Abstract #

Both appropriate DNA methylation and Histone modifications play a crucial role in the maintenance of normal cell function and cellular identity.

In cancerous cells these "epigenetic belts" become massively pertubed, leading to significant changes in expression profiles which confer advantage to the development of a malignant phenotype.

DNA-methyltransferase 1 (DNMT1), DNMT3A, DNMT3B are the enzymes responsible for setting up and maintaing DNA metylation patterns in eukaryotic cells.

Intriguingly, DNMTs were found to be overexpressed in cancerous cells, which is believed to partly explain the hypermethylation phenomenon commonly observed in tumors.

However, several lines of evidence indicate that further layers of gene regulation are critical coordinators of DNMT expression, catalytic activity and target specificity.

Splice variants of DNMT transcripts have been detected with seem to modulate methyltransferase activity. Also, the DNMT mRNA 3'UTR as well as the coding sequence harbors multiple binding sites for trans-acting factors guiding post-transcriptional regulation and transcript stabilization.

However, microRNAs targeting DNMT transcripts have recently been discovered in normal cells, yet expression of these microRNAs was found to be diminished in Breast cancer tissues.

In this review we summarize the current knowledge on mechanisms which potentially lead to the establishment of a DNA hypermetylome in cancer cells.

Introduction #

The molecular mechanisms underlying the development and progression of Breast cancer are far from being understood. It is evident that the initiation of breast cancer as well as its transition toward distinct breast cancer subtypes is triggered by the accumulation of pathologically altered gene function.

Like in other cancers, the increasing number of deregulated genes subsequently affects virtually any import cellular network, such as cell cycle control, Apoptosis, DNA repair, Detoxification, Inflammation, Cell adhesion or migration.

According to the somatic mutation theory cancer has long been considered as a genetic disorder of fatal acquisition of multiple mutations in key genes, which coordinate these functional networks. Such mutations can result either in inactivation of Tumor suppressor genes (e.g. TP53, BRCA1) or activation of proto-Oncogenes (e.g. MYC), both of which contributes to the malignant state of a transformed cell.

During the past decade, the somatic mutation theory of cancer has been revolutionized for it became evident that epigenetic malfunction plays a role as equally important as genetics in cancer development.

The concept of epigenetics describes mitotically stable states and changes of gene activity that do not involve alterations of the primary DNA sequence, thus provide a second layer of information above the pure genomic blueprint.

Epigenetic mechanisms coordinate crucial biological processes, like X-inactivation, Genomic imprinting, Position-effect variegation, Reprogramming of genomes during differentiation and development, or RNA interference leading to postranscriptional gene silencing.

It is not surprising that defects in the dynamics of these key functions were found to be associated with many human disorders, including breast cancer.

In recent years, two epigenetic mechanisms have emerged as the most critical players of transcriptional regulation: The methylation of DNA and chemical histone tail modifications.

DNA methylation #

It refers to the covalent post-replicative addition of a methyl group (-CH3) onto the 5-carbon of the cytosine ring within CpG dinucleotides.

This enzymatic reaction is conferred by DNA methyltransferases (DNMT), which catalyze the transfer from the methyl group donor S-adenosyl methionine.

Typically, such CpG dinucleotides are enriched in gene promoters or the first exon where they cluster to form a so called CpG island. Approximately 60% of protein-coding mammalian genes harbor CpG islands in their promoter region.

These are normally unmethylated in transcriptionally active genes like housekeeping genes, whereas developmental and tissue-specific genes mostly appear to be methylated and silenced in differentiated tissues.

In Cancer, however, numerous genes which are unmethylated in the non-malignant tissue become aberrantly methylated in the tumor.

Since the first discovery of a hypermethylated gene in cancer, retinoblastoma tumor suppressor (RB1), many tumor suppressor genes have been identified being hypermethylated in tumorours tissues as compared to their normal counterparts, e.g. VHL, CDKN2A, or BRCA1.

Although our knowledge on epigenetically inactivated genes in cancer is constantly increasing, the basic methanisms underlying aberrant DNA methylation as well as the selection of genes that become methylated are only rudimentary understood, and shall be reviewed further on.

Histone modification #

The second key player in chromatin conformation and transcriptional regulation are histone modifications. Histone proteins constitute the nucleosome around which DNA is tightly packaged.

Their N-terminal tails reach out of the nucleosomal core and harbor numerous spots for protein modifications, such as acetylation, methylation, phosphorylation, sumoylation, ubiquitination or ADP ribosylation.

