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Keywords:

  • epigenome;
  • methylation;
  • DNMT;
  • molecular marker

Abstract

  1. Top of page
  2. Abstract
  3. DNMT Genes as Molecular Markers
  4. DNMT Genes as Molecular Targets
  5. Paradoxical Effect of DNMT1(−/−) and DNMT3B(−/−) Cells Treated with 5-Aza-CdR
  6. Conclusion
  7. REFERENCES

Tumor cell proliferation, de-differentiation, and progression depend on a complex combination of altered cell cycle regulation, excessive growth factor pathway activation, and decreased apoptosis. The understanding of these complex mechanisms should lead to the identification of potential molecular markers, targets, and molecular profiles that should eventually expand and improve therapeutic intervention.

It now appears clear that methylation plays a central role in transformation, both in vitro and in vivo. However, the exact targets and mechanism(s) are not yet fully understood. This is partly due to the significant number of genes altered by changes in intracellular methyltransferase activity and the chemical agents used to modulate gene expression. The complex nature of methylation's role in regulating gene expression suggests that in addition to investigating individual genes, researchers should develop more comprehensive methods to examine gene expression patterns and their predictive value as this will likely be necessary in the future.

If methylation plays a role in transformation, then it seems logical that genes regulating intracellular methylation status may be used as molecular markers to profile tumors by any new methods currently being developed. Perhaps more noteworthy is that DNMT genes may be found to be novel molecular targets for new factor-specific anticancer agents. This idea will be addressed. Cancer 2005. Published 2005 by the American Cancer Society.

The complex nature regulating the epigenome involves three primary mechanisms: 1) DNA methylation; 2) posttranslational modification of histones, including methylation, phosphorylation, or acetylation; and 3) chromatin alterations such as modifications of specific proteins binding to insulator sequences.1, 2 DNA methylation occurs almost exclusively at CpG nucleotides, and this subsequent pattern is transmitted through mitosis and maintained after DNA replication by DNA methyltransferase gene-1 (DNMT1), which has a greater affinity for hemimethylated than for unmethylated DNA.3, 4

Since the initial discovery of altered methylation in human cancer,5 a host of epigenetic alterations has been found, including global hypomethylation, gene hypomethylation and hypermethylation, and loss of imprinting.6, 7 Overexpression of DNMT and increased methylation of promoters of tumor suppressor genes and their associated silencing have been found in retinoblastoma8, 9 as well as many other genes.7 As a result of this, it has been suggested that silencing tumor suppressor genes by hypermethylation results in a cellular environment permissive to the development of chromosomal instability or genomic instability.7, 10 Although methylation itself may be one mechanism for silencing gene expression, other plausible processes include alterations in chromatin structure resulting in altered gene activity.7

A relatively new idea in cancer research involves the concept that many of the same genes or pathways that are altered in the process of transformation are subsequently used by tumor cells to evade damaging and cytotoxic effects of anticancer agents.11 The potential relation of these intracellular factors, which are altered as a result of malignant progression, may be critical to predicting how tumor cells respond to therapeutic intervention. Thus, the overarching theme of this review is an extension of the hypothesis that elevated DNMT activity, up-regulated as a result of transformation or a disorganized tumor microenvironment, alters gene expression and results in a phenotype resistant to anticancer agents. As such, DNMT genes may be molecular markers that can be profiled in tumors to either idealize cancer therapies or determine potential treatment outcome or, perhaps more importantly, be targeted (or inhibited) by novel factor-specific chemical agents to improve tumor cell death.

DNMT Genes as Molecular Markers

  1. Top of page
  2. Abstract
  3. DNMT Genes as Molecular Markers
  4. DNMT Genes as Molecular Targets
  5. Paradoxical Effect of DNMT1(−/−) and DNMT3B(−/−) Cells Treated with 5-Aza-CdR
  6. Conclusion
  7. REFERENCES

With the ever-expanding discovery of molecular components and metabolic mechanisms underlying the pathways involved in carcinogenesis and tumor cell resistance to anticancer agents, it is now possible to identify potential molecular markers that can be initially validated both in vitro and in vivo.12 These factors, or molecular markers, may be used to predict tumor histology, subtype, metastatic potential and, most importantly, to provide ideal therapy to individual patients.

