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Supplementary Table 1 from Epigenetic Inactivation of the HOXA Gene Cluster in Breast Cancer

Supplementary Figure 2 from Agglomerative Epigenetic Aberrations Are a Common Event in Human Breast Cancer

The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16INK4A expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that may prove useful in breast cancer risk assessment. [Cancer Res 2009;69(12):5251–8]

Supplementary Figure 1 from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Supplementary Table 2 from Epigenetic Inactivation of the HOXA Gene Cluster in Breast Cancer

Supplementary Figure 2 from Epigenetic Inactivation of the HOXA Gene Cluster in Breast Cancer

Supplementary Table 1 from Agglomerative Epigenetic Aberrations Are a Common Event in Human Breast Cancer

Using an integrated approach of epigenomic scanning and gene expression profiling, we found aberrant methylation and epigenetic silencing of a small neighborhood of contiguous genes—the HOXA gene cluster in human breast cancer. The observed transcriptional repression was localized to ∼100 kb of the HOXA gene cluster and did not extend to genes located upstream or downstream of the cluster. Bisulfite sequencing, chromatin immunoprecipitation, and quantitative reverse transcription-PCR analysis confirmed that the loss of expression of the HOXA gene cluster in human breast cancer is closely linked to aberrant DNA methylation and loss of permissive histone modifications in the region. Pharmacologic manipulations showed the importance of these aberrant epigenetic changes in gene silencing and support the hypothesis that aberrant DNA methylation is dominant to histone hypoacetylation. Overall, these data suggest that inactivation of the HOXA gene cluster in breast cancer may represent a new type of genomic lesion—epigenetic microdeletion. We predict that epigenetic microdeletions are common in human cancer and that they functionally resemble genetic microdeletions but are defined by epigenetic inactivation and transcriptional silencing of a relatively small set of contiguous genes along a chromosome, and that this type of genomic lesion is metastable and reversible in a classic epigenetic fashion. (Cancer Res 2006; 66(22): 10664-70)

Supplementary Table 1 from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Supplementary Figure 2 from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Supplementary Figure 1 from Agglomerative Epigenetic Aberrations Are a Common Event in Human Breast Cancer

Supplementary Table 3 from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Supplementary Figure 6 from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Coordinates of All Identified DRMs from Agglomerative Epigenetic Aberrations Are a Common Event in Human Breast Cancer

Supplementary Table 4 from Epigenetic Inactivation of the HOXA Gene Cluster in Breast Cancer

Supplementary Figure 3 from Agglomerative Epigenetic Aberrations Are a Common Event in Human Breast Cancer

Supplementary Figure 3 from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Supplementary Figure 3 from Epigenetic Inactivation of the HOXA Gene Cluster in Breast Cancer

Supplementary Figure 5 from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Supplementary Table 2 from Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Supplementary Table 3 from Epigenetic Inactivation of the HOXA Gene Cluster in Breast Cancer

We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreatic cancer cases from benign pancreatic cyst patients and to monitor tumor dynamics using quantitative DNA methylation analysis of cell-free DNA (cfDNA) from blood samples. The biomarkers were able to distinguish malignant cases from benign disease with high sensitivity and specificity (AUC = 0.999). Furthermore, the biomarkers detected a consistent decline in tumor-derived cfDNA in samples from patients undergoing chemotherapy. The study indicates that our liquid biopsy assay could be useful for management of pancreatic cancer patients.

Identification of cancer-specific methylation of DNA released by tumours can be used for non-invasive diagnostics and monitoring. We previously reported in silico identification of DNA methylation loci specifically hypermethylated in common human cancers that could be used as epigenetic biomarkers. Using DNA methylation specific qPCR we now clinically tested a group of these cancer-specific loci on cell-free DNA (cfDNA) extracted from the plasma fraction of blood samples from healthy controls and non-small cell lung cancer (NSCLC) patients. These DNA methylation biomarkers distinguish lung cancer cases from controls with high sensitivity and specificity (AUC = 0.956), and furthermore, the signal from the markers correlates with tumour size and decreases after surgical resection of lung tumours. Presented observations suggest the clinical value of these DNA methylation biomarkers for NSCLC diagnostics and monitoring. Since we successfully validated the biomarkers using independent DNA methylation data from multiple additional common carcinoma cohorts (bladder, breast, colorectal, oesophageal, head and neck, pancreatic or prostate cancer) we predict that these DNA methylation biomarkers will detect additional carcinoma types from plasma samples as well.