Both the type of modification and the affected amino acid residue determine the tightness of the DNA-histone interaction, leading to either an open chromatin state allowing active transcription (e.g. acetylation of lysine) or to a compact chromatin state associated with transcriptional repression (e.g. deacetylation of the same residue).

Altered histone modifications in breast cancer will be reviewed in a further article of this issue, but one essential relationship ought to be mentioned: DNA methylation and histone modifications interact with each other in the regulation of gene expression.

It is generally believed that DNA methylation is the initiating event that marks certain genomic sites for the establishment of a transcriptionally inactive chromatin state.

DNA methylation, however, may also depend on prior methylation of histone 3 at lysine 9 (H2K9), and is followed by binding of methyl-CpG binding domain proteins (MBDs) which contribute to gene repression by the recruitment of histone deacetylases (HDACs) to the nucleosome.

Also, for certain genes it has been shown that the initial recruitment of DNMTs to target sequences is mediated by Enhancer of zeste homologue 2 (EZH2) as a part of the repressive polycomb group (PcG) of proteins, increasing the complexity of relations between various epigenetic repression systems.

microRNA #

In recent years, the discovery of a class of small non-coding RNAs, so called microRNAs (miRNAs), has gained much attention in oncological research.

miRNAs are regulatory RNAs 20-30 nucleotides in length, that perfectly match the 3' untranslated regions (3'UTR) of target mRNAs, resulting in its degratation or inhibition of mRNA translation.

It is the function of the target mRNA that determines a miRNA acting either tumor suppressive (if directed against proto-oncogene transcripts) or oncogenic (if directed against proto-oncogene transcripts).

Prominent members of miRNAs include the let-7 family (containing at least 11 homologous miRNAs), whose depletion in breast, lung, and colon cancer causes enhanced tumorigenicity. Another example is miR-21, whose overexpression in breast cancer confers increased invasion capacities and promotes tumor metastasis to the lung.

The number of genes known to be regulated by miRNAs is growing rapidly. The latest release of the Sanger miRNA registry currently annotates more than 800 human miRNAs (miRBase), yet many more miRNAs are expected to be identified in the future.

It is not surprising that miRNAs, just like protein-coding genes, have to be tightly regulated in order to contribute to a distinct transcriptome of a normal cell.

In cancer, however, miRNAs were found to be massively deregulated. Recent genome-wide approaches revealed that miRNAs are globally downregulated in Breast cancer.

Signatures of deregulated miRNAs were shown to be useful in subtyping mammary carcinomas, or determining their aggressiveness, e.g. in node-negative estrogen receptor-positive tumors.

Like protein-coding genes, DNA sequences encoding miRNAs were found to be a target of aberrant DNA methylation, explaining in part how miRNAs may be upregulated (through DNA hypermethylation) in cancer.

Besides DNA methylation, a failure of post-transcriptional regulation may also lead to impaired miRNA biogenesis, as has been shown for the miRNA maturation responsible endoribonuclease Dicer, which is commonly expressed at lower levels e.g. in progressive breast cancer.

The production of mature miRNA underlies a complex process of subsequent modifications of the primary transcript, termed pri-miRNA. The primary transcript contains a stem-loop structure representing the active miRNA species. This stem-loop is liberated by the nuclear ribonuclease III Drosha, and then termed premiRNA.

After export to the cytoplasm the precursor miRNA is further processed by the ribonuclease Dicer, resulting in the mature miRNA.

Finally, this miRNA is loaded into the RNA induced silencing complex (RISC) where it exhibits translational repression of its target mRNA. In tumourous cells, discrepancies between the levels of primary transcript, precursor and mature miRNA have been reported, strongly arguing for defects in the maturation pathways of miRNAs on various levels, such as Drosh or Dicer processing.

The second part of this article the current knowledge on defects in miRNA processing shall be highlighted.

Mechanisms of Altered DNA Methylation #

DNA methylation patterns differ largely between tumor tissues and corresponding normal tissues. A paradoxon commonly observed in carcinomas is that dispite of the regional hypermethylation of tumor suppressor genes, the global 5-methylcytosine content is drastically decreased in the bulk of the tumor genome.

Less frequent than regional DNA hypermethylation, also regional DNA hypomethylation occurs in cancer, resulting in the activation of potential oncogenes.

The existence of specific enzyme conferring active demethylation of methylated DNA is still unclear. However, enzymes conferring methylation of DNA have been well characterized.

DNA Methyltransferases #

The C-terminal catalytic domain of DNMTs transfers methyl groups onto cytosine residues within the DNA, thus methyltransferases represent the crucial enzyme class responsible for hypermethylation of tumor suppressor genes.

