Vol.

10, 4589 4596, July 15, 2004

Clinical Cancer Research 4589

Review

The Clinical Application of Targeting Cancer through Histone Acetylation and Hypomethylation
Jill Gilbert,1 Steve D. Gore,2 James G. Herman,3 and Michael A. Carducci4
1

Division of Oncology and Hematology, Stanley S. Scott Cancer Center, Louisiana State University, New Orleans, Louisiana, and Divisions of 2Hematologic Malignancies, 3Cancer Biology, and 4 Medical Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland

ABSTRACT
Methods of gene inactivation include genetic events such as mutations or deletions. Epigenetic changes, heritable traits that are mediated by changes in DNA other than nucleotide sequences, play an important role in gene expression. Two epigenetic events that have been associated with transcriptional silencing include methylation of CpG islands located in gene promoter regions of cancer cells and changes in chromatin conformation involving histone acetylation. Recent evidence demonstrates that these processes form layers of epigenetic silencing. Reversal of these epigenetic processes and up-regulation of genes important to prevent or reverse the malignant phenotype has therefore become a new therapeutic target in cancer treatment.

INTRODUCTION
Multiple genetic aberrations are observed in the progression from the normal phenotype to the malignant phenotype. The particular mutations and stepwise progression vary between malignancies, but several mutational events are common among the various cancers. Such well-described changes include mutations of the p53 tumor suppressor gene and abnormalities of p16, a tumor suppressor gene and cyclin D kinase inhibitor (1). A common event in the progression toward malignancy involves transcriptional silencing of key nonmutated genes such as tumor suppressor genes or mismatch repair genes (2). Silencing of the mismatch repair gene MLH1 has been well described in colon cancer (35). Additionally, the class glutathione S-transferase (GSTP1) gene, which encodes the glutathione S-transferase detoxification enzyme, is silenced in the majority of prostate adenocarcinomas (6). Transcriptional silencing is not limited to

solid tumors but has also been found in hematological malignancies. Evidence points to transcriptional alterations as the major contributor to the characteristic maturational block of acute promyelocytic leukemia. Furthermore, silencing of the tumor suppressor gene, p15 (INK4B), is a frequent abnormality in acute myelogenous leukemia (2, 7, 8). Methods of gene inactivation include genetic events such as mutations or deletions. However, epigenetic changes, heritable traits mediated by changes in DNA other than nucleotide sequences, play an important role in gene expression (9). Two interactive epigenetic modifications that culminate in a change in chromatin information resulting in transcriptional silencing include methylation of CpG islands located in gene promoter regions of cancer cells and changes in chromatin conformation through histone acetylation status. Recent evidence demonstrates that these processes form layers of epigenetic silencing. Reversal of these epigenetic processes and up-regulation of genes important to prevent or reverse the malignant phenotype has therefore become a potential new therapeutic target in cancer treatment. Depending on the particular gene, transcriptional restoration or up-regulation may prevent the development of cancer, halt disease progression, or delay the appearance of metastases. Well-designed clinical trials are therefore needed to explore this therapeutic strategy.

METHYLATION
In mammalian cells, methylation of DNA nucleotides occurs at the cytosines 5 to guanosine (10). Methylation serves several purposes in the nonmalignant cell. Methylation is involved in early embryonic gene regulation. This process plays a role in dictating which genes are activated during differentiation to form a committed cell (10 12). Additionally, methylation has a protective role as it silences incorporated viral genomes, including EBV and HIV (13, 14). In the cell, gene regions rich in CpG dinucleotides are known as CpG islands. CpG islands are commonly found in gene promoter regions, exons, and 3 -regions of genes (15, 16). With some exception (X-linked gene promoters and imprinted genes among others), CpG islands are usually protected from methylation in normal cells (1721). However, this protection may be lost in the cancer cell. Sixty to 90% of cytosines in gene promoter-linked CpG islands are methylated in cancer cells (22). Many important genes have been found to be hypermethylated in malignancy. For example, Table 1 reviews genes reported to be methylation silenced in prostate cancer as a model tumor type. Multiple tumor suppressor genes have been studied and found to be hypermethylated in cancer. The p16 gene, designated as CDKN2A, has been the most extensively studied tumor suppressor gene for promoter hypermethylation. CDKN2A regulates the phosphorylation status of retinoblastoma protein and therefore plays a role in regulation of cell proliferation. Several investigations have demonstrated low rates of mutational inactivation of this gene in multiple tumor types (23,

Received 10/3/03; revised 3/24/04; accepted 4/7/04. Grant support: NIH Grants CA UO1-70095 and CA-RO1 75525 (to M. Carducci), NIH CA-RO1 87760 (to S. Gore), CA P50-58236, and AEGON International Research Fellowship (to J. Gilbert). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Jill Gilbert, Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar Street, Room 521, New Orleans, LA 70112. Phone: (504) 568-5613; Fax: (504) 568-6888; E-mail: jgilbe@lsuhsc.edu.

4590 Clinical Application of Targeting Cancer

Table 1 Gene GST- (Ref. 103) Androgen receptor (Refs. 104108) Estrogen receptor (Refs. 108, 109) CDKN2A p16/MTS1 (Refs. 110112) Retinoic acid receptor (Ref. 13) Endothelin-B receptor (Refs. 114116) Neutral endopeptidase (Refs. 117, 118)

Known genes silenced by promoter methylation and potential role in prostate cancer progression % methylation silenced 100% 828% 6095% 15% 7990% Function Protects cells from cytotoxic stresses Nuclear steroid receptor for androgen effects Nuclear steroid receptor for estrogen effects Tumor suppressor genecell cycle inhibitor Retinoic acid suppresses cancer cell growth and induces apoptosis Mediates ET-1 clearance and inhibits secretion Inactivates neuropeptides implicated in prostate cancer progression Cellular adhesion molecule Effect if silenced Absence promotes carcinogenesis Promote androgen independence May promote androgen resistance Allows cancer cell to cycle without checkpoints Unchecked growth and tumor progression Increased ET-1 levels promoting PCA progression Neuropeptides increase promoting continued growth Increased risk for metastasis Potential effect if reactivated by therapy Slows accumulation of genetic hits/slows progression Restore sensitivity to androgen ablative therapies May provide opportunity to use estrogens as therapy Slows cancer progression by forcing cancer cell to stop in cell cycle Receptor mediated inhibition of cancer growth and induction of cancer cell apoptosis Puts brake on prostate cancer progression by increasing clearance of ET-1 Neuropeptide growth factors are inactivated and cancer growth slows Reduced risk for metastasis