The Kaneuchi et al. article published in this issue of Cancer is a prime example of the use of tumor genetics to identify potential molecular markers in the epigenome. The research by Kaneuchi et al.13 demonstrated that expression or nonexpression of the WT1 and WT1-AS genes is an important biochemical feature that distinguishes serous from clear cell ovarian adenocarcinoma, respectively. In addition, Kaneuchi and colleagues showed that altered methylation of the WT1 and WT1-AS promoters is central to gene inactivation. These results are significant because treatment, clinical outcome, and follow-up are substantially different between serous and clear cell ovarian adenocarcinoma.

Their results are consistent with those from other groups who have shown that methylation of specific genes may be a potential molecular marker in thyroid,14 breast,15 prostate,16 gastric,17 and colon carcinomas.3 As the technology to study both the genome and epigenome increases and improves, it seems likely that the role of epigenetics in transformation and tumor cell resistance will expand, as will the number of methylation-related molecular markers.

DNMT Genes as Molecular Targets

  1. Top of page
  2. Abstract
  3. DNMT Genes as Molecular Markers
  4. DNMT Genes as Molecular Targets
  5. Paradoxical Effect of DNMT1(−/−) and DNMT3B(−/−) Cells Treated with 5-Aza-CdR
  6. Conclusion
  7. REFERENCES

Molecular-targeted therapies hold the promise of being highly selective for cancer cells versus healthy cells, thus drastically reducing side effects in patients while increasing cytotoxicity of tumor cells. With the discovery of new drugs that can be designed to target specific molecular targets, it should be possible to profile tumors and determine the most appropriate therapy for each patient. Such therapeutic decisions will be based on unique characteristics of each tumor that include gene mutations, protein structure changes, signaling pathway alterations, and, perhaps, changes in regulation of the epigenome. Thus, it seems logical to hypothesize that DNMT genes may be potential molecular targets in specific tumor types.

With this in mind, we have established criteria for an ideal molecular target. Theoretically, an ideal molecular target should: 1) be overexpressed or constitutively active in tumor cells; 2) enhance tumor proliferation; 3) inhibit watchdog or fidelity genes; 4) incite a prosurvival effect; and 5) enhance resistance to therapeutic modalities (e.g., irradiation and chemotherapy). DNMT genes clearly meet several of these criteria. Alterations in methylation patterns and activity have been observed in countless tumors and tumor cell lines,3 and agents such as 5-Aza-CdR that inhibit DNMT activity have been shown to also inhibit tumor cell proliferation and, to a lesser extent, induce cell death.3, 11 On the basis of these findings, it seems logical to assume that DNMT genes may be effective molecular targets.

The one criterion not met by DNMT genes is up-regulation of prosurvival signaling factors. However, in this regard, there are recent data suggesting that overexpression of DNMT may activate as well as silence genes in tumor cells.18 Although these results are not obvious, two studies have been published that describe the use of microarray analysis to examine changes in gene expression, and these studies suggest that hypermethylation may activate specific genes as well as gene families.11, 18 In published work by Ordway et al.,18 of Dr. Tom Curran's laboratory at St. Jude Children's Hospital, Rat-1 cell lines overexpressing DNMT1 were investigated by microarray analysis for changes in gene expression. These results showed that slightly more genes (roughly 375 genes) were increased in cells lines overexpressing DNMT1 than were decreased (roughly 325 genes). An examination of these data shows several candidate genes that are considered prosurvival or antideath genes.

A similar microarray analysis was done on somatic HCT116 cell line knockout for DNMT1(−/−), DNMT3B(−/−), and double knockouts (DKOs).11 Previous studies have shown that both DNMT1 and DNMT3B cooperatively maintain DNA methylation and that genetic disruption of both genes reduced genomic DNA methylation by roughly 95%.19 Similar to results shown by Ordway et al., a significant number of genes decreased in cells lacking DNMT activity, and roughly an equal number of genes increased in the DNMT1(−/−), DNMT3B(−/−), and DKO cell lines.11 The genes that decreased after genetic inhibition of DNMT activity appear to be involved in a diverse range of critical intracellular processes including cell cycle regulation, DNA repair, and most importantly, programmed cell death, proliferation, and several prosurvival signaling cascades.

One of the most interesting observations from these microarray data is that several prosurvival or proproliferative factors appear to be regulated by methylation status, i.e., their expression is decreased by hypomethylation. This raises an interesting question: Does overexpression of DNMT genes silence tumor suppressor genes as well as activate prosurvival or antideath genes?