In mammals, five members of the DNMT protein family have been discovered (Dnmt1, Dnmt2, Dnmt3a, Dnmt3b, Dnmt3L), of which only three were shown to possess catalytic methyltransferase activity (1, 3A, 3B).

Dnmt1 exhibits a strong preference for hemimethylated over unmethylated DNA, and its particular targeting of replication foci, as shown by co-localiation with proliferating cell nuclear antigen (PCNA), is thought to allow copying of the parental DNA methylation pattern onto the newly synthesized DNA daughter strand. Therefore, Dnmt1 is regarded as a maintenance methyltransferase.

The DNMT3 family consists of two catalytic members, 3A, 3B, both of which exibit increased methyltransferase activity towards unmethylated over hemi-methyltransferase activity towards unmethylated over hemi-methylated DNA, which is why they were termed de novo methyltransferases.

DNMT3A -/- knock-out mice appear to be normally developed, but die shortly after birth.

Homozygous inactivation of DNMT3B leads to embryonic lethality due to multiple developmental disorders including growth impairment and rostral neural tube defects.

In human, a specific mutation in the DNMT3B gene is responsible for a syndrome referred to as ICF (Immuno-deficiency, Centromere instability and Facial abnormalities), which is characterized by global hypomethylation of centromeric DNA repeat sequences, chromatin decondensation and genomic instability in tissues of affected patients.

The remaining members of the DNMT family, Dnmt2 and Dnmt3L, lack cytosine methyltransferase activity, although Dnmt3L was shown to be capable of stimulating de novo DNA methylation mediated by Dnmt3a.

DNA Hypomethylation in Breast Cancer #

Among solid tumor types, global DNA hypomethylation in most evident in breast cancer with up to 50% of cases showing reduced 5-methylcytosine content when compared with normal tissue counterparts.

Hypomethylation in breast carcinomas mainly affects repetitive DNA sequences and pericentromeric satellite DNA, which are normally heavily methylated in non-malignant cells.

For instance, long interspersed nuclear elements (LINEs) represent retrotransposons that are methylated in all mammalian cell types.

Cancer-related hypomethylation of these transposable elements induces transcriptional reactivation, thus they can relocate and integrate into other sites of the genome, leading insertional mutagenesis and contributing to genomic instability.

Hypomethylation of the Sat2 and Satα repeats frequently occurs in certain cancers, such as ovarian and breast cancer. In the latter, Sat2 hypomethylation was shown to affect 50% and SatR-1 hypomethylation 86% of breast tumors.

In contrast to ovarian cancer, where increased satellite DNA hypomethylation is associated with tumor progression, satellite DNA hypomethylation in breast cancer is involved in early tumor development.

Though being a relatively rare event, DNA hypomethylation can also affect individual genes. In breast cancer, this is the case for the Melanoma associated cancer/testis antigens MAGE, which are methylated and silenced in adult tissues, but hypomethylated and expressed in several tumors and breast cancer cells.

Other hypomethylated genes in breast tumors include the gene encoding the plasminogen activator uPA (PLAU), the breast cance specific protein 1/synuclein-𝛄 gene (SNCG), and more recently reported, the multidrug resistance 1 gene (MDR1)

The underlying mechanisms leading to DNA hypomethylation in cancer have not yet been clearly elucidated. There is no reduction of DNA methyltransferase activity in cancer cells.

Despite, in animal models knockdown or deficencies in the activities of DNMTs lead to genome-wide DNA hypomethylation and chromosomal instability.

Reports from hepatocellular carcinoma and leukaemia suggested that enzymatically inactive DNMT splice variants compete with enzymatically active forms for the same binding site of pericentromeric satellite DNA, thereby inhibiting DNA methylation. However, this association could not be confirmed in other tumor types., so further investigations are needed.

De Novo Gene Methylation is a Non-random Process #

As stated before, many unmethylated tumor suppressor genes in normal tissues acquire hypermethylation during tumor development, so the key question here is: does aberrant hypermethylation of genes in cancer follow a random process that is accompanied by clonal selection of those cells which gained growth advantages, or do DNMTs specifically recognize target genes, which implies that the repertoire of potential targets for invactivation is already encoded by intrinsic or extrinsic factor?

Currently, research results seem to support the latter hypothesis. First to mention, it was shown that distinct tumor types may habor methylation in several common genes, but also in numerous different genes. This finding lead to the first hypermethylation profiles of human cancer.

