DNA methylation considered as one of the most significant epigenetic mechanisms that considered for long term suppressing of gene expression and mainly characterized by the binding of methyl group to the cytosine residue of DNA molecules which mostly occurs in CpG islands that not fully dispersed in the whole genome (67). The human genome composed of long stretches of CpG islands, with curiously high level of CpG dinucleotides mainly increased in the promoters regions of the genes. In general, the CpG islands in the promoter of normal gene are unmethylated and transcribed in a normal way (68).
The recognition of methylation status in mammal mostly occurs at developmental stage and is normally preserve throughout life span. DNA methyltransferase (DNMT) and demethylase enzymes are largely associated with the methylation pattern of gene. Aberrant expression of these enzymes might disregulated the methylation pattern that lead to genomic instability and promote tumorigenesis or maintain the malignant state of tumor cells (69). Eukaryotes DNMTs are classified as Dnmt1, Dnmt2, Dnmt3aDnmt3b (70). DNMT1 play essential role in methylation patterns especially responsible for maintaining DNA methylation levels, while DNMT2 help in methylation of transfer RNAs (tRNAs).
DNMT3a/DNMT3b is responsible for setting up of DNA methylation levels during embryogenesis as well as established genomic imprints during germ cell development (71) (72). Mostly cancer occurs as a result of transcription inactivation of a gene that is mainly associated with promoter hypermethylation. Abberant DNA methylation mediated activation or silencing of genes which may affects all cell signaling pathway , such as regulation of cell cycle, DNA repair, , activation of apoptosis or control of signaling networks related to tumor development (73).
127 our experts are availableright now
The inactivation of tumor suppressor genes as a result of methylation has been reported in osteosarcoma. Although silencing of TP53 and RB genes are not frequently occur by methylation, alteration in p53 and pRB pathway have been revealed as pathogenic methylation targets, particulary the locus of CDKN2A gene, which encodes the inhibitor of Mdm2, p14ARF, cyclin‑dependent kinase inhibitor and p16INK4a(7). Most of the gene exhibits as either targets of p53 or having p53- modulating activity have been identified in the methylated status and mostly silenced in OS or xenograft cell lines such as CDKN1A, GADD45 and HIC1(74).
Moreover, promoter hypermethylation silenced the transcription profile of several other tumor suppressor genes in OS-derived cell lines including DAPK1, RASSF1A, TIMP3 etc (Fig. 1) (6). (75) investigated that GADD45 gene encoding proteins of the 5-hydroxymethylcytosine (5 hmC) family, which mediates the methylation pattern in osteogenic differentiation, is mainly suppressed by PI3K/Akt and c-Myc pathways in OS cells (75). The suppression of GADD45 gene may be occurs as a result of aberrant promoter methylation.
IRX1 gene has been identified as pro-metastatic associated gene in OS when it poses little methylation. IRX1 gene encodes a protein of the Iroquois homeobox protein 1 family, a transcription factor which plays an important role in embryogenesis and was previously identified as a potential tumor suppressor in gastric cancer (78). It was investigated that the hypomethylation of IRX1 gene enhance pulmonary metastasis of the OS, since upregulation of this gene was mostly associated with the hypomethylation of its promoter in both OS-derived cell lines as well as in clinical samples resulted from the tumor.
IRX1 also plays a vital role in the regulation of NF‑kB / CXCL14 cell signaling pathway that associated with pro- metastatic ability of cancer. In addition, it has been revealed that IRX1 can enhance the metastatic properties of tumor cells both in vitro and in vivo, promote invasion and migration, as well as mediating resistance to anoikis in the murine model (Fig. 1) (22). Methylation analysis of more than 1.1 million loci was studied on biopsy sample taken from OS patients. It was revealed that patients having tumor recurrence poses high degree of methylation. A large number of oncogenes including, RAF1, PAK1 and SEMA4D are hypomethylated and highly expressed in human OS as compared to normal osteoblasts (79).
However, most of these epigenetic changes such aberrant activation or suppression are correlated with loss of expression ability at target loci in OS cells (66,79). A brief study to compare the DNA methylation patterns of OS samples with normal samples demonstrated that the promoters of particular genes are mostly methylated in the tumor samples. The essential function and pathways that targeted by these genes were described through protein‑protein interaction (PPI), resulted by the detection of cancer related genes which posses their promoters differentially methlated, in which 169 hypomethylated loci and 1379 hypermethylated loci were identified. Toll receptor signaling pathway constrained high degree of differential hypomethylation.
Differential hypermethylation was significantly greater in the transcription activator STAT3, the MAXI interactor signals transducer 1 (MXI1) and the T-cell acute lymphocytic leukemia 1 (TAL1) of PPI network. These genes were hypermethylated in OS cells and being classified as cancer related candidates (80). It has been revealed that HOTAIR gene is overexpressed in human OS cells, while inhibiting the expression of this gene results in the suppression of DNMT1 with a decreased in overall DNA methylation status.
Furthermore it has also been shown that the HOTAIR product reduced CDKN2A expression via blocking CDKN2A promoter activity through DNA hypermethylation. Mechanistically, HOTAIR gene plays crucial role in OS by downregulating the expression of miR-126, which acts as a negative regulator of DNMT1. Thus the activity of DNMT1 occurred in the lack of miR-126 overexpression, corresponding to the suppressing of CDKN2A due to DNA hypermethylation of its promoter, thus errand tumor development (81).
Moreover, large numbers of differentially methylated regions have been associated with up-regulation of genes such as CDK4, CEP72, KIRREL and SEZ6L2, which may play essential role in the pathogenesis of OS (82).
In addition, miR-449c acts as negative regular of c-Myc oncogene. The inhibition of miR-449c expression occurred as results of hypermethylation of two CpG island located next to miR-449c genomic region. The silencing of miR-449c contributed to enhance the expression of c-Myc that results to promote the activation of downstream targets that involved in OS tumorigenesis (83).