The forced genetic overexpression of DNMT1 transforms mammalian cells, and conversely, the chemical inhibition of DNMT1 activity reverses the phenotypic transformation.20 This observation, along with those discussed above, suggest that DNMT1 may be a potential molecular target for therapeutic intervention. In this regard, several clinical studies including Phase II trials of DNMT inhibitors have been completed.21 However, results of these studies are unclear, and it has been suggested that this is because of a lack of specificity and targeting of these agents to specific potentially responsive tumor subtypes.

Paradoxical Effect of DNMT1(−/−) and DNMT3B(−/−) Cells Treated with 5-Aza-CdR

  1. Top of page
  2. Abstract
  3. DNMT Genes as Molecular Markers
  4. DNMT Genes as Molecular Targets
  5. Paradoxical Effect of DNMT1(−/−) and DNMT3B(−/−) Cells Treated with 5-Aza-CdR
  6. Conclusion
  7. REFERENCES

Recently, we addressed this issue by treating the somatic HCT116 knockout DNMT1(−/−) and DNMT3B(−/−) cell lines with 5-Aza-CdR followed by use of microarray technology to perform genomic expression analysis.11 Analysis of these experiments presented several unusual results suggesting that tumor cells with aberrant DNMT expression or activity may respond to agents that inhibit either methyltransferase activity or chromatin compaction in an unexpected or paradoxical manner. This effect is most predominantly observed in DNMT1(−/−) cells exposed to 5-Aza-CdR or TSA where eight areas were identified (Fig. 1). For example, in area VII, a significant number of genes are up-regulated (green color) in the DNMT1(−/−) somatic knockout cells. However, after addition of either 5-Aza-CdR or TSA, these genes are observed to be down-regulated. In contrast, in area III, genes down-regulated in the DNMT1(−/−) cells are up-regulated after exposure to 5-Aza-CdR or TSA. To a lesser extent, this paradoxical effect is also observed in DNMT3B(−/−) knockout cells treated with either 5-Aza-CdR or TSA (Fig. 1).

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Figure 1. Paradoxical effect of DNMT1(−/−) and DNMT3B(−/−) cells treated with 5-Aza-CdR or TSA. Ratios of gene expression values were generated and clustered hierarchically. (A) DNMT1(−/−) and (B) DNMT3B(−/−) cell lines and areas of interest are identified with an arrow and Roman numeral. Areas of interest that demonstrated a significant paradoxical change in gene expression after exposure to 5-Aza-CdR (I–VII) or TSA (I–VI) are shown.

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The 11 genes in area IV that changed from up-regulated in the DNMT1(−/−) cells to down-regulated after subsequent chemical exposure (Table 1) include FK506 binding protein 8 (FKBP8) and forkhead box F2 (FOXF2). Of particular interest is IGF2, whose expression is linked to abnormal imprinting in cancer, and surprisingly, DNMT3B itself.3 In addition, 33 genes changed from down-regulated in the DNMT1(−/−) cells to up-regulated after chemical exposure (Fig. 1, area VII; Table 1), which include breast cancer antiestrogen resistance 3 (BCAR3), Williams Beuren syndrome gene (WBSCR22), arsenate resistance protein 2 (ARS2), and the xanthine dehydrogenase gene (XDH). Taken together, these results reinforce observations described earlier that pharmacologic manipulation is not the same as genetic knockout of DNMT.

Table I. DNMT1(−/−) and DNMT3B(−/−) Show a Paradoxical Effect to Chemical Modification
Region IV (11 genes)Region VII (33 genes)
  1. The genes encompassed in regions IV and VII of figure 1 are shown.