5070% of samples tested 1520% 3068%

E-Cadherin (Refs. 119121)

24). However, hypermethylation associated with loss of expression of this gene has been found to be one of the most frequent alterations in non-small cell lung cancer, squamous cell carcinoma of the head and neck, gliomas, colorectal, breast, nonHodgkins lymphomas, and multiple myeloma. In fact, hypermethylation and transcriptional silencing has been found in 33% breast, 60% prostate, 23% renal cell carcinoma, and 92% colon cancer cell lines (2535). However, it should be noted that these numbers represent a limited number of examined cell lines and tumor samples. Importantly, it should be noted that hypermethylation may be a more frequent event in cell lines than in the respective primary human malignancies. Although colon cancer cell lines demonstrate only a 5-fold increase in CpG island hypermethylation than the respective primary malignancy, head and neck squamous cell carcinoma cell lines demonstrate a 93-fold increase. Additionally, methylation patterns differ between tumor types. However, although methylation frequency and patterns differ between tumors types and between cell lines and tumors, the percentage of methylated genes and the particular genes that experience epigenetic silencing within a methylation pattern and tumor type still need to be elucidated (36, 37).

METHYLTRANSFERASE INHIBITION
Inhibition of DNA methyltransferases (DNMTs), the enzymes responsible for maintaining specific methylation of CpG islands at gene promoter regions, leading to transcriptional silencing, promotes expression of previously silenced genes in vitro. It should be noted, however, that different malignancies have different levels of methyltransferase activity. Moreover, at least three different DNMTs exist, and many of the available methyltransferase inhibitors (MTIs) are not specific for a particular methyltransferase or gene. The clinical implication of MTI depends on the function of the re-expressed gene(s). Using

prostate cancer as a model, Table 1 predicts potential clinical effects that may be noted should a silenced gene be reexpressed. 5-Azacytidine (5 AC) and 5-aza-2 -deoxycytidine (5dAC) are cytosine analogues that have been shown to demethylate DNA with resultant effects on gene expression and cell differentiation. Once incorporated into DNA, a marked dosedependent and time-dependent decrease ( 95%) in DNMT activity occurs. 5 AC has shown the capacity to induce differentiation in murine erythroleukemia cells as well as HL-60 cells (38 41). In contrast to most genetic events, gene silencing due to epigenetic processes may be reversible using pharmacological agents, including MTIs. In human lung cancer cell lines containing methylated and silenced CDKN2A (p16), the treatment of cells with 2 -deoxy-5-azacytidine, an inhibitor of DNA MTI, resulted in a dose- and duration-dependent expression of CDKN2A. Examples involving genes other than p16 abound (42 45). Clinically, MTI has been most extensively studied in patients with hemoglobinopathies and myeloid disorders. Ley et al. (46) treated patients with severe -thalassemia with 5 AC at a dose of 2 mg/kg/day for 7 days by continuous i.v. infusion. This resulted in marked increase in -chain synthesis and increased levels of hemoglobin F. In comparison to pretreatment methylation levels, hypomethylation of bone marrow DNA was demonstrated near the regions of the -globin genes and the genes (46). Multiple studies have examined the use of demethylating agents in the treatment of sickle cell disease. Koshy et al. (47) treated 8 adult sickle cell patients in a dose-escalating Phase I/II study with 2 -deoxy-5-azacytidine. Significant increases in hemoglobin F and -globin after treatment with the demethylating agent with maximum hemoglobin F were attained within 4 weeks of treatment. The mechanism by which this effect is mediated is not entirely clear but may be caused by

Clinical Cancer Research 4591

hypomethylation of the -globin gene promoter and possible drug-induced differentiation of a pluripotential stem cell due to altered gene regulation (47, 48). The myelodysplastic syndromes (MDSs) are often characterized by multiple chromosomal abnormalities. Recent evidence suggests that epigenetic events such as hypermethylation may also play a role in their pathogenesis. The cyclindependent kinase inhibitor, p15INK4b, has been found to be progressively hypermethylated and silenced in high-grade MDSs. In fact, hypermethylation of p15 is significantly correlated with blastic bone marrow involvement, and the prevalence of hypermethylation may increase from low-grade MDSs to acute myeloid leukemia (49). Treatment with MTIs has demonstrated evidence of both biological and clinical activity in MDS. Treatment with 5dAC resulted in a decrease in p15 promoter methylation in 9 of 12 patients with MDS. This was associated with clinical response (50). Silverman et al. (51) treated 43 patients with refractory anemia with excess blasts (RAEB) and RAEB in transformation (RAEB-T) with a continuous infusion of 5 AC at 75 mg/m2/day for 7 days every 4 weeks. The cytological response rate was 49% with 37% demonstrating trilineage response (51). Cancer and Leukemia Group B additionally evaluated 5 AC and performed a Phase III trial involving 191 patients with MDS. Patients were randomized to observation and supportive care or to 5 AC at a dose of 75 mg/m2/day for 7 days every 4 weeks. Statistically significant differences were seen in favor of the azacytidine group for response rate, improved quality of life, time to death, or leukemic transformation (52). Although the potential reversal of epigenetic silencing of key genes holds promise as a novel treatment target, the potential role of hypomethylation in tumorigenesis remains controversial. In fact, a variety of cancers display global genomic hypomethylation (5356). It remains uncertain whether hypomethylation precedes or is a result of gene-specific hypermethylation. Furthermore, although some studies link DNA hypomethylation to chromosomal instability and tumorigenesis, other studies do not support this finding (57 64). Although no increase in incidence of malignancies has been reported in patients who have received MTIs, investigators should be aware of a theoretical risk of tumorigenesis as patient follow-up matures. Additionally, most MTIs are not specific for a particular DNMT. Such lack of specificity may ultimately result in an unfavorable toxicity profile. Importantly, several agents such as 5 AC and 5dAC have been associated with significant toxicity, including severe nausea and vomiting, as well as cytopenias and local tissue reaction (if given s.c.). Agents that are more specific for a particular DNMT may demonstrate a more favorable toxicity profile. Newer compounds with specificity for particular DNMTs are being developed and hold promise for a more targeted approach toward methylation. Of these newer compounds, MG98, an antisense oligonucleotide, is a specific inhibitor of human DNMT1 mRNA. MG98 produces a dosedependent reduction in the DNMT1 protein and results in demethylation of the p16 gene promoter and re-expression of the p16 protein in tumor cell lines. In a Phase I study of MG98 in patients with refractory solid tumors, the drug was well tolerated at the maximum-tolerated dose with mild to moderate fatigue, nausea, and vomiting as the most common toxicities.