BackgroundSLIT2 has been shown to serve as a tumor suppressor in breast, lung, colon, and liver cancers. Additionally, expression of SLIT2 has been shown to be epigenetically regulated in prostate cancer. Therefore, we sought to determine transcriptional regulation of SLIT2 in pancreatic ductal adenocarcinoma.MethodsRNA expression of SLIT2, SLIT3, and ROBO1 was examined in a panel of pancreatic ductal adenocarcinoma cell lines while protein expression of ROBO1 and SLIT2 was examined in tumor tissue. Methylation of the SLIT2 promoter was determined using Sequenom while histone modifications were queried by chromatin immunoprecipitation. Reexpression of SLIT2 was tested by treatment with 5-aza-2’deoxycytidine and Trichostatin A.ResultsPancreatic cancer cell lines fall into three distinct groups based on SLIT2 and ROBO1 expression. The SLIT2 promoter is methylated in pancreatic ductal adenocarcinoma and SLIT2 expression is dependent on the level of methylation at specific CpG sites. Treatment with 5-aza-2’deoxycytidine (but not Trichostatin A) led to SLIT2 reexpression. The SLIT2 promoter is bivalent in pancreatic ductal adenocarcinoma and histone marks around the transcriptional start site are responsible for transcription.ConclusionsLoss of SLIT2 expression modulated by epigenetic silencing may play a role in pancreatic ductal adenocarcinoma progression.

BackgroundmiRNAs are small, endogenous non-coding RNAs approximately 22 nucleotides in length that account for approximately 1% of the genome and play key regulatory roles in multiple signaling pathways. mir-218-1 is an intronic miRNA located within intron 15 of the SLIT2 gene. Public datasets showed enrichment of H3K4me3 within intron 4 of the SLIT2 gene. Therefore, we sought to determine the genomic location and transcriptional regulatory elements of the mir-218-1 candidate alternative promoter in pancreatic ductal adenocarcinoma.MethodsExpression of mir-218 was evaluated in a panel of pancreatic ductal adenocarcinoma cell lines. The mir-218-1 candidate alternative promoter was characterized by chromatin immunoprecipitation, Sequenom, and luciferase assays. Transcriptional regulation of the mir-218-1 candidate alternative promoter was assessed using chromatin immunoprecipitation and an inhibitor to NF-kB.ResultsWe found that expression of mir-218-1 does not correlate with SLIT2 expression and that mir-218-1 has a novel transcriptional start site separate from the SLIT2 promoter. This novel transcriptional start site showed transcriptional activity and was regulated by NF-kB.Conclusionsmir-218-1 is transcribed from an independent and novel transcriptional start site located within intron 4 of the SLIT2 gene in pancreatic ductal adenocarcinoma. Additionally, mir-218-1 expression is regulated by Nf-kB at this alternative transcriptional start site in pancreatic cancer.

Cancer-specific DNA methylation from the tumor derived fraction of cell free DNA found in blood samples could be used for minimally invasive detection and monitoring of cancer. The knowledge of marker regions with cancer-specific DNA methylation is necessary to the success of such a process. We analyzed the largest cancer DNA methylation dataset available—TCGA Illumina HumanMethylation450 data with over 8,500 tumors—in order to find cancer-specific DNA methylation markers for most common human cancers. First, we identified differentially methylated regions for individual cancer types and those were further filtered against data from normal tissues to obtain marker regions with cancer-specific methylation, resulting in a total of 1,250 hypermethylated and 584 hypomethylated marker CpGs. From hypermethylated markers, optimal sets of six markers for each TCGA cancer type were chosen that could identify most tumors with high specificity and sensitivity [area under the curve (AUC): 0.969-1.000] and a universal 12 marker set that can detect tumors of all 33 TCGA cancer types (AUC >0.84). In addition to hundreds of new DNA methylation markers, our approach also identified markers that are in current clinical use, SEPT9 and GSTP1, indicating the validity of our approach and a significant potential utility for the newly discovered markers. The hypermethylated markers are linked to polycomb associated loci and a significant fraction of the discovered markers is within noncoding RNA genes; one of the best markers is MIR129-2. Future clinical testing of herein discovered markers will confirm new markers that will improve minimally invasive diagnosis and monitoring for multiple cancers.

Understanding what drives breast tumor progression is of utmost importance for blocking tumor metastasis; we have identified that semaphorin 7a is a potent driver of ductal carcinoma in situ (DCIS) progression. Semaphorin 7a is a glycophosphatidylinositol membrane-anchored protein that promotes attachment and spreading in multiple cell types. Here, we show that increased expression of SEMA7A occurs in a large percentage of breast cancers and is associated with decreased overall and distant metastasis-free survival. In both in vitro and in vivo models, short hairpin-mediated silencing of SEMA7A reveals roles for semaphorin 7a in the promotion of DCIS growth, motility and invasion as well as lymphangiogenesis in the tumor microenvironment. Our studies also uncover a relationship between COX-2 and semaphorin 7a expression and suggest that semaphorin 7a promotes tumor cell invasion on collagen and lymphangiogenesis via activation of β1-integrin receptor. Our results suggest that semaphorin 7a may be novel target for blocking breast tumor progression.