FKBP8: FK506 binding protein 8BCAR3: Breast cancer anti-estrogen resistance 3UBE2G2: Ubiquitin -conjugating enzyme:
FOXF2: Forkhead box F2GNB5: Guanine nucleotide binding protei, β 5THOP1: Thirnet oligopeptidase 1
AHSG: α-2-HS-glycoproteinARS2: Arsenate resistance protein 2SC65: Synaptonemal complex protein SC65
PPP2R5A: PP2, regulatory subunit B,TIEG2: TGF β inducible early growth response 2MAN1A1: Mannosidase, a, class 1A, member 1
CA9: Carbonic anhydrase IXMCSP: Mitochondrial capsule selenoproteinEFNB1: Ephrin -B1
CDH13: Cadherin 13, H -cadherin (heart)MLLT6: Myeloid/lymphoid; translocated to, 61312232A: EST
IGF2: Insulin-like growth factor 2MYO1C: Myosin 1CDSC1: Desmocollin 1
13CDNA73: Hypothetical protein CG003FLJ11126: Hypothetical protein FLJ11126 ACTA1: Actin, a 1, skeletal muscle
DNMT3B: DNA-methyltransferase 3 βNOVA2: Neuro-oncological ventral antigen 2ACTA1: Actin, a 1, skeletal muscle
TRB@: T cell receptor, β locusCD84: CD84 antigen (leukocyte antigen) ACY1: Aminoacylase 1
FLOT1: Flotillin 1MYO1C: Myosin ICC22ORF3: Chromosome 22 open reading frame 3ACY1: Aminoacylase 1
 NM23-H6: Nucleoside diphosphate kinase F0811: Homo sapiens DNA sequence
 WBSCR22: Williams Beuren syndromeF0811: Homo sapiens DNA sequence
 TCCCIA00427: Homo sapiens clone TCCCIA00427 SLC31A1: Solute carrier family 31 (Cu transporters)
 NT5B-5′-nucleotidase, cytosolic IISLC31A1: Solute carrier family 31 (Cu transporters)
 MYST1: MYST histone acetyltransferase 1 CCL16: Chemokine (C -C motif) ligand 16
  CCL16: Chemokine (C -C motif) ligand 16
   KIAA1276: KIAA1276 protein
  KIAA1276: KIAA1276 protein
   EHHADH: Enoyl -Co A, hydratase/3 -hydroxyacyl Co A
  EHHADH: Enoyl -Co A, hydratase/3 -hydroxyacyl Co A
   REL: v -Rel reticuloendotheliosis viral oncogene homolog
  REL: v -Rel reticuloendotheliosis viral oncogene homolog
   ERCC1: excision repair cross-complementing deficiency
  ERCC1: excision repair cross-complementing deficiency
   XDH: Xanthine dehydrogenase
  XDH: Xanthine dehydrogenase

One possible explanation for this effect comes from analysis of STAT1 and IL-6 gene families, both of which regulate the transcription of other downstream genes. Hierarchical cluster maps identified STAT1 as significantly up-regulated in all three knockout cell lines but not after drug treatment.11 In contrast, IL-6 was up-regulated in cells treated with 5-Aza-CdR or TSA but not in the three knockout cell lines. Thus, it appears STAT1 and IL-6 may be regulated by different mechanisms and, in turn, activate separate classes of genes. Regardless of the exact mechanism(s), these results show a paradoxical effect that is critical to validating DNMT genes as potential molecular targets. Results of these experiments very strongly suggest how tumor cells respond to agents that inhibit DNMT activity, and the subsequent changes in gene expression patterns may vary widely depending on the inherent genetic and functional activity of intracellular DNMT genes. This is of central importance and suggests that clinical studies using methylated proteins as molecular makers, or DNMT genes as molecular targets, will require tumor profiling much like other factor-specific anticancer agents. This may explain the uneven nature of previous clinical studies with methyltransferase inhibitors and suggests that more detailed analysis of the effect(s) of these inhibitors on gene expression and cell survival will be required of future translational research and clinical trials.

Conclusion

  1. Top of page
  2. Abstract
  3. DNMT Genes as Molecular Markers
  4. DNMT Genes as Molecular Targets
  5. Paradoxical Effect of DNMT1(−/−) and DNMT3B(−/−) Cells Treated with 5-Aza-CdR
  6. Conclusion
  7. REFERENCES

It is becoming increasingly clear that changes in the epigenome play a critical role in both cellular transformation and carcinogenesis as well as in the mechanism by which tumor cells defend themselves against damaging and cytotoxic effects of therapeutic modalities. These observations would suggest a role for testing epigenetic alterations in tumors to determine any potential molecular markers or targets. The recent functional and molecular characterization of properties of DNMT genes and their enzyme properties should allow a more systematic and rational approach to new drug discovery and design. However, similar to many of the new agents currently in clinical study, a rigorous set of translational work must also be performed to establish and validate molecular markers with such clinically significant endpoints as clinical complete response, local and distant control, and disease-free and overall survival.

REFERENCES

  1. Top of page
  2. Abstract
  3. DNMT Genes as Molecular Markers
  4. DNMT Genes as Molecular Targets
  5. Paradoxical Effect of DNMT1(−/−) and DNMT3B(−/−) Cells Treated with 5-Aza-CdR
  6. Conclusion
  7. REFERENCES