Moreover, one partial response was seen in a patient with renal cell carcinoma. Interestingly, no consistent changes in DNMT1 mRNA levels were noted in peripheral blood mononuclear cells. However, DNMT1 was not measured in tumor tissue pre- and posttherapy and may have proved more informative for evidence of biological effect (65, 66).

HISTONE ACETYLATION
The nucleosome, consisting of 146 bp of DNA wrapped around a core of histone octamers, inhibits gene transcription through prevention of access of DNA-binding transcriptional regulators to promoter regions of genes (67). Chromatin structure is plastic, and chromatin remodeling can lead to activation or repression of transcription. Acetylation of conserved lysine residues on the NH2-terminal tails of the core histones, H2A, H2B, H3, and H4, represents an important mechanism by which chromatin structure is altered (68). In vivo, histone acetylation depends on the balance between the enzymes with histone acetylase activity and enzymes that deacetylate histones, histone deacetylase (HDAC). Acetylated histones associate preferentially with transcriptionally activated chromatin; such histone acetylation may decrease the affinity of histone binding to DNA through partial neutralization (69, 70). Histone acetylation may also facilitate binding of transcription factors to the promoters and disrupt higher order chromosome structure, promoting transcription (71, 72). Agents that inhibit HDACs lead to maintenance of histones in the hyperacetylated state and promote transcription of a variety of genes (73). As noted earlier, histone acetylation is associated with an open chromatin conformation, allowing for gene transcription, whereas HDACs maintain the chromatin in the closed, nontranscribed state. The retinoic acid receptor, RAR, serves as a model for the effect of acetylation modulation on gene transcription. RAR binds specific retinoic acid response elements in DNA as a heterodimer with a related protein, RXR. In the presence of a ligand (retinoic acid), the complex allows DNA transcription to occur at the promoter regions of retinoic acid-responsive genes. In the absence of a ligand, transcription does not occur. However, presence of a ligand is just one necessary step in the process of gene transcription. In fact, in the absence of a ligand, transcriptional silencing is a multistep process. Transcriptional regulators such as Mad and E2F bind to the DNA and recruit a corepressor molecule, nuclear receptor core repressor, as well as nuclear receptor core repressor DNA-binding proteins such as Sin3, which in turn recruits a HDAC, promoting transcriptional silencing. In the presence of a ligand, a conformational change occurs in the RAR, and the nuclear receptor core repressor/Sin3/ HDAC complex does not bind to the RAR, thus removing a block on transcription of the target gene. In addition to removal of the inhibitory complex, binding and activation of the ligandreceptor complex with resultant transcription of target genes is also dependent on coactivators. Such coactivators include the p160 family members such as the cyclic AMP-responsive element binding proteins and CBP/p300, which possess intrinsic histone acetylase activity (74). One of the most interesting evaluations of histone deacetylase inhibitors (HDACI) has been in acute promyelocytic leukemia. In acute promyelocytic leukemia, the fusion protein

4592 Clinical Application of Targeting Cancer

PML-RAR results in a transcriptional block due to altered dose-response characteristics of all-trans-retinoic acid on the hybrid protein compared with wild-type RAR . Physiological concentrations of all-trans-retinoic acid do not release the transcriptional repression complex from the promoter-bound RARRXR complex (75). Both in vitro and in vivo data have demonstrated that both the transcriptional block and refractoriness to all-trans-retinoic acid therapy can be overcome with the use of a HDACI (76). Phenylbutyrate, a first generation HDACI, has demonstrated intriguing in vitro and clinical results. The ability of HDAC inhibitors to modulate gene expression may explain the differentiating properties of this class of agents. In solid tumor cell lines, phenylbutyrate (PB), an aromatic fatty acid with HDACI activity, was shown to induce expression of p21waf1/cip1, a cell cycle checkpoint protein, and G0-G1 arrest, within 24 h of treatment. Growth arrest was also accompanied by induction of p57kip1, another protein associated with differentiation (77, 78). PBs multiple mechanisms of action for its observed effects include inhibition of HDACs, modification of lipid metabolism, and activation of peroxisome proliferation activator receptor. Clinically, PB (buphenyl) has been Food and Drug Administration approved for use clinically in patients with urea cycle disorders. PB also increases fetal hemoglobin production in patients with sickle cell anemia or B-thalassemia (77, 79 90). In terms of cancer, PB has also been evaluated clinically in hematological malignancies and solid tumor malignancies. A trial of continuous infusion PB given consecutively for 7 of 14 days or consecutively for 21 of 28 days in patients with MDS and acute myelogenous leukemia demonstrated partial hematological responses in 2 patients who received the 21 consecutive day schedule. Additionally, fetal erythropoiesis, a surrogate marker of PB activity, increased in 4 of 11 MDS patients and 7 of 11 acute myelogenous leukemia patients (91). In solid tumor malignancies, PB was evaluated as a 120-h continuous infusion. No responses were noted. However, 90% of patients with prostate cancer had a rise in their prostate-specific antigen during the 5-day infusion, perhaps reflecting alterations in gene re-expression (92). In a Phase I evaluation of oral PB in patients with refractory solid tumor malignancies, no complete or partial responses were noted. However, 25% of patients studied had stable disease for 6 months while on the drug (93). In addition to PB, multiple classes of histone deacetylase inhibitors exist and are presently being tested in clinical trials. One example, suberoylanilide hydroxamic acid, proves especially interesting. Suberoylanilide hydroxamic acid, a hydroxamic acid-based hybrid polar compound with HDAC inhibitory activity, has also demonstrated differentiating effects. In T24 bladder carcinoma cells, the addition of suberoylanilide hydroxamic acid resulted in an accumulation of acetylated histones H3 and H4 and a 9-fold increase in p21waf1 mRNA and protein (93). Depsipeptide, a bicyclic peptide derived from Chrombacterium violaceum has demonstrated potent cytotoxic activity through several different mechanisms including histone deacetylase inhibition. This agent had demonstrated activity against chronic lymphocytic leukemia cells in which treatment with depsipeptide resulted in histone H3 and H4 acetylation as well as expression of apoptotic proteins involving the caspase 8 and

