SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

DNA methylation (referred to as methylation) is a major epigenetic modification which regulates gene expression mainly by binding methyl-CpG binding proteins (MBDs) and their associated chromatin remodeling factors to DNA [1, 2]. It has been shown that methylation plays an important role in various molecular and cellular processes including embryonic development and genomic imprinting as well as in the pathogenesis of malignant diseases [3‐7]. Methylation occurs by the covalent addition of a methyl group to the 5′ carbon of cytosines within CpG dinucleotides which is catalysed by DNA methyltransferases (DNMTs) [8, 9]. Unlike genetic changes, methylation may be reversed by DNMT inhibitors such as 5-aza-2′-deoxycytidine (5-AzadC) and 5-azacytidine (5-AzaC). A synergistic effect on re-expression of by methylation silenced genes by DNMT inhibitors in combination with histone deacetylase inhibitors like trichostatin A (TSA) was reported [10].

In non-small cell lung cancers (NSCLCs) numerous tumor suppressor genes (TSGs) are already known which are frequently methylated. Besides other epigenetic and genetic factors methylation may be responsible for transcriptional inactivation of these genes [11‐15]. However, from the vast majority of ~500 genes which were identified to be tumor-specifically methylated in a genome-wide approach, tumor-specific methylation was unknown so far [12]. When we additionally analysed publically available microarray data in primary tumors (TU) compared to non-malignant lung tissue (NL) samples of NSCLC patients, we observed tumor-specifically downregulated expression of many of these genes [11, 16, 17]. An extensive PubMed search revealed that many of these genes are functionally uncharacterised in NSCLCs and in other cancer types. Based on all these observations, we selected the genes SPAG6 (Sperm Associated Antigen 6) and L1TD1 (LINE-1 Type Transposase Domain Containing 1) for detailed investigation. SPAG6 is located in the chromosomal region 10p12.2 and is thought to be a cancer-testis antigen (CTA) [18]. CTAs represent a large family of cancer-associated antigens which are expressed in immunoprivileged tissues such as testis but were also detected in tumor tissues of various origins including lung cancer [19]. SPAG6 is also expressed in normal lung tissues where it is associated with ciliary function [20]. It encodes a microtubule-associated protein which either functions as microtubule itself or binds to microtubules to form the cytoskeleton of the cell (www.​pantherdb.​org). There is increasing evidence that the expression of CTAs might be involved in tumorigenesis, however, so far there are no reports available about an involvement of SPAG6 in malignant disease biology or cancer cell invasiveness [21]. L1TD1 is located in the chromosomal region 1p31.3 where frequent loss of heterozygosity (LOH) was observed in NSCLCs [22]. This gene encodes a stem-cell specific RNA-binding protein required for self-renewal of human embryonic stem cells and for cancer cell proliferation [23]. Since the mechanism(s) of inactivation of both, SPAG6 and L1TD1, were not studied in detail and their role in the pathogenesis of NSCLCs was unclear so far, we were interested to further investigate these genes.

Thus, we determined gene expression, methylation and re-expression of SPAG6 and L1TD1 in various NSCLC cell lines to elucidate if methylation is associated with the transcriptional inactivation of these genes. Moreover, we investigated tumor-specific methylation of these genes in a large number of NSCLC patients and compared these data as well as mRNA expression data with clinico-pathological characteristics of NSCLC patients. We also analysed protein expression of both genes in a subset of NSCLC patients and compared these results with SPAG6 and L1TD1 methylation. In addition, potential tumor-cell growth suppressing properties of these genes were investigated in in vitro studies and, for L1TD1, also in in vivo xenograft experiments.

Overall, we identified methylation as a mechanism involved in the regulation of transcriptional activity of SPAG6 and L1TD1 in NSCLCs. Furthermore, our results indicate that L1TD1 functions as a tumor cell growth suppressor in NSCLC cells.

In a recent study, we performed a genome-wide screen for CpG island methylation in NSCLC patients and identified more than 400 tumor-specifically methylated genes. Two of them, SPAG6 and L1TD1, were selected for detailed investigation of gene expression, gene-specific methylation and potential tumor-cell growth suppressing properties in NSCLCs. So far, only little information about a potential impact of these 2 genes on the development of lung cancer was available.

SPAG6 regulates proliferation and differentiation of certain cell types and belongs to the family of CTAs [35, 36]. Normally CTAs are expressed by gametes and trophoblasts but are aberrantly expressed in a variety of tumors [21]. Their transcriptional activity is mainly regulated by epigenetic modifications including methylation and histone acetylation [21, 37]. The role of CTAs in germ line as well as in tumor tissues is poorly understood, however, since germ and cancer cells share certain characteristics including immortalization, invasion and migration, an involvement of CTAs in the development of different tumor types is suggested [37].

L1TD1 is involved in the regulation of self-renewal and pluripotency of human embryonic stem cells. It is highly expressed in medulloblastoma cells where it is associated with cell viability, chemotherapeutic drug resistance and stem cell-like properties [38].

To determine a potential role of SPAG6 and/or L1TD1 in various malignancies, we analysed RNA-seq datasets from the TCGA database. While tumor-specific downregulation of SPAG6 mRNA expression was observed in all tumor types investigated except hepatocellular and prostate carcinomas, downregulated L1TD1 mRNA expression was found in NSCLCs, breast, colorectal and prostate carcinomas but not in head and neck, kidney and hepatocellular carcinomas. Overall, these data suggest that deregulated expression of SPAG6 and L1TD1 may play a role not only in the pathogenesis of NSCLCs but also in tumors of other entities and that expression of these 2 genes differs between certain tumor types.

