb from the CpG island (shores) are marked in dark gray; regions from 2 to 4 kb from the CpG island (shelves) are highlighted in intermediate gray; and the rest of the gene is colored in light gray. The blue histogram represents the CpG percentage in this chromosomal region. Cancers 2020,12, 2075 4 of 18 2.2. Expression Levels of the Imprinted SLC22A18 and SLC22A18AS Genes in NSCLC Tissue To study the effect of SLC22A18 and SLC22A18AS methylation status on transcriptional regulation, we analyzed the expression levels of both genes by qPCR in the second cohort of NSCLC patients. The results confirm an inverse association between the methylation status and the expression levels of both genes, which were significantly increased for SLC22A18 (p<0.001) and SLC22A18AS (p=0.022) in the NSCLC tissue compared to the matched nontumor lung tissue (Figure 2a). Next, we analyzed their expression levels according to histological subtypes. We found that SLC22A18 and SLC22A18AS were significantly overexpressed in adenocarcinoma (p=0.001) relative to the nontumor lung tissue. On the other hand, no significant differences were found for these genes in the SCC tissue compared with the expression in the nontumor tissue; however, an upward trend was observed (p=0.074 and p=0.088, respectively) (Figure 2b). We also compared the expression levels of the SLC22A18 and SLC22A18AS genes in tissues of the main histological subtypes of NSCLC. Both genes showed higher expression levels in the lung adenocarcinoma samples than they did in the SCC samples, but these differences did not reach a level of significance. Cancers 2020, 12, 2075 4 of 18 (shelves) are highlighted in intermediate gray; and the rest of the gene is colored in light gray. The blue histogram represents the CpG percentage in this chromosomal region. 2.2. Expression Levels of the Imprinted SLC22A18 and SLC22A18AS Genes in NSCLC Tissue To study the effect of SLC22A18 and SLC22A18AS methylation status on transcriptional regulation, we analyzed the expression levels of both genes by qPCR in the second cohort of NSCLC patients. The results confirm an inverse association between the methylation status and the expression levels of both genes, which were significantly increased for SLC22A18 (p < 0.001) and SLC22A18AS (p = 0.022) in the NSCLC tissue compared to the matched nontumor lung tissue (Figure 2a). Next, we analyzed their expression levels according to histological subtypes. We found that SLC22A18 and SLC22A18AS were significantly overexpressed in adenocarcinoma (p = 0.001) relative to the nontumor lung tissue. On the other hand, no significant differences were found for these genes in the SCC tissue compared with the expression in the nontumor tissue; however, an upward trend was observed (p = 0.074 and p = 0.088, respectively) (Figure 2b). We also compared the expression levels of the SLC22A18 and SLC22A18AS genes in tissues of the main histological subtypes of NSCLC. Both genes showed higher expression levels in the lung adenocarcinoma samples than they did in the SCC samples, but these differences did not reach a level of significance. Owing to SLC22A18 and SLC22A18AS are in the same cluster, we analyzed whether their expression was correlated in the NSCLC patient samples. We found a significant positive correlation between the SLC22A18 and SLC22A18AS genes (r = 0.641; p < 0.001) (Figure 2c). Figure 2. Expression levels of the SLC22A18 and SLC22A18AS genes in the tumor and matched nontumoral samples from patients with lung cancer. (a) Comparison of the SLC22A18 and SLC22A18AS gene expression levels in the NSCLC tissue. (b) Comparison of the expression levels of both genes according to histological subtypes. NSCLC: non-small cell lung cancer; ADC: Figure 2. Expression levels of the SLC22A18 and SLC22A18AS genes in the tumor and matched nontumoral samples from patients with lung cancer. ( a ) Comparison of the SLC22A18 and SLC22A18AS gene expression levels in the NSCLC tissue. ( b ) Comparison of the expression levels of both genes