effector caspase 3 pathways. This was accompanied by a reduction of expression of c-FLIP. Together, these biological markers suggest potential endpoints for correlative studies of this class of agents. Early-phase clinical trials in patients with refractory solid tumor malignancies have been promising with acceptable toxicities (94 97). Additional trials using this agent are ongoing.

HYPERMETHYLATION AND HISTONE DEACETYLATION

Both hypermethylation and histone deacetylation result in transcriptional silencing. Evidence suggests that these processes are often not independent of each other and, in fact, result in layers of epigenetic silencing. Jones et al. (98) demonstrated that the repressive chromatin structure associated with dense methylation is associated with histone deacetylation. Methylated DNA binds the transcriptional repressor, MeCP2, at the methylated CpG binding domain. MeCP2 then recruits and binds the Sin3/histone deacetylase complex additionally supporting the closed chromatin structure (98). Theoretically, the reversal of both processes would lead to greater gene transcription than the reversal of one epigenetic layer alone. Cameron et al. (99) examined the colorectal carcinoma cell line, RKO, which is characterized by CpG island hypermethylation and transcriptional silencing of MLH1, TIMP3, and CDKN2A. The histone deacetylase inhibitor, trichostatin A (TSA), failed to reactivate expression of MLH1, TIMP3, or CDKN2A. However, genes that were not hypermethylated and silenced such as CDKN2B demonstrated increased expression with the addition of TSA. After partial demethylation with of 5dAC, the addition of TSA resulted in robust expression of MLH1, TIMP3, and CDKN2A (99). Using a microarray-based technique that evaluates gene expression in combination with epigenetic change, investigators evaluated the RKO cell line after treatment with 5dAC and TSA. In concordance with the finding of Cameron et al. (99), 74 genes demonstrated upregulation of expression after treatment with 5dAC and/or TSA. These genes were additionally subdivided into group 1 genes or group 2 genes. Group 1 genes demonstrated no increased expression using TSA alone, minimal increased expression using 5dAC alone, but significantly increased expression using combined 5dAC and TSA. A subset of the genes showed some basal expression by reverse transcription-PCR before treatment with any agent. Group 2 genes demonstrated up-regulation of expression after TSA alone. Methylation analysis of the above genes demonstrated dense methylation of the 5 -CpG islands for the group 1 genes that did not demonstrate baseline reverse transcription-PCR expression. The genes that demonstrated baseline expression were found to have partial methylation in these regions. In contrast, genes in group 2 did not demonstrate methylation of their 5 -CpG islands. Thus, this innovative analysis provided additional information about the promoters of the genes that are re-expressed using the various agents alone or in combination (100). The synergistic effects of progressively longer exposure to the agents was also examined. Minimal demethylation produced by 24 h of 5dAC treatment required HDACI for effective re-expression, whereas with 72 h treatment with the MTI, an approximate doubling of cells re-expressing MLH1 was ob-

Clinical Cancer Research 4593

served. After the more complete demethylation accomplished by 5-day treatment with 5dAC, no increase was produced by the HDACI. DNA methylation is an S-phase-specific process. Therefore, longer exposures to MTIs may be necessary to expose as many cells as possible to the MTI during the S phase of the cell cycle. Additionally, many HDACI lead to G0-G1 growth arrest within 96 h of exposure. Thus, a balance must be attained between optimal exposure to the MTI during S phase and exposure to the HDACI before G0-G1 arrest. However, the mechanism by which the reversal of these processes results in gene expression has not been completely elucidated. Bisulfite genomic sequencing revealed that most CpG sites/allele of the targeted silenced genes were still methylated after treatment with the two agents and that chromatin structure was not altered. However, it is possible that important alleles not detected by bisulfite-sequencing analysis were extensively demethylated. Furthermore, only a small number of alleles may have undergone complete conversion to an open chromatin structure and likely was below the sensitivity of the assay (unpublished data).

