Aberrantly methylated-differentially expressed genes and pathways in colorectal cancer

Colorectal cancer (CRC) is one of the most common malignant tumors with incidence ranked third in men and second in women worldwide [1]. Multiple factors might cause CRC including alcohol, smoking, obesity and insufficient physical exercise [2]. The accumulation of various genetic and epigenetic alternations in colorectal epithelial cells is also regarded as essential processes driving the initiation and progression of CRC [3].

Epigenetics is known as heritable alterations in gene expression that are not mediated by changes within the DNA sequence [4]. Cancer epigenetic covers aspects of DNA methylation, noncoding RNA and histone modification, of which the most widely investigated epigenetic changes is aberrant DNA methylation [5]. Aberrant methylation could influence the functions of key genes especially tumor suppressor genes through altering their expression, thus making it involved in various processes of CRC development [6]. Although multiple studies have demonstrated certain genes with aberrant DNA hypermethylation or hypomethylation in CRC, the comprehensive profile and pathways of the interaction network remain largely elusive.

In recent years, micoarrays based on high-throughput platforms emerge as a promising and efficient tool to screen significant genetic or epigenetic alternations in carcinogenesis and identify biomarkers for diagnosis and prognosis of cancer [7]. A number of gene expression profiling microarrays have been conducted to find various differentially expressed genes (DEGs) in CRC [8]. In addition, some studies concerning aberrant methylation in CRC have also been performed to indicate differentially methylated genes (DMGs) [9]. However, individual investigations possess limited numbers of overlapping gene profiling and insufficient power to identify key genes and pathways involved in multiple cellular process and biological function. Now, by the means of advanced bioinformatics analysis of available microarray data, it is possible to come up with more reliable and precise screening results via overlapping relevant data sets.

Until now, no research has been performed to jointly analyze information of both gene expression profiling microarray and gene methylation profiling microarray in the development of CRC. In the present study, data of gene expression profiling microarrays (GSE68468, GSE44076) and gene methylation profiling microarrays (GSE29490, GSE17648) were integrated and analyzed by a series of bioinformatics tools. Aberrantly methylated-DEGs and pathways were identified in CRC. Protein–protein interaction network was constructed and hub genes were revealed. By this means, we expect to find novel aberrantly methylated genes and pathways in CRC and shed light on the underlying molecular mechanisms that orchestrate colorectal carcinogenesis.

Elucidating the underlying mechanisms of the initiation and development of CRC would greatly benefit the diagnosis, treatment and prognosis evaluation. In this study, we identified 411 hypomethylation-high expression genes and 239 hypermethylation-low expression genes through analyzing available data of gene expression (GSE68468, GSE44076) and gene methylation (GSE29490, GSE17648) microarrays in CRC by multiple bioinformatics tools. Enrichment of these genes demonstrated certain pathways and hub genes affected by aberrant methylation, which may provide novel insights for unraveling pathogenesis of CRC.

As was suggested by DAVID analysis, hypomethylation-high expression genes in CRC were enriched in biological processes of response to wounding, inflammatory response, cell proliferation, cell adhesion and biological adhesion. Molecular function of GO analysis showed enrichment in identical protein binding, cytokine activity, endopeptidase activity, heparin binding, protein binding and bridging. It is reasonable because frequent cell proliferation and loss of cell adhesion is an apparent hallmark of cancers including CRC [13]. Chronic inflammatory and wounding conditions in the intestinal tract have also been found to increase CRC risk [14]. As main inflammatory mediators, cytokines critically determine the pro- or anti-tumorigenic signals within the environment of CRC [15]. KEGG pathway enrichment analysis suggested significant enrichment in pathways including cytokine–cytokine receptor interaction, p53 signaling and cell cycle. It was consistent with the fact that p53 signaling was frequently dysregulated in CRC [16].

PPI network of hypomethylation-high expression genes illustrated the overview of their functional connections, of which top 5 hub genes were also selected: CAD, CCND1, ATM, RB1 and MET. CAD gene encodes a trifunctional protein which is associated with the enzymatic activities of the first 3 enzymes in the 6-step pathway of pyrimidine biosynthesis [17]. Regulatory component of the cyclin D1-CDK4 (DC) complex that phosphorylates and inhibits members of the retinoblastoma (RB) protein family including RB1 and regulates the cell-cycle during G1/S transition [18]. Therefore, CCND1 and RB1 might be candidate genes affected by aberrant methylation that modulate cell cycle during CRC progression. ATM gene encodes a protein named serine/threonine protein kinase, which is a crucial cell cycle checkpoint kinase responsible for regulating a wide variety of downstream proteins including tumor suppressor protein p53 [19]. Aberrant methylation of the promoter region of ATM has been found to associate with increased radiosensitivity in human CRC cell-line HCT-116 [20]. Besides, aberrant methylation of ATM was also detected in colorectal cancers and adenomas [21, 22]. MET gene encodes a member of the receptor tyrosine kinase family of proteins which regulate various physiological processes including proliferation, morphogenesis and survival, but little is known about its role in CRC.