according to histological subtypes. NSCLC: non-small cell lung cancer; ADC: adenocarcinoma; SCC: squamous cell carcinoma; NT: nontumor. Boxes show the interquartile range (IQR) and median (thick line); whiskers indicate the range. Outliers >1.5 × IQR but <3 × IQR from the nearest edge of the box are represented by open circles and those >3 × IQR from the nearest edge of the box are represented by * symbols. (c) Spearman’s correlation of the SLC22A18 and SLC22A18AS expression levels. Cancers 2020,12, 2075 5 of 18 Owing to SLC22A18 and SLC22A18AS are in the same cluster, we analyzed whether their expression was correlated in the NSCLC patient samples. We found a significant positive correlation between the SLC22A18 and SLC22A18AS genes (r=0.641; p<0.001) (Figure 2c). 2.3. Validation of the Expression Profiles of the SLC22A18 and SLC22A18AS Genes in the NSCLC Samples in Public Databases We expanded the expression analysis to eight different lung cancer datasets (GSE3141, GSE8894, GSE14814, GSE19188, GSE29013, GSE31210 GSE37745 and GSE68465) by obtaining the expression data for SLC22A18 and SLC22A18AS (Figure 3and Figure S2). In datasets where both nontumor and tumor lung tissue expression data were available (GSE31210, GSE19188 and GSE68465), SLC22A18 and SLC22A18AS expression was analyzed, and overexpression of both genes was found in NSCLC tissues (p<0.001) in all three of these datasets (Figure 3a). One of these datasets, GSE19188, also allowed us to compare the expression levels of SLC22A18 and SLC22A18AS for each histological NSCLC subtype (lung adenocarcinoma and SCC) independently with respect to the nontumor tissues. The results are similar to those previously observed for the second patient cohort analyzed, showing SLC22A18 and SLC22A18AS overexpression in lung adenocarcinoma (p<0.001 for both genes) and SCC (p=0.009 and p=0.048, respectively) with respect to the nontumoral tissues. We also compared the expression levels of both genes in the two main histological subtypes of NSCLC (adenocarcinoma and SCC). The analyses were performed in those databases that had expression data for both genes differentiated specifically by histological subtypes (GSE3141, GSE14814, GSE8894, GSE19188, GSE29013, and GSE37745). Most of the datasets showed significantly higher expression of SLC22A18 and SLC22A18AS in the adenocarcinoma relative to the SCC subtype (Figure 3b). After confirming that NSCLC patients had aberrant expression of the SLC22A18 and SLC22A18AS genes, as identified through public databases, we investigated whether there was a genetic correlation between both genes, as observed in the second patient cohort. The Spearman’s correlation analysis showed a significant positive association for all except one of the consulted datasets (Figure 4). Cancers 2020, 12, 2075 6 of 19 Figure 3. Analysis of the SLC22A18 and SLC22A18AS expression levels from NCBI Gene Expression Omnibus (GEO) datasets. (a) Differential expression of the SLC22A18 and SLC22A18AS genes in the NSCLC tissue and nontumor tissue. (b) Comparison of SLC22A18 and SLC22A18AS expression in the lung adenocarcinoma and SCC tissue. NSCLC: non-small cell lung cancer; ADC: adenocarcinoma; SCC: squamous cell carcinoma; and NT: nontumor. Boxes show the interquartile range (IQR) and median (thick line); whiskers indicate the range. Outliers > 1.5 × IQR but < 3 × IQR from the nearest edge of the box are represented by open circles and those > 3 × IQR from the nearest edge of the box are represented by * symbols. Figure 3. Cont. Cancers 2020,12, 2075 6 of 18 Cancers 2020, 12, 2075 6 of 19 Figure 3. Analysis of the SLC22A18 and SLC22A18AS expression levels from NCBI Gene Expression Omnibus (GEO) datasets. (a) Differential expression of the SLC22A18 and SLC22A18AS genes in the NSCLC tissue and nontumor tissue. (b) Comparison of SLC22A18 and SLC22A18AS expression in the lung adenocarcinoma and SCC tissue. NSCLC: non-small cell lung cancer; ADC: adenocarcinoma; SCC: squamous cell carcinoma; and NT: nontumor. Boxes show the