CLINICAL APPLICATION
The clinical application of targeting cancer through demethylation and histone acetylation is an exciting strategy for cancer therapy. Investigators at the Kimmel Cancer Center at Johns Hopkins have applied the compelling preclinical data to two open, ongoing active clinical trials in patients with refractory solid tumor malignancies and in patients with high-grade MDS or refractory acute myelogenous leukemia. The studies involve the DNA MTI, 5 AC and the HDAC inhibitor, and sodium PB. The ultimate goal of the studies is to re-express cancer-specific targeted silent genes. The studies are Phase I evaluations using biological end points from peripheral blood, bone marrow, and tumor tissue. In designing the trial, several points were considered. Incorporation of 5 AC into DNA occurs predominantly during S phase. Thus, longer exposure to 5 AC may lead to a greater number of cells exposed during S phase and greater gene reexpression. Additionally, although higher doses of 5 AC may lead to a direct cytotoxic effect (and higher toxicity), lower doses given over longer periods (more cells exposed during S phase) of time may allow MTI with minimal toxicity. Secondly, PB effects histone acetylation within 6 h but leads to G0-G1 growth arrest or apoptosis within 96 h. Although 5 AC acts during the S phase, the optimum cell cycle period for exposure to PB remains uncertain. If the efficacy of PB also depends on cells that are actively in the cell cycle, maximal re-expression of key genes may require continuous infusion PB (24 48 h) rather than intermittent bolus. Thus, the first dose level of the trial in patients with solid tumor malignancies incorporates these principles. Patients are exposed to low doses of 5 AC for extended periods (up to 14 days). PB is given as a 24-h continuous infusion once/week during 5 AC administration. In the trial of hematological malignancies and MDSs, patients receive 5 AC daily s.c. for 5 days followed by a 7-day continuous infusion of PB at its maximal tolerated dose. The initial dose cohort was treated at 75 mg/m2/day of 5 AC s.c., the dose studied by Cancer and Leukemia Group B in MDS. Subsequent cohorts receive lower doses of 5 AC if correlative studies demonstrate inhibition

of DNMT activity. Once the minimal effective pharmacodynamic dose for MTI is determined, subsequent cohorts will receive prolonged schedules of 5 AC. Tumor biopsies have been obtained pre- and posttherapy on patients with solid tumor malignancies. Bone marrow biopsies have been performed on all hematological malignancy patients pre- and posttherapy. Dose adjustments have been made according to toxicity and biological end points. Correlative studies to determine these biological end points are still underway. It proves important to add correlative studies in future evaluations of this class of agents. That is, exploration of surrogate markers of biological effect such as changes in histone acetylation in peripheral blood mononuclear cells will aid in detailed pharmacodynamic studies of HDACI and may provide evidence of drug activity even if clinical response is not seen. In conclusion, the targeting of cancer through demethylation and histone acetylation proves to be an exciting area of cancer therapy. At this time, second generation MTIs and HDAC inhibitors are being developed and will ultimately require evaluation in monotherapy and in combination (65, 94 98, 101, 102) The manipulation of gene expression through epigenetic modification heralds a new era of gene-targeted therapy and holds promise as both therapeutic and preventative strategies.

REFERENCES
1. Vogelstein B, Kinzler KW, editors. The genetic basis of human cancer. New York: McGraw-Hill; 1998. 2. Warrell RP, He LZ, Richon V, Calleja E, Pandolfi PP. Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl Cancer Inst (Bethesda) 1998;90: 16215. 3. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG Island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA 1999;96:8681 6. 4. Plumb JA, Strathdee G, Sludden J, Kaye SB, Brown R. Reversal of drug resistance in human tumor xenografts by 2 -deoxy-5-azacytidineinduced demethylation of the MLH1 gene promoter. Cancer Res 2000; 60:6039 44. 5. Ricciardiello L, Goel A, Montovani V, et al. Frequent loss of hMLH1 by promoter hypermethylation leads to microsatellite instability in adenomatous polyps of patients with a single first-degree member affected by colon cancer. Cancer Res 2003;63:78792. 6. Lee WH, Morton RA, Epstein JI, et al. Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci USA 1994;91:117337. 7. Stirewalt DP, Radich JP. Malignancy: tumor suppressor gene aberrations in acute myelogenous leukemia. Hematology 2000;5:1525. 8. Chim CS, Liang R, Tam CY, Kwong YL. Methylation of p15 and p16 genes in acute promyelocytic leukemia: potential diagnostic and prognostic significance. J Clin Oncol 2001;19:2033 40. 9. Baylin SB, Herman JG. DNA. hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000;16:168 74. 10. NG HH, Bird A. DNA methylation and chromatin modification. Curr Opin Genet Dev 1999;9:158 63. 11. Oligny LL. Human molecular embryogenesis: an overview. Pediatr Dev Pathol 2001;4:324 43. 12. Paulen M, Ferguson-Smith AC. DNA. methylation in genomic imprinting, development, and disease. J Pathol 2001;195:97110. 13. Gutenkunst KA, Kashanchi F, Brady JN, Bednarik DP. Transcription of HIV-1 LTR is regulated by the density of DNA CpG methylation. J Acquir Immune Defic Syndr 1993;6:5419.