Multiple studies report poor survival rates in postpartum women with breast cancer[1, 2]. A recent study from our group has specifically shown that incidence of recurrence and death from breast cancer is increased approximately 3-fold in postpartum patients compared to nulliparous patients[3]. We define postpartum breast cancers as those diagnosed within 5 years of birth and as a highly metastatic subset of young women’s breast cancer[4]. We have recently shown increased incidence of lymph node involvement in patients diagnosed less than 2 years postpartum and have utilized tissues from our clinical cohort to show increased lymphatic vessel density in stage II postpartum patient samples compared to nulliparous. Using xenograft and isograft murine models we have modeled postpartum breast cancer to show that postpartum tumors are larger[5], exhibit increased lymphatic vessel density, and more frequently spread to mouse lymph node and lung tissues[5] compared to tumors in nulliparous mice. In order to determine whether postpartum tumor cells specifically promote lymphangiogenesis, we utilized postpartum tumor cells ex vivo to show that lymphatic structure formation is promoted via increased postpartum tumor cell secretion of PGE2, which acts on the EP2 receptor on lymphatic endothelial cells. We have also performed expression analyses on postpartum tumor cells isolated from our xenograft model to reveal increased expression of COX-2, VEGF-C, and Semaphorin 7a; all three of these molecules have reported, and possibly interrelated, roles in promotion of lymphangiogenesis[6, 7]. Importantly, both in vivo inhibition and knockdown of COX-2 decrease postpartum tumor associated lymphangiogenesis in pre-clinical models. These results, along with our results indicating that COX-2 inhibition during the postpartum period decreases metastasis in our pre-clinical models, have led us to propose that COX-2 dependent promotion of lymphangiogenesis may facilitate, in part, metastasis of postpartum tumors. 1. Stensheim, H., et al., Cause-specific survival for women diagnosed with cancer during pregnancy or lactation: a registry-based cohort study. J Clin Oncol, 2009. 27(1): p. 45-51. 2. Johansson, A.L., et al., Increased Mortality in Women with Breast Cancer Detected during Pregnancy and Different Periods Postpartum. Cancer Epidemiol Biomarkers Prev, 2011. 20(9): p. 1865-72. 3. Callihan, E.B., et al., Postpartum diagnosis demonstrates a high risk for metastasis and merits an expanded definition of pregnancy-associated breast cancer. Breast Cancer Res Treat, 2013. 138(2): p. 549-59. 4. Lyons, T.R., P.J. Schedin, and V.F. Borges, Pregnancy and breast cancer: when they collide. J Mammary Gland Biol Neoplasia, 2009. 14(2): p. 87-98. 5. Lyons, T.R., et al., Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nat Med, 2011. 17(9): p. 1109-15. 6. Timoshenko, A.V., et al., COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer. Br J Cancer, 2006. 94(8): p. 1154-63. 7. Bender, R.J. and F. Mac Gabhann, Expression of VEGF and semaphorin genes define subgroups of triple negative breast cancer. PLoS One, 2013. 8(5): p. e61788. Citation Format: Traci R Lyons, Sarah A Black, Bernard W Futscher, Virginia F Borges, Pepper J Schedin. Promotion of lymphangiogenesis by postpartum breast tumor cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-11.

Background: Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS. Methods: We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS. Results: Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers a1-antitrypsin and a1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models. Conclusion: Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS. J. Surg. Oncol. fl 2015 Wiley Periodicals, Inc.

Author(s): Cornish-Bowden, Athel; Cornish-Bowden, Athel; Rasnick, David; Heng, Henry H; Horne, Steven; Abdallah, Batoul; Liu, Guo; Ye, Christine J; Bloomfield, Mathew; Vincent, Mark D; Aldaz, C Marcelo; Karlsson, Jenny; Valind, Anders; Jansson, Caroline; Gisselsson, David; Graves, Jennifer A Marshall; Stepanenko, Aleksei A; Andreieva, Svitlana V; Korets, Kateryna V; Mykytenko, Dmytro O; Huleyuk, Nataliya L; Baklaushev, Vladimir P; Kovaleva, Oksana A; Chekhonin, Vladimir P; Vassetzky, Yegor S; Avdieiev, Stanislav S; Bakker, Bjorn; Taudt, Aaron S; Belderbos, Mirjam E; Porubsky, David; Spierings, Diana CJ; de Jong, Tristan V; Halsema, Nancy; Kazemier, Hinke G; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline SJM; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M; Tatche, Maria Colome; Foijer, Floris; Liehr, Thomas; Baudoin, Nicolaas C; Nicholson, Joshua M; Soto, Kimberly; Quintanilla, Isabel; Camps, Jordi; Cimini, Daniela; Durrbaum, M; Donnelly, N; Passerini, V; Kruse, C; Habermann, B; Storchova, Z; Mandrioli, Daniele; Belpoggi, Fiorella; Silbergeld, Ellen K; Perry, Melissa J; Skotheim, Rolf I; Lovf, Marthe; Johannessen, Bjarne; Hoff, Andreas M; Zhao, Sen; SveeStromme, Jonas M; Sveen, Anita; Lothe, Ragnhild A; Hehlmann, R; Voskanyan, A; Fabarius, A; Bocking, Alfred; Biesterfeld, Stefan; Berynskyy, Leonid; Borgermann, Christof; Engers, Rainer; Dietz, Josef; Fritz, A; Sehgal, N; Vecerova, J; Stojkovicz, B; Ding, H; Page, N; Tye, C; Bhattacharya, S; Xu, J