interquartile range (IQR) and median (thick line); whiskers indicate the range. Outliers > 1.5 × IQR but < 3 × IQR from the nearest edge of the box are represented by open circles and those > 3 × IQR from the nearest edge of the box are represented by * symbols. Figure 3. Analysis of the SLC22A18 and SLC22A18AS expression levels from NCBI Gene Expression Omnibus (GEO) datasets. ( a ) Differential expression of the SLC22A18 and SLC22A18AS genes in the NSCLC tissue and nontumor tissue. ( b ) Comparison of SLC22A18 and SLC22A18AS expression in the lung adenocarcinoma and SCC tissue. NSCLC: non-small cell lung cancer; ADC: adenocarcinoma; SCC: squamous cell carcinoma; and NT: nontumor. Boxes show the interquartile range (IQR) and median (thick line); whiskers indicate the range. Outliers >1.5 × IQR but <3 × IQR from the nearest edge of the box are represented by open circles and those >3 × IQR from the nearest edge of the box are represented by * symbols. Cancers 2020, 12, 2075 6 of 18 Figure 3. Analysis of the SLC22A18 and SLC22A18AS expression levels from NCBI Gene Expression Omnibus (GEO) datasets. (a) Differential expression of the SLC22A18 and SLC22A18AS genes in the NSCLC tissue and nontumor tissue. (b) Comparison of SLC22A18 and SLC22A18AS expression in the lung adenocarcinoma and SCC tissue. NSCLC: non-small cell lung cancer; ADC: adenocarcinoma; SCC: squamous cell carcinoma; and NT: nontumor. Boxes show the interquartile range (IQR) and median (thick line); whiskers indicate the range. Outliers > 1.5 × IQR but < 3 × IQR from the nearest edge of the box are represented by open circles and those > 3 × IQR from the nearest edge of the box are represented by * symbols. Figure 4. Spearman’s correlation analysis of SLC22A18 and SLC22A18AS expression based on different datasets. 2.4. Rescue of DNA Methylation Status of the SLC22A18 and SLC22A18AS Genes In Vitro by Treatment with Ademetionine Figure 4. Spearman’s correlation analysis of SLC22A18 and SLC22A18AS expression based on different datasets. Cancers 2020,12, 2075 7 of 18 2.4. Rescue of DNA Methylation Status of the SLC22A18 and SLC22A18AS Genes In Vitro by Treatment with Ademetionine After observing, on the one hand, a DNA hypomethylation state of SLC22A18 and SLC22A18AS and, on the other, an increase in the expression of both genes in NSCLC tissues, we hypothesized that it was due to a direct relationship. As a proof of concept, we examined whether ademetionine, a universal and ubiquitous methyl donor that increases DNA methylation, could decrease the expression of these genes in a panel of 10 NSCLC cell lines (seven adenocarcinoma and three SCC). After 24 h of treatment, SLC22A18 and SLC22A18AS expression levels were significantly rescued for both histological subtypes (Figure 5). Thus, the methylation status influences in the expression of the imprinted SLC22A18 and SLC22A18AS genes in NSCLC. Cancers 2020, 12, 2075 7 of 18 After observing, on the one hand, a DNA hypomethylation state of SLC22A18 and SLC22A18AS and, on the other, an increase in the expression of both genes in NSCLC tissues, we hypothesized that it was due to a direct relationship. As a proof of concept, we examined whether ademetionine, a universal and ubiquitous methyl donor that increases DNA methylation, could decrease the expression of these genes in a panel of 10 NSCLC cell lines (seven adenocarcinoma and three SCC). After 24 h of treatment, SLC22A18 and SLC22A18AS expression levels were significantly rescued for both histological subtypes (Figure 5). Thus, the methylation status influences in the expression of the imprinted SLC22A18 and SLC22A18AS genes in NSCLC. Figure 5. Effect of ademetionine supplementation on the expression of SLC22A18 and SLC22A18AS. Real-time PCR (qPCR) was conducted to quantify SLC22A18 (a) and SLC22A18AS (b) mRNA expression relative to Dimethyl Sulfoxide (DMSO)-treated controls at 24 h after ademetionine treatment (200 μM). ADC: adenocarcinoma; SCC: squamous cell carcinoma. *** p-value < 0.0001; ** pvalue < 0.001; and * p-value < 0.05. 