4594 Clinical Application of Targeting Cancer

14. Robertson KD. The role of DNA methylation in modulating Epstein-Barr virus gene expression. Curr Top Microbiol Immunol 2000; 249:2134. 15. Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol 1987;196:261 82. 16. Takai D, Jones PA. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA 2002;99: 3740 5. 17. Razin A, Riggs AD. DNA. methylation and gene function. Science (Wash. DC) 1980;210:604 10. 18. Razin A, Shemer R. DNA. methylation in early development. Hum Mol Genet 1995;4:17515. 19. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:41528. 20. Birger Y, Shemer R, Perk J, Razin A. The imprinting box of the mouse Igf2r gene. Nature (Lond.) 1999;397:84 8. 21. Hershko A, Razin A, Shemer R. Imprinted methylation and its effect on expression of the mouse Zfp127 gene. Gene (Amst.) 1999; 234:3237. 22. Herman JG, Baylin SB. Promoter-region hypermethylation and gene silencing in human cancer. Curr Top Microbiol Immunol 2000; 249:3554. 23. Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science (Wash. DC) 1994;264:436 40. 24. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature (Lond.) 1994;368:753 6. 25. Herman JG, Merlo A, Mao L, et al. Inactivation of the CDKN2/ p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res 1995;55:452530. 26. Merlo A, Herman JG, Mao L, et al. 5 -CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/ CDKN2/MTS1 in human cancers. Nat Med 1995;1:686 92. 27. Otterson GA, Khleif SN, Chen W, Coxon AB, Kaye, FJ. CDKN 2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza-2 -deoxycytidine. Oncogene 1995; 11:1211 6. 28. Martinez-Delgado B, Fernandez-Piqueras J, Garcia MJ, et al. Hypermethylation of 5 -CpG island of p16 is a frequent event in nonHodgkins lymphoma. Leukemia (Baltimore) 1997;11:425 8. 29. Herman JG, Civin CI, Issa JP, Collector MI, Sharkis SJ, Baylin SB. Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies. Cancer Res 1997; 57:837 41. 30. Pinyol M, Cobo F, Bea S, et al. p16(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkins lymphomas. Blood 1998;91:2977 84. 31. Klangby U, Okan I, Magnusson KP, Wendland M, Lind P, Wiman KG. p16/INK4a and p15/INK4b gene methylation and absence of p16/ INK4a mRNA and protein expression in Burkitts lymphoma. Blood 1998;91:1680 7. 32. Villuendas R, Sanchez-Beato M, Martinez JC, et al. Loss of p16/ INK4a protein expression in non-Hodgkins lymphoma is a frequent finding associated with tumor progression. Am J Pathol 1998;153: 88797. 33. Ng MH, Chung YF, Lo KW, Wickham NW, Lee JC, Huang DP. Frequent hypermethylation of p16 and p15 genes in multiple myeloma. Blood 1997;89:2500 6. 34. Tasaka T, Asou H, Munker R, et al. Methylation of the p16INK4A gene in multiple myeloma. Br J Haematol 1998;101:558 64. 35. Paz MF, Fraga MF, Avila S, et al. A systematic profile of DNA methylation in human cancer cell lines. Cancer Res 2003;63:1114 21. 36. Smiraglia DJ, Rush LJ, Fruhwals MC, et al. Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies. Hum Mol Genet 2001;10:14139.

37. Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpGisland methylation has non-random and tumor type-specific patterns Nat Genet 2000;25:132 8. 38. Paces V, Doskocil J, Sorm F. Incorporation of 5-azacytidine into nucleic acids of Escherichia coli. Biochim Biophys Acta 1968;161: 352 60. 39. Creusot F, Acs G, Christman JK. Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2 -deoxycytidine. J Biol Chem 1982;257: 2041 8. 40. Christman JK, Weich N, Schoenbrun B, Schneidermen N, Acs G. Hypomethylation of DNA during differentiation of Friend erythroleukemia cells. J Cell Biol 1980;86:366 70. 41. Christman JK, Mendelsohn N, Herzog D, Schneiderman N. Effect of 5-azacytidine on differentiation and DNA methylation in human promyelocytic leukemia cells (HL-60). Cancer Res 1983;43:7639. 42. Zhu WG, Dai Z, Ding H, et al. Increased expression of unmethylated CDKN2D by 5-aza-2 -deoxycytidine in human lung cancer cells Oncogene 2001;20:778796. 43. Ferguson AT, Vertino PM, Spitzner JR, Baylin SB, Muller MT, Davidson NE. Role of estrogen receptor gene demethylation and DNA methyltransferase. DNA adduct formation in 5-aza-2 deoxycytidineinduced cytotoxicity in human breast cancer cells. J Biol Chem 1997; 272:32260 6. 44. Claus R, Lubbert M. Epigenetic targets in hematopoietic malignan cies. Oncogene 2003;22:6489 96. 45. Vidanes GM, Paton V, Wallen E, Peehl DM, Navone N, Brooks JD. Silencing of pi-class glutathione S-transferase in MDA Pca 2a and MDA Pca 2b cells. Prostate 2002;51:22530. 46. Ley TJ, DeSimone J, Anagnou NP, et al. 5-Azacytidine selectively increases gamma-globin synthesis in a patient with beta thalassemia. N. Engl J Med 1982;307:1469 75. 47. Koshy M, Dorn L, Bressler L, et al. 2-Deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia. Blood 2000;96:2379 84. 48. Dover GJ, Charache S. Increasing fetal hemoglobin production in sickle cell disease: results of clinical trials. Prog Clin Biol Res 1987; 251:455 66. 49. Uchida T, Kinoshita T, Nagai H, et al. Hypermethylation of the p15INK4B gene in myelodysplastic syndromes. Blood 1997;90:14039. 50. Daskalakis M, Nguyen TT, Nguyen C, et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-aza-2 -deoxycytidine (decitabine) treatment. Blood 2002; 100:2957 64. 51. Silverman LR, Holland JF, Weinberg RS, et al. Effects of treatment with 5-azacytidine on the in vivo and in vitro hematopoiesis in patients with myelodysplastic syndromes. Leukemia (Baltimore) 1993;7:219. 52. Silverman LR, Demakos EP, Bercedis LP, et al. Randomized controlled trial of azacytidine in patients with the myelodysplastic syndrome: a study of the Cancer and Leukemia Group B. J Clin Oncol 2002;20:2429 40. 53. Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene 2002;21:5400 13. 54. Bedford MT, van Helden PD. Hypomethylation of DNA in pathological conditions of the human prostate. Cancer Res 1987;47:5274 6. 55. Wahlfors J, Hiltunen H, Keionen K, Hamalainen E, Alhonen L, Janne J. Genomic hypomethylation in human chronic lymphocytic leukemia. Blood 1992;80:2074 80. 56. Esteller M, Fraga MF, Guo M, et al. DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis Hum Mol Genet 2001;10:30017. 57. Jones PA. DNA methylation and cancer. Cancer Res 1986;46: 461 6. 58. Qu GZ, Grundy PE, Narayan A, Ehrlich M. Frequent hypomethylation in Wilms tumors of pericentromeric DNA in chromosomes 1 and 16. Cancer Genet Cytogenet 1999;109:34 9. 59. James SJ, Pogribny IP, Pogribna M, Miller BJ, Jernigan S, Melynk S. Mechanisms of DNA damage, DNA hypomethylation, and tumor