Investigation of more than 11,000 genes for differential methylation status and more than 3,000 genes for differential expression in the OS disclosed that the functions of genes associated to this tumor were mostly enriched in biological processes correlated to immune /inflammatory response, hematopoietic cell lineage pathways and Pertussis pathways. NRF and UBS were identified to be regulated by various genes in the OS. According to Kaplan Meier analysis of OS‑associated genes identified DNALI1, BHMT2, DOCK2 and RIPK3 as essential survival-related genes. PRAME and SEMA3A are constrained in the 40 genes and within the top 10 of the mainly differentially expressed genes in OS (84).
Histone Modification
Covalent modifications of the amino acid termini of the histones residues in nucleosomes play a crucial role in the regulation of gene expression (85). Histone modifications are classified as methylation, acetylation, sumoylation, phosphorylation and ubiquitination that make its more complex than DNA methylation(86). The amino termini modification of the histones disturbed the affinities of the chromatin‑ associated proteins and enhanced regulation of the vibrant transitions between transcriptionally active or repress chromatin levels. The level of acethylation of histones and other transcription residues are regulated by action of acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes. These both enzymes work in a dynamic equilibrium states. Aberrant acetylation with histone modifications are associated with the abnormal expression of tumor suppressor genes and oncogenes which ultimately results to tumorigenesis (87).
Histone deacetylases (HDACs) regulate modification in nucleosome conformation and play significant role in the regulation of gene expression1. HDACs are associated with cell-cycle differentiation and progression, and their aberrant expression is associated with various cancers2,3. The main functions of HDACs are removal of acetyl groups from amino terminus of nucleosomal histones and are implicated in the transcription repression9. HDAC inhibitors including suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), have anti-tumor effects, as they can induce terminal differentiation4,5 inhibit cell growth4±6, and prevent the formation of tumors in mice models7,8, and they have synergetic effect in the diagnosis of promyelocytic leukemia3. (histone deacetylation paper).
Histone deacetylase complex are also associated with RB tumour suppressor gene. RB mediates the initiation of G1 arrest by silencing the function of S-phase specific genes controlled by E2F. This suppression of gene is reliant on the association of deacetylase complex with the RB. The significance of the binding of HDAC to RB is described by two observations: first, the many tumor-specific mutations present in RB disrupt its association with the histone deacetylase [65••–67••] and second, the ability of viral oncoproteins including HPV16 E7 protein or the SV40 T-Antigen to disturb deacetylase activity from RB [65••,66••].(histone deacetylase 1 paper).
The various HDAC proteins, when tethered to the promoter by a GAL4 DNA binding domain, can repress transcription. In addition, different transcriptional repressors such as RB, Mad, YY1, nuclear receptors and the yeast Ume6 protein [51,59,61,63,64,65••–67••,68] are associated with a histone deacetylase complex. These proteins repress transcription through inhibiting the activity of deacetylase by trichostatin A (TSA) as of histone deacetylase inhibitor. These repressor proteins are thought to recruit the deacetylase complex to the promoter by either binding the enzyme directly, as in the case of RB (TKouzarides, A Brehm, unpublished data) or by contacting a component of the complex, such as Sin3 in the case of Mad [64].
Histones hypoacetylation is mosly observed in tumors cells (88). Furthermore, Histone methylation may activate or silence gene expression, depending on where methylation takes place. Generally, methylations of H3K4, H3K36 and H3K79 are associated with activation of gene expression while H3K9, H3K27 and H4K20 methylations are related to gene silencing. Thus, DNA methylation interacts with histones modification and collective action of both mechanisms plays significant role in gene expression (89). WNT5A is characterized as a family of genes which encode signaling glycoprotein and its distorted expression is related with different types of cancer. The transcription profile of promoters A and B of the WNT5A gene was studied in human osteosarcoma cell lines (SaOS-2 and U2OS), normal human osteoblasts, and in tumor tissue. It has been shown that both promoters A and B of WNT5A gene are active in normal osteoblasts, being that promoter B was nearly 11 times more active than promoter A. The promoter B constitutes three adjacent regions that enriched with CpG islands of exon 1β are highly methylated in both U2OS and SaOS-2 cells.
During studying the association of Histone modifications with the transcription activity of promoters A and B in U20S cell. it was identified that H3K4me3, a marker of histone activation, posses increased level of histone modifications in promoter A and a decreased level of histone modifications in promoter B, suggesting that H3K4me3 acts a repressive role via decreasing the activity of the promoter B. It has also been shown that promoter A is highly enriched with active H3K4me3 compared with promoter B in SaOS-2 and U2OS cells.
Moreover, the promoter B is highly enriched with repressive H3K27me3 in SaOS-2 cells. Embarrassment of promoter B of the WNT5A gene lead to developed OS characteristic and also been associated with both histone modifications and DNA methylation. These results point out that histone modification at the promoter B of WNT5A gene results to decrease it transcription activity in OS cells (90). The upregulation of demethylases KDM6A and KDM6B in OS cells as result of cisplatin treatment were mainly associated with demethylation of histone H3 lysine trimethylation (H3K27me3).
Cisplatin-resistant tumors cells had lower levels of H3K27me3 than susceptible OS specimens. In vitro inhibition of histone methyltransferase EZH2 in OS cells reduced H3K27me3 levels and led to cisplatin resistance. On the other hand, inhibition of demethylases KDM6A and KDM6B enhance H3K27me3 levels and turn back cisplatin resistance both in vitro and in vivo. This study indicates that H3K27me3 acts in decreasing KDM6A and KDM6B expression activity by increasing tumor cell susceptibility to cisplatin (91).