2.5. Prognostic Roles of the Imprinted SLC22A18 and SLC22A18AS Genes for NSCLC Patients To evaluate whether SLC22A18 and SLC22A18AS expression levels were associated with clinical outcomes for patients with the main histological subtypes of NSCLC, we analyzed SLC22A18 and SLC22A18AS expression levels by first progression of disease and overall survival data using the KM Plotter website [18]. For lung adenocarcinoma patients, we found that a higher expression of SLC22A18 and SLC22A18AS was significantly associated with worsening disease progression (hazard ratio (HR) = 1.51, 95% confidence interval (CI) = 1.09–2.08, p = 0.011; and HR = 2.43, 95% CI = 1.77– 3.34, p < 0.001, respectively). Then, low levels of SLC22A18AS were also associated with a better overall survival (HR = 1.95, 95% CI = 1.03–3.69, p = 0.037) (Figure 6a). However, we found nonsignificant differences for the SLC22A18 expression levels with respect to overall survival in the patients with lung adenocarcinoma. In the case of SCC, the trend was similar, and lower expression of both genes trended with a better clinical outcome. Nevertheless, these differences were significant only for the time to first disease progression (HR = 1.72, 95% CI = 1.33–2.23, p < 0.001) (Figure 6b). Figure 5. Effect of ademetionine supplementation on the expression of SLC22A18 and SLC22A18AS. Real-time PCR (qPCR) was conducted to quantify SLC22A18 ( a ) and SLC22A18AS ( b ) mRNA expression relative to Dimethyl Sulfoxide (DMSO)-treated controls at 24 h after ademetionine treatment (200 µ M). ADC: adenocarcinoma; SCC: squamous cell carcinoma. *** p-value <0.0001; ** p-value <0.001; and *p-value <0.05. 2.5. Prognostic Roles of the Imprinted SLC22A18 and SLC22A18AS Genes for NSCLC Patients To evaluate whether SLC22A18 and SLC22A18AS expression levels were associated with clinical outcomes for patients with the main histological subtypes of NSCLC, we analyzed SLC22A18 and SLC22A18AS expression levels by first progression of disease and overall survival data using the KM Plotter website [ 18 ]. For lung adenocarcinoma patients, we found that a higher expression of SLC22A18 and SLC22A18AS was significantly associated with worsening disease progression (hazard ratio (HR) =1.51, 95% confidence interval (CI) =1.09–2.08, p=0.011; and HR =2.43, 95% CI =1.77–3.34, p <0.001, respectively). Then, low levels of SLC22A18AS were also associated with a better overall survival (HR =1.95, 95% CI =1.03–3.69, p=0.037) (Figure 6a). However, we found nonsignificant differences for the SLC22A18 expression levels with respect to overall survival in the patients with lung adenocarcinoma. In the case of SCC, the trend was similar, and lower expression of both genes trended with a better clinical outcome. Nevertheless, these differences were significant only for the time to first disease progression (HR =1.72, 95% CI =1.33–2.23, p<0.001) (Figure 6b). Cancers 2020,12, 2075 8 of 18 Cancers 2020, 12, 2075 8 of 18 Figure 6. Five-year clinical outcomes for the two main histological subtypes of NSCLC from the Cancer Biomedical Informatics Grid (caBIG), GEO and The Cancer Genome Atlas (TCGA) repositories according to the expression levels of (a) SLC22A18 (probeset 204981_at) and (b) SLC22A18AS (probeset 206097_at). HR: hazard ratio. 2.6. SLC22A18 and SLC22A18AS Knockdown Impairs Tumor Cell Proliferation The observed association between SLC22A18 and SLC22A18AS gene overexpression and impaired patient survival made us hypothesize about whether there was a functional relationship between the regulation of these genes and tumorigenesis. For this, we tested the proliferative activity of SLC22A18 and SLC22A18AS genes and their functional effect on lung cancer regulation by small interfering RNA (siRNA) knockdown of each gene in adenocarcinoma and SCC cell lines. Effective downregulation of each gene expression was confirmed by qPCR (Figure 7a,b). The silenced expression of both genes significantly impaired cell proliferation in almost all tested