Clinical Cancer Research 4595

progression in the folate/methyl-deficient rat model of hepatocarcinogenesis. J Nutr 2003;133:3740S7S. 60. Pogribny IP, Jernigan S, James SJ. Reduction of DNA methyltransferase capacity by DNA lesions. FASEB J 15:401. 61. Pogribny IP. A sensitive new method to rapidly and sensitively detect abnormal methylation patterns in global DNA and within CpG islands. Biochem Biophys Res Commun 1999;262:624 8. 62. Dunn BK. Hypomethylation: one side of a larger picture. Ann NY Acad Sci 2003;938:28 42. 63. Eads CA, Nickel AE, Laird PW. Complete genetic suppression of polyp formation and reduction of CpG-island hypermethylation in Apc(Min/ ) Dnmt1-hypomorphic mice. Cancer Res 2002;62:1296 9. 64. Laird PW, Jackson-Grusby L, Fazeli A, et al. Suppression of intestinal neoplasia by DNA hypomethylation. Cell 1995;81:197205. 65. Stewart DJ, Donehower RC, Eisenhauer EA, et al. A Phase I pharmacokinetic and pharmacodynamic study of the DNA methyltransferase 1 inhibitor MG98 administered twice weekly. Ann Oncol 2003; 14:766 74. 66. Fournel M, Przemyslaw S, Normand B, Besterman JM, MacLeod AR. Down-regulation of human DNA-(Cytosine-5) methyltransferase induces cell cycle regulators p16ink4A and p21WAF/Cip1 by distinct mechanisms. J Biol Chem 1999;274:24250 6. 67. Davie JR. Covalent modifications of histones: expression from chromatin templates. Curr Opin Genet Dev 1998;8:173 8. 68. Luo RX, Dean DC. chromatin remodeling and transcriptional regulation. J Natl Cancer Inst (Bethesda) 91:1288 94. 69. Hebbes TR, Clayton AL, Thorne AW, Crane-Robinson C. Core histone hyperacetylation co-maps with generalized Dnase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J 1994;13: 182330. 70. Struhl K. Histone acetylation and transcriptional regulatory mechanism. Genes Dev 1998;12:599 606. 71. Lee KY, Hayes JJ, Pruss D, Wolffe AP. A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell 1993;72:73 84. 72. Vettese-Dadley M, Grant PA, Hebbes TR, Crane-Robinson C, Allis CD, Workman JL. Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro. EMBO J 15:2508 18. 73. Yoshida M, Horinouchi S, Beppu T. Trichostatin A and trapoxin: novel chemical proves for the role of histone acetylation in chromatin structure and function. Bioassays 1995;17:42330. 74. Xu W, Chen H, Du K, et al. A transcriptional switch mediated by cofactor methylation. Science (Wash. DC) 2001;294:250711. 75. Cote S, Zhou D, Bianchini A, Nervi C, Gallagher RE, Miller WH. Altered ligand binding and transcriptional regulation by mutations in the PML/RARalpha ligand-binding domain arising in retinoic acid-resistant patients with acute promyelocytic leukemia. Blood 2000;96:3200 8. 76. Warrell RP, He LZ, Richon V, Calleja E, Pandolfi PP. Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl Cancer Inst (Bethesda) 90: 16215. 77. Liu L, Hudgins WR, Miller AC, Chen LC, Samid D. Transcriptional up-regulation of TGF-alpha by phenylacetate and phenylbutyrate is associated with differentiation of human melanoma cells. Cytokine 1995;7:449 56. 78. Davis T, Kennedy C, Chiew YE, Clarke CL, deFazio A. Histone deacetylase inhibitors decrease proliferation and modulate cell cycle gene expression in normal mammary epithelial cells. Clin Cancer Res 2000;6:4334 42. 79. Hudgins WR, Fibach E, Safaya S, Rieder RF, Miller AC, Samid D. Transcriptional up-regulation of gamma-globin by phenylbuytyrate and analogous aromatic fatty acids. Biochem Pharmacol 1996;52:122733. 80. Tong KP, David-Beabes G, Meeker A, Bucci J, Dewesse T, Carducci MA. Phenylbutyrate has pleiotropic effects on gene transcription and inhibits telomerase activity in human prostate cancer. Anticancer Res 1997;17:3953 62.

81. Samid D, Shack S, Myers CE. Selective growth arrest and phenotypic reversion of prostate cancer cells in vitro by nontoxic pharmacological concentrations of phenylacetate. J Clin Investig 1991;91: 2288 95. 82. Pineau T, Hudgins WR, Liu L, et al. Activation of the human peroxisome proliferator-activated receptor by the antitumor agent phenylacetate and its analogues. Biochem Pharmacol 1996;52:659 67. 83. Richon VM, Emiliani S, Verdin E, et al. A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases. Proc Natl Acad Sci USA 1998;95:30037. 84. Lea MA, Tulsyan N. Discordant effects of butyrate analogues on erthroleukemia cell proliferation, differentiation and histone deacetylase. Anticancer Res 1995;15:879 83. 85. Rifkind RA, Richon VM, Marks PA. Induced differentiation, the cell cycle and the treatment of cancer. Pharmacol Ther 1996;69:97102. 86. Brusilow SW. Phenylacetylglutamine may replace urea as a vehicle for waste nitrogen excretion. Pediatr Res 1991;29:14750. 87. Brusilow S, Finkelstein J. Restoration of nitrogen homeostasis in a man with ornithine transcarbamylase deficiency. Metabolism 1993;42: 1336 9. 88. Brusilow SW, Danney M, Waber LJ, et al. Treatment of episodic hyperammonnemia in children with inborn errors of urea synthesis. N Engl J Med 1984;310:630 4. 89. Mitchell RB, Wagner JE, Karp JE, et al. Syndrome of idiopathic hyperammonemia after high dose chemotherapy: a review of nine cases. Am J Med 1988;85:6627. 90. Dover G, Brusilow S, Charache S. Induction of fetal hemoglobin production in subjects with sickle cell anemia with oral sodium phenylbutyrate. Blood 1994;84:339 43. 91. Gore SD, Weng LJ, Figg WD, et al. Impact of prolonged infusions of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukemia. Clin Cancer Res 2002;8:96370. 92. Carducci MA, Gilbert J, Bowling MK, et al. A Phase I clinical and pharmacological evaluation of sodium phenylbutyrate on a 120-h infusion schedule Clin Cancer Res 2001;7:304755. 93. Gilbert J, Baker SD, Bowling MK, et al. A Phase I dose escalation and bioavailability study of oral sodium phenylbutyrate in patients with refractory solid tumor malignancies. Clin Cancer Res 2001;7:2292300. 94. Aron JL, Parthun MR, Marcucci G, et al. Depsipeptide (FR901228) induces histone acetylation and inhibition of histone deacetylase in chronic lymphocytic leukemia cells concurrent with activation of caspase 8-mediated apoptosis and down-regulation of c-FLIP protein. Blood 2003;102:652 8. 95. Sandor V, Bakke S, Robey RW, et al. Phase I trial of the histone deacetylase inhibitor, depsispeptide (FR901228, NSC 630176), in patients with refractory neoplasms. Clin Cancer Res 2002;8:718 28. 96. Byrd JC, Shinn C, Ravi R, et al. Depsipeptide (FR901228): a novel therapeutic agent with selective, in vitro activity against human B-cell chronic lymphocytic leukemia cells. Blood 1999;94:1401 8. 97. Marshall JL, Rizvi N, Kauh J, et al. A Phase I trial of depsipeptide (FR901228) in patients with advanced cancer. J Exp Ther Oncol 2002; 2:32532. 98. Jones PL, Veenstra GJ, Wade PA, et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 1998;19:18791. 99. Cameron EE, Backman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 1999;21:1037. 100. Suzuki H, Gabrielson E, Chen W, et al. A genomic screen for genes up-regulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet 2002;31:1419. 101. Richon VM, OBrien JP. Histone deacetylase inhibitors: a new class of potential therapeutic agents for cancer treatment. Clin Cancer Res 2002;8:662 4.