The analysis of 12 tumor suppressor genes in 600 primary tumors representing 15 tumor entities revealed tumor-characteristic methylation changes were shared and others were cancer type-specific.

As an example, the gene encoding secreted fizzled-related proteins (SFRP) 1 and 2, both encoding inhibitors of the Wnt signaling pathway, were found to be high-frequently methylated in virtually any human tumor type, including breast and colon cancer.

Furthermore, SFRP methylation occurs already in early tumor stages, suggesting that epigenetic inactivation of SFRP genes may be a common hallmark of human neoplasia following non-random but targeted gene selection.

Second, specific nulceotide sequence patterns within gene promoters were identified that are more prone to hypermethylation than other sequences, which tend to be methylation-resistant.

Feltus and co-workers have demonstrated in an ectopic Dnmt1 overexpression model that CpG islands differ in their susceptibility to de novo methylation, suggesting the existence of cis-acting intrinsic factors that facilitate methylation of specific target sequences.

Indeed, in a further sutdy Feltus and colleagues were able to identify 13 short DNA sequence patterns between 11 bp and 37 bp in length, which were able to distinguish between methylation-prone and methylation-resistant promoters with 87% accuracy.

Interestingly, only the methylation-prone DNA sequences were closely associated with CpG islands, whereas methylation-resistant sequences were randomly distributed along the analyzed chromosome, further supporting the idea of an intrinsic hypermethylation code in the DNA sequence of affected gene promoters.

However, this study used a model of Dnmt1 overexpression, which exerts only limited de novo methylation capacity. It remains to be shown whether Dnmt3a or Dnmt3b overexpression models many also identify similar methylation-prone DNA sequences in the mammalian genome.

At least in part, another finding supports the hypothesis that aberrant de novo methylation is a non-random process.

It has been recently demonstrated that chromatin repressive proteins, such as PcG, can mark certain target genes for hypermethylation by DNMTs.

In an approach to identify the functional relation between DNMTs and EZH2, Vire and colleagues showed that the presence of EZH2 is tightly associated with the presence of Dnmt1, Dnmt3a and Dnmt3b proteins at gene promoters, e.g. those of the MYT1 or KCNA1 gene.

They found that after siRNA-mediated depletion of EZH2 expression DNMTs were no more bound to the respective promoters, while depletion of DNMT expression did not prevent EZH2 from endowing target sequences with the repression mark H3K27.

In another study, Schlesinger and co-workers investigated whether the H3K27 mark is specifically associated with de novo methylated CpG islands in the colon tumor cell line Caco-2.

Supporting the previous idea, all analyzed genes found to be hypermethylated in the cell line were also found being eriched for H2K27. Interestingly, the acquisition of the H3K27 mark turned out to occur not only in the tumor cells themselves, but already existed in normal control tissues, as determined by chromatin immunoprecipitation (ChIP) analysis using an antibody against trimethylated H3K27.

Since unmethylated control genes in normal tissues, such as blood lymphocytes, embryonic stem cells and fibroblasts were lacking this histone mark, it is conceivable that a subset of target genes may first become "primed" by the H3K27 mark through EZH2 in normal tissues, which then could represent a favored substrate for hypermethylation during cancer development.

Among the identified PcG target genes were the previously mentioned SFRP1 and SFRP2 gene, suggesting the EZH2-mediated de novo methylation may be affecting in particular developmental regulator genes that are occupied by polycomb repressor complexes in embryonic stem cells, increasing their susceptibility to non-random methylation-mediated silencing in cancer.

DNMTs are Differentially Expressed in Human Cancer #

DNMTs are ubiquitously expressed at distinct level in normal human tissues. In cancer, they are overexpressed in varous tumor types, e.g. in leukemia, colorectal cancer, prostate cancer, ovarian cancer, endometrial cancer and breast cancer.

Surprisingly, the mean levels of Dnmt1, Dnmt3a, and Dnmt3b overexpression turned out to be quite similar among different tumor type. However, these levels were not strikingly high, ranging from 1.8 to 2.9-fold in breast cancer up to 4-fold in colon cancer.

In breast cancer, approximately 30% of patients revealed overexpression of Dnmt3b in the tumor tissue as compared to normal breast tissue. Taken only these overexpressing tumors into account, the Dmt3b expression change was 82-fold, thus being significantly higher.

Interestingly, Dnmt1 and Dnmt3a were overexpressed in only 5% and 3% of breast carcinomas, exhibiting also a lower expression change of 17- and 14-fold in the affected tumors, respectively.