cell lines of both histological subtypes (Figure 7c). Only one squamous cell carcinoma cell line (H520) showed no statistically significant difference, despite a relative decline in proliferation of about 10%. These results evidence an oncogenic role of SLC22A18 and SLC22A18AS overexpression caused by epigenetic imbalances that promote tumor cell proliferation. Figure 7. SLC22A18 and SLC22A18AS knockdown inhibit cell proliferation . qPCR analysis of SLC22A18 (a) and SLC22A18AS (b) mRNA expression at 72 h post-transfection of each siRNA Figure 6. Five-year clinical outcomes for the two main histological subtypes of NSCLC from the Cancer Biomedical Informatics Grid (caBIG), GEO and The Cancer Genome Atlas (TCGA) repositories according to the expression levels of ( a ) SLC22A18 (probeset 204981_at) and ( b ) SLC22A18AS (probeset 206097_at). HR: hazard ratio. 2.6. SLC22A18 and SLC22A18AS Knockdown Impairs Tumor Cell Proliferation The observed association between SLC22A18 and SLC22A18AS gene overexpression and impaired patient survival made us hypothesize about whether there was a functional relationship between the regulation of these genes and tumorigenesis. For this, we tested the proliferative activity of SLC22A18 and SLC22A18AS genes and their functional effect on lung cancer regulation by small interfering RNA (siRNA) knockdown of each gene in adenocarcinoma and SCC cell lines. Effective downregulation of each gene expression was confirmed by qPCR (Figure 7a,b). The silenced expression of both genes significantly impaired cell proliferation in almost all tested cell lines of both histological subtypes (Figure 7c). Only one squamous cell carcinoma cell line (H520) showed no statistically significant difference, despite a relative decline in proliferation of about 10%. These results evidence an oncogenic role of SLC22A18 and SLC22A18AS overexpression caused by epigenetic imbalances that promote tumor cell proliferation. Cancers 2020, 12, 2075 8 of 18 Figure 6. Five-year clinical outcomes for the two main histological subtypes of NSCLC from the Cancer Biomedical Informatics Grid (caBIG), GEO and The Cancer Genome Atlas (TCGA) repositories according to the expression levels of (a) SLC22A18 (probeset 204981_at) and (b) SLC22A18AS (probeset 206097_at). HR: hazard ratio. 2.6. SLC22A18 and SLC22A18AS Knockdown Impairs Tumor Cell Proliferation The observed association between SLC22A18 and SLC22A18AS gene overexpression and impaired patient survival made us hypothesize about whether there was a functional relationship between the regulation of these genes and tumorigenesis. For this, we tested the proliferative activity of SLC22A18 and SLC22A18AS genes and their functional effect on lung cancer regulation by small interfering RNA (siRNA) knockdown of each gene in adenocarcinoma and SCC cell lines. Effective downregulation of each gene expression was confirmed by qPCR (Figure 7a,b). The silenced expression of both genes significantly impaired cell proliferation in almost all tested cell lines of both histological subtypes (Figure 7c). Only one squamous cell carcinoma cell line (H520) showed no statistically significant difference, despite a relative decline in proliferation of about 10%. These results evidence an oncogenic role of SLC22A18 and SLC22A18AS overexpression caused by epigenetic imbalances that promote tumor cell proliferation. Figure 7. SLC22A18 and SLC22A18AS knockdown inhibit cell proliferation . qPCR analysis of SLC22A18 (a) and SLC22A18AS (b) mRNA expression at 72 h post-transfection of each siRNA Figure 7. SLC22A18 and SLC22A18AS knockdown inhibit cell proliferation. qPCR analysis of SLC22A18 Cancers 2020,12, 2075 9 of 18 ( a ) and SLC22A18AS ( b ) mRNA expression at 72 h post-transfection of each siRNA relativized to negative control ( c ) Relative cell proliferation assay in SLC22A18 and SLC22A18AS knockdown versus negative control after specific transfection. ADC: adenocarcinoma; SCC: squamous cell carcinoma. siSLC22A18: knockdown siRNA against SLC22A18; siSLC22A18AS: knockdown siRNA against SLC22A18AS. *** p-value <0.0001; ** p-value <0.001; and * p-value <0.05. 