4596 Clinical Application of Targeting Cancer

102. Cheng JC, Matsen CB, Gonzales FA, et al. Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst (Bethesda) 2003;95:399 409. 103. Millar DS, Ow KK, Paul CL, Russell PJ, Molloy PL, Clark SJ. Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer. Oncogene 1999;18:131324. 104. Jarrard DF, Kinoshita H, Shi Y, et al. Methylation of the androgen receptor promoter CpG island is associated with loss of androgen receptor expression in prostate cancer cells. Cancer Res 1998;58: 5310 4. 105. Izbicka E, MacDonald JR, Davidson K, Lawrence RA, Gomez L, Von Hoff D. 5,6-Dihydro-5 -azacytidine (DHAC) restores androgen responsiveness in androgen-insensitive prostate cancer cells. Anticancer Res 1999;19:128591. 106. Nakayama T, Watanable M, Suzuki H, et al. Epigenetic regulation of androgen receptor gene expression in human prostate cancers. Lab Investig 2000;80:1789 96. 107. Kinoshita H, Shi Y, Sandefur C, et al. Methylation of the androgen receptor minimal promoter silences transcription in human prostate cancer. Cancer Res 2000;60:362330. 108. Sasaki M, Tanaka Y, Perinchery G, et al. Methylation and inactivation of estrogen, progesterone, and androgen receptors in prostate cancer. J Natl Cancer Inst (Bethesda) 2002;94:384 90. 109. Li LC, Chui R, Nakajima K, Oh BR, Au HC, Dahiya R. Frequent methylation of estrogen receptor in prostate cancer: correlation with tumor progression. Cancer Res 2000;60:702 6. 110. Jarrard DF, Bova GS, Ewing CM, et al. Deletional, mutational, and methylation analyses of CDKN2 (p 16/MTS1) in primary and metastatic prostate cancer. Genes Chromosomes Cancer 1997;19:90 6. 111. Chi SG, DeVere White RW, Muenzer JT, Gumerlock PH. Frequent alteration of CDKN2 (p16(INK4A)/MTS1) expression in human primary prostate carcinomas. Clin Cancer Res 1997;3:1889 97.

112. Nguyen TT, Nguyen CT, Gonzales FA, Nichols PW, Yu MC, Jones PA. Analysis of cyclin-dependent kinase inhibitor expression and methylation patterns in human prostate cancers. Prostate 2000;43:233 42. 113. Nakayama T, Watanable M, Yamanaka M, et al. The role of epigenetic modifications in retinoic acid receptor beta2 gene expression in human prostate cancers. Lab Investig 2001;81:1049 57. 114. Nelson JB, Lee WH, Nguyen SH, et al. Methylation of the 5 -CpG island of the endothelin B receptor gene is common in human prostate cancer. Cancer Res 1997;57:357. 115. Pao MM, Tsutsumi M, Liang G, Uzvolgyi E, Gonzales FA, Jones PA. The endothelin receptor B (EDNRB) promoter displays heterogeneous, site specific methylation patterns in normal and tumor cells. Hum Mol Genet 2001;10:90310. 116. Jeronimo C, Henrique R, Campos PF, et al. Endothelin B receptor gene hypermethylation in prostate adenocarcinoma. J Clin Pathol 2003; 56:525. 117. Sumitomo M, Shen R, Walburg M, et al. Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling. J Clin Investig 2000;106:1399 407. 118. Usmani BA, Shen R, Janeczko M, et al. Methylation of the neutral endopeptidase gene promoter in human prostate cancers. Clin Cancer Res 2000;6:1664 70. 119. Graff JR, Herman JG, Lapidus RG, et al. E-Cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res 1995;55:51959. 120. Graff JR, Gabrielson E, Fujii H, Baylin SB, Herman JG. Methylation patterns of the E-cadherin 5 -CpG Island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression. J Biol Chem 2000;275:272732. 121. Kallakury BV, Sheehan CE, Winn-Deen E, et al. Decreased expression of catenins, p120 CTN, and e-cadherin cell adhesion proteins and e-cadherin gene promoter methylation in prostatic adenocarcinomas. Cancer (Phila.) 2001;92:2786 95.