These results have two implications: First, it appears that Dnmt3b plays the predominant role over Dnmt3a and Dnmt1 in breast tumorigenesis.

This is consistent with a recent study in breast cancer cell lines, which demonstrated a strong correlation between total DNMT activity and overexpression of Dnmt3b, but not with the expression of Dnmt3a or Dnmt1.

Second, it is noteworthy that due to the lack of a specific antibody against Dnmt3b almost any expression analysis so far has been performed on the RNA level.

Only recently, a study of colon cancer employing 765 primary colorectal carcinomas revealed that Dnmt3b protein overexpression affects 15% of cases, in consistency with the relatively low frequency of overexpression reported in other tumor types.

The fact that only a subgroup of tumors is affected by Dnmt3b overexpression raises the question whether this can be regarded as a universal feature of tumorous cells.

In general, it is possible that further regulators of DNMT activity may play an important role in the dysfunction of the DNA methylation machinery.

For instance, DNMT target sequence specificity may be impaired by genetic or regulatory factors, such as DNMT-associated proteins or protein complexes in which DNMTs reside.

Furthermore, DNMT3B is the only DNA methyltransferase whose mRNA is expressed as several alternative splice variants. Although DNMT3B1 and DNMT3B3 are the most abundantly expressed transcripts, only DNMT3B1 and DNMT3B2 were shown to be catalytically active, while the remaining splice variants do not possess methyltransferases activity due to the lack of a C-terminal catalytic domain.

Despite this, Ostler et al. identified over 20 novel DNMT3B transcripts from various cancer cell lines that are aberrantly spliced at the 5'-end and lacked the C-terminal catalytic domain.

Suprisingly, forced expression of one of the variant transcripts (DNMT3B7) significantly changed the DNA methylation pattern in kidney HEK293 cells.

Since no catalytic domain could be responsible for the DNA methylation changes, it was proposed that the truncated Dnmt3b7 protein could interfere with DNA methylation processes by binding of Dnmt3b interaction partners, or that the truncated version of DNMT3b affects the activity of catalytic DNMTs by directly binding to the DNA.

Adding more comprlexity to this, the catalytically inactive Dnmt3L directly binds Dnmt3b and positively stimulates its methylation activity.

Taking into account the large number of physiological and aberrant splice variants of the DNMT3B transcript, it can only be speculated that the precise mechanisms of Dnmt3b action and the biological function of the many diverse transcripts still have to be identified.

Despite the low-frequent overexpression of DNMTs in tumors, a direct evidence for Dnmt3 involvement in cancer has been previously described.

Soejima and colleagues demonstrated that Dnmt3b contributes to the oncogenic phenotype in a lung cancer model. In their study, Dnmt3b was able to promote oncogenic transformation induced by SV40 T antigen in bronchial epithelial cells, whereas antisense suppression of Dnmt3b prevented tumor growth in soft agar assays.

In a study on colon tumorigenesis the impact of a conditional inactivation of DNMT3B in APC (Min/+) mice was investigated.

Although loss of Dnmt3b expression had no impact on micro-adenoma formation, it significantly descreased the formation of macroscopic colonic adenomas, suggesting a role of Dnmt3b in the transition from one to the other stage.

In breast cancer, elevated expression of Dnmt3b was shown to be significantly associated with higher histological grade, absense of estrogen receptor-α and presence of the proliferation marker Ki67, pointing to a potential involvement of Dnmt3b in breast tumor progression and aggressiveness.

In the same study, an association of high Dnmt3b expression and reduced relapse-free patient survival was detected, although it was only significant in a subgroup of patients receiving adjuvant hormone therapy, while in patients receiving adjuvant chemotherapy no difference could be detected.

A question of particular interest here is: Is a more maligant phenotype, which seems to be associated with higher Dnmt3b expression, indeed related to increased hypermethylation of genes in these tumors? The answer to this is still not clear.

While clear associations have been demonstrated in some in vivo studies, in other studies this relationship could not be confirmed. For instance, in colorectal tumors no significant correlation could be found between the level of Dnmt3b overexpression and the methylation status of the four indicator genes adenomatous polyposis coli (APC), estrogen receptor α (ESR1), cyclin-dependent kinase inhibitor 2A (CDKN2A), and mutL homolog 1 (MLH1).

Similar results have been described in hepatocarcinoma, lung cancer and gastric carcinoma, although different indicator genes were used.

Dnmt3b is Post-Transcriptionally Regulated #

MicroRNAs and Breast Cancer #

Concluding Remarks #

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