Module analysis of the PPI network for hypomethylation-high expression genes suggested that chemokine signaling, ribosome, nucleic acid binding, carbon metabolism, cell cycle and proteasome might be involved in CRC development. Ribosome, nucleic acid binding and cell cycle were all critical cellular processes during DNA replication and translation, which tend to be disordered in cancer. Chemokine signaling has been found to influence CRC invasion and/or metastasis via changing tumor microenvironment [23]. As an essential process for the degradation of proteins and maintenance for homeostasis in eukaryotic cells, ubiquitin proteasome system (UPS) alternations have been linked to gastrointestinal malignancies including CRC [24]. Our findings highlighted the probable importance of the regulation of these key biological behaviors by aberrantly hypomethylation in CRC, which warranted further investigations to confirm.

For hypermethylation-low expression genes in CRC, GO analysis showed that enriched biological processes were cell–cell signaling, transmission of nerve impulse, regulation of neurological system process and neuron differentiation. Recently, nerve ablation was found to delay development of precancerous lesions and inhibit tumor growth and metastasis [25], but relevant studies were still limited in CRC. The regulation of hypermethylation for nerve related genes might become promising research direction for CRC development in the future. Molecular function enrichment revealed transcription factor activity, channel activity and passive transmembrane transporter activity. KEGG analysis displayed enrichment in pathways of calcium signaling, maturity onset diabetes of the young, cell adhesion molecules (CAMs), neuroactive ligand-receptor interaction, chemokine signaling and Fc gamma R-mediated phagocytosis. These results outlined the important roles of transcription factor, cellular channel and adhesion in CRC, which were candidate study targets for their underlying mechanisms of hypermethylation and dysregulation.

After constructing PPI network for hypermethylation-low expression genes, top 5 hub genes appeared to be EGFR, ACTA1, SST, ESR1 and DNM2. As the most significant hub gene, promoter hypermethylation of EGFR gene has been proved to be related with worse survival of CRC patients who received cetuximab treatment [26]. Additionally, upregulation of EREG expression through promoter demethylation might activate the EGFR pathway during the genesis of CRC [27], which also emphasize the role of aberrant methylation in EGFR pathway. Somatostatin, encoded by SST gene, is a hormone important for regulation of the endocrine system and proliferation of both normal and tumorigenic cells [28]. Promoter hypermethylation-related decreased somatostatin production was found to promote uncontrolled cell proliferation in CRC [29], and methylation of serum SST gene might be an independent prognostic marker in CRC [30]. These results all suggested that SST gene methylation may serve as a critical regulator during CRC development. Estrogen receptor, encoded by ESR1 gene, is important for hormone binding, DNA binding and activation of transcription. And aberrant methylation of ESR1 gene in CRC has previously been discovered by several studies [31, 32]. ACTA1 gene encodes a protein belonging to actin family, which exert functions in cell motility, structure and integrity [33]. DNM2 gene encodes dynamin-2, which is subfamily of GTP-binding proteins and implicated in cell processes such as endocytosis and cell motility [34]. The roles of ACTA1 and DNM2 and their methylation status in CRC require further explorations.

Core modules within PPI network of hypermethylation-low expression genes possessed functions including synaptic transmission, gastric acid secretion, glutamatergic synapse and cell fate specification. Up to now, little is known concerning the effect of glutamatergic synapse and synaptic transmission in CRC as well as its regulation by aberrant methylation. Cell fate specification and differentiation were key procedure during cancer development [13]. According to our analysis, hypermethylation might modulate key genes responsible for cell fate determination, thus influencing the progression of CRC. It is worth noting that biological process of gastric acid secretion might also play a critical role in colorectal carcinogenesis. Gastrin has been identified as a major regulator of gastric acid secretion [35] and it also exerts effects in different tissues promoting cell division and inhibiting apoptosis [36]. Non-amidated gastrins have been proved to accelerate the development of CRC [37]. Moreover, progastrin expression level was higher in CRC than that in normal colorectal mucosa [38], and CRC patients have elevated circulating concentrations of Gamide and total gastrins [39]. The specific roles of these key modules affected by hypermethylation in the occurrence and progression of CRC still need future experiments to elucidate.

Several limitations should be acknowledged in this investigation. The clinical parameters and prognosis were not analyzed in this study due to the availability of data. Besides, the influence of aberrantly methylation on gene expression was only validated in TCGA database. Further molecular experiments are needed to better confirm the findings of the identified genes and pathways in CRC of our investigation.

In summary, our study indicated a series of aberrantly methylated-differentially expressed genes and pathways in CRC by joint bioinformatics analysis of both gene expression and gene methylation microarrays, which may contribute to the finding of molecular mechanisms underlying the initiation and development of CRC. Hub genes including CAD, CCND1, ATM, RB1, MET, EGFR, ACTA1, SST, ESR1 and DNM2 might serve as aberrantly methylation-based biomarkers for precise diagnosis and treatment of CRC in the future. Compared with individual investigation, this study is possible to come up with more reliable and accurate screening results via overlapping relevant data sets. Further molecular experiments are needed to confirm the findings of the identified candidate genes in CRC.