Besides other mechanisms, methylation is involved in the regulation of transcriptional gene activity [1, 2]. In NSCLCs, many TSGs are already known which are frequently inactivated by methylation [11, 13, 15, 39]. To determine if downregulation of SPAG6 and L1TD1 may be caused by methylation, we performed gene-specific approaches to detect mRNA/protein expression and methylation of these genes in NSCLC cell lines and in primary TU and NL samples from NSCLC patients. Indeed, we found a correlation between downregulated SPAG6 and L1TD1 mRNA expression and methylation of these genes in all NSCLC cell lines analysed. Treatment of these cells with epigenetically active drugs which inhibit DNA methyltransferases and histone deacetylases resulted in upregulated expression of both genes. In addition, we found an association between SPAG6 and L1TD1 methylation and loss of SPAG6 and L1TD1 protein expression when we performed IHC of FFPE tissue samples from NSCLC patients. While most of the SPAG6 or L1TD1 methylated TU samples did not express these proteins, not methylated TU samples mostly expressed SPAG6 or L1TD1. However, these results did not reach statistical significance, probably because of the low sample number available for these analyses and the fact that tissue microarrays only represented small parts of the primary tumor samples. Our hypothesis that methylation is one of the mechanisms responsible for inactivation of these genes is further supported by the fact that SNVs and homozygous deletions of SPAG6 and L1TD1 were rarely detected in LUAD and LUSC SNP and in aCGH datasets of NSCLC patients. However, other mechanisms including deregulation of histone modifications and non-coding RNAs may also contribute to the transcriptional regulation of these genes. All these findings indicate that methylation is indeed involved in the transcriptional regulation of SPAG6 and L1TD1 in NSCLCs.

Methylation of certain TSGs in NSCLCs is tumor-subtype specific [40]. While for instance APC and CDH13 methylation was detected more frequently in lung adenocarcinomas compared to lung squamous cell carcinomas, p16 methylation was detected more frequently in lung squamous cell carcinomas than in lung adenocarcinomas [40, 41]. By comparing SPAG6 and L1TD1 methylation with histological classification of our tumor samples, we did not find a difference between the frequencies of methylation of these genes in lung adenocarcinomas or lung squamous cell carcinomas. These observations suggest that methylation of SPAG6 and L1TD1 is a common feature in all histological subtypes of NSCLCs. Methylation of several genes was shown to be associated with shorter survival of NSCLC patients (e.g. APC, CDH1, DAPK or p16) [13, 42, 43]. In our study, we did not find a correlation between SPAG6 or L1TD1 methylation and OS as well as DFS of NSCLC patients or any other clinico-pathological characteristic of these patients. However, analyses of gene expression microarray data indicate that low SPAG6 expression is associated with a shorter OS of lung squamous cell carcinoma patients and low L1TD1 expression with a shorter OS of lung adenocarcinoma patients. Nevertheless, these findings need to be studied in larger patient cohorts.

TSGs are characterised by a variety of molecular features including frequent CpG island methylation, downregulated expression and that they are often located in regions of LOH. Since SPAG6 and L1TD1 were found to be frequently methylated and downregulated in NSCLC cells and are located in chromosomal regions where frequent LOH was observed, we hypothesized that these genes may have tumor-cell growth suppressing properties. Indeed, in vitro experiments showed reduced cell growth, proliferation, viability and colony-forming abilities of pCMV6-L1TD1 transfected cells suggesting that it may be a potential TSG in NSCLCs. Because of these encouraging results and to further support our hypothesis that L1TD1 has tumor-cell growth suppressing properties, we additionally performed xenograft experiments to investigate the growth of tumors induced by L1TD1-overexpressing and wildtype NSCLC cells. Interestingly, we observed significantly smaller tumors induced by pCMV6-L1TD1 compared to pCMV6-ENTRY transfected NCI-H1975 cells indicating that L1TD1 indeed has tumor-cell growth suppressing properties. Based on the literature this is the first report which describes a potential impact of L1TD1 expression in the pathogenesis of NSCLCs. However, further studies are necessary to elucidate molecular mechanisms affected by L1TD1 in NSCLC cells.

Although it was reported that SPAG6 is involved in proliferation and differentiation of neuronal progenitor cells, in our in vitro studies ectopic SPAG6 expression did not affect the behavior of NSCLC cells [35, 36]. Since no reports are currently available about a potential role of SPAG6 in malignant diseases further studies are necessary to determine cell-type specific functions of SPAG6 and to elucidate if besides tumor-specific methylation other functions of SPAG6 may be involved in the pathogenesis of NSCLCs.

Our results demonstrate that tumor-specific methylation of SPAG6 and L1TD1 is a frequently occurring event in NSCLCs and they suggest that methylation plays an important role in the transcriptional regulation of these genes. Protein expression of both genes was frequently downregulated in primary NSCLCs. In addition, or findings indicate that methylation of these genes may be of relevance not only in NSCLCs but also in other malignancies. Moreover, in vitro and in vivo experiments showed that L1TD1 has tumor-cell growth suppressing properties in NSCLC cells. Taken together we identified methylation as a potential mechanism for frequent downregulation of SPAG6 and L1TD1 in NSCLCs and suggest a putative role of L1TD1 in tumor cell development.