2.7. Reactome Pathway Analysis for the Imprinted SLC22A18 and SLC22A18AS Genes A bioinformatic approach was used to visualize the possible effects of the roles of SLC22A18 and SLC22A18AS in the NSCLC context. SLC22A18 plays a vital role as a membrane transporter. Under physiological conditions, SLC22A18, an SLC-mediated transmembrane transporter (false discovery rate, FDR =3.92 × 10 −2 ), has been specifically reported to be an organic cationic transporter (FDR = 6.01 × 10 −3 ) based on a proton efflux (H + ) antiport process (Figure 8a). On the other hand, defective SLC22A18 has been described in lung cancer (LNCR) and other types of tumors, such as embryonal rhabdomyosarcoma 1 (RMSE1) (FDR =8.09 × 10 −4 ) (Figure 8b). However, although two biological pathways have been identified to date for SLC22A18, none have been described for its antisense gene. Cancers 2020, 12, 2075 9 of 18 relativized to negative control (c) Relative cell proliferation assay in SLC22A18 and SLC22A18AS knockdown versus negative control after specific transfection. ADC: adenocarcinoma; SCC: squamous cell carcinoma. siSLC22A18: knockdown siRNA against SLC22A18; siSLC22A18AS: knockdown siRNA against SLC22A18AS. *** p-value < 0.0001; ** p-value < 0.001; and * p-value < 0.05. 2.7. Reactome Pathway Analysis for the Imprinted SLC22A18 and SLC22A18AS Genes A bioinformatic approach was used to visualize the possible effects of the roles of SLC22A18 and SLC22A18AS in the NSCLC context. SLC22A18 plays a vital role as a membrane transporter. Under physiological conditions, SLC22A18, an SLC-mediated transmembrane transporter (false discovery rate, FDR = 3.92 × 10 −2 ), has been specifically reported to be an organic cationic transporter (FDR = 6.01 × 10 −3 ) based on a proton efflux (H + ) antiport process (Figure 8a). On the other hand, defective SLC22A18 has been described in lung cancer (LNCR) and other types of tumors, such as embryonal rhabdomyosarcoma 1 (RMSE1) (FDR = 8.09 × 10 −4 ) (Figure 8b). However, although two biological pathways have been identified to date for SLC22A18, none have been described for its antisense gene. Figure 8. Representation of the enhanced reactome of the SLC22A18 gene. (a) Solute carrier (SLC)- mediated transmembrane transport. (b) SLC transporter disorder. SLC: solute-carrier superfamily. 3. Discussion In this study, we analyzed the methylation and expression status of the imprinted SLC22A18 and SLC22A18AS genes in the context of NSCLC. We have shown here that both genes were in a hypomethylated status in NSCLC, which is a characteristic feature of the main histological subtypes, such as adenocarcinoma and SCC. SLC22A18 and SLC22A18AS promoter DNA hypomethylation contributed to their overexpression. In addition, we have also provided evidence that the determination of SLC22A18 and SLC22A18AS expression levels suggested a prognostic role for the time to first progression of the disease and overall survival of patients. The epigenetic instability of imprinted genes is receiving more attention in the cancer research community [19]. These genes are particularly vulnerable to having one specifically silenced parental Figure 8. Representation of the enhanced reactome of the SLC22A18 gene. ( a ) Solute carrier (SLC)-mediated transmembrane transport. ( b ) SLC transporter disorder. SLC: solute-carrier superfamily. 3. Discussion In this study, we analyzed the methylation and expression status of the imprinted SLC22A18 and SLC22A18AS genes in the context of NSCLC. We have shown here that both genes were in a hypomethylated status in NSCLC, which is a characteristic feature of the main histological subtypes, such as adenocarcinoma and SCC. SLC22A18 and SLC22A18AS promoter DNA hypomethylation contributed to their overexpression. In addition, we have also provided evidence that the determination Cancers 2020,12, 2075 16 of 18 8. 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