Genome-wide disruption of the epigenetic code is a hallmark of malignancy that encompasses many distinct, highly interactive modifications. Delineating the aberrant epigenome produced during toxicant-mediated malignant transformation will help identify the underlying epigenetic drivers of environmental toxicant-induced carcinogenesis. Gene promoter DNA methylation and gene expression profiling of arsenite-transformed prostate epithelial cells showed a negative correlation between gene expression changes and DNA methylation changes; however, less than 10% of the genes with increased promoter methylation were downregulated. Studies described herein confirm that a majority of the DNA hypermethylation events occur at H3K27me3 marked genes that were already transcriptionally repressed. In contrast to aberrant DNA methylation targeting H3K27me3 pre-marked silent genes, we found that actively expressed C2H2 zinc finger genes (ZNFs) marked with H3K9me3 on their 3' ends, were the favored targets of DNA methylation linked gene silencing. DNA methylation coupled, H3K9me3 mediated gene silencing of ZNF genes was widespread, occurring at individual ZNF genes on multiple chromosomes and across ZNF gene family clusters. At ZNF gene promoters, H3K9me3 and DNA hypermethylation replaced H3K4me3, resulting in a widespread downregulation of ZNF gene expression, which accounted for 8% of all the downregulated genes in the arsenical-transformed cells. In summary, these studies associate toxicant exposure with widespread silencing of ZNF genes by DNA hypermethylation-linked H3K9me3 spreading, further implicating epigenetic dysfunction as a driver of toxicant associated carcinogenesis.

Background: Inflammatory breast cancer (IBC) is a rare, highly aggressive form of breast cancer. The mechanism of IBC carcinogenesis remains unknown. We sought to evaluate potential genetic risk factors for IBC and whether or not the IBC cell lines SUM149 and SUM190 demonstrated evidence of viral infection.Methods: We performed single nucleotide polymorphism (SNP) genotyping for 2 variants of the ribonuclease (RNase) L gene that have been correlated with the risk of prostate cancer due to a possible viral etiology. We evaluated dose-response to treatment with interferon-alpha (IFN-α); and assayed for evidence of the putative human mammary tumor virus (HMTV, which has been implicated in IBC) in SUM149 cells. A bioinformatic analysis was performed to evaluate expression of RNase L in IBC and non-IBC.Results: 2 of 2 IBC cell lines were homozygous for RNase L common missense variants 462 and 541; whereas 2 of 10 non-IBC cell lines were homozygous positive for the 462 variant (p= 0.09) and 0 of 10 non-IBC cell lines were homozygous positive for the 541 variant (p = 0.015). Our real-time polymerase chain reaction (RT-PCR) and Southern blot analysis for sequences of HMTV revealed no evidence of the putative viral genome.Conclusion: We discovered 2 SNPs in the RNase L gene that were homozygously present in IBC cell lines. The 462 variant was absent in non-IBC lines. Our discovery of these SNPs present in IBC cell lines suggests a possible biomarker for risk of IBC. We found no evidence of HMTV in SUM149 cells. A query of a panel of human IBC and non-IBC samples showed no difference in RNase L expression. Further studies of the RNase L 462 and 541 variants in IBC tissues are warranted to validate our in vitro findings.

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenitetransformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. Aberrant DNA methylation in general occurred more often within H3K27me3 stem cell domains. We found a striking association between enrichment of H3K9me3 stem cell domains and toxicant-induced agglomerative DNA methylation. Global gene expression profiling of the arsenite-transformed prostate epithelial cells showed that gene expression changes and DNA methylation changes were negatively correlated, but less than 10% of the hypermethylated genes were down-regulated. These studies confirm that a majority of the DNA hypermethylation events occur at transcriptionally repressed, H3K27me3 marked genes. In contrast to aberrant DNA methylation targeting H3K27me3 premarked silent genes, we found that actively expressed ZNF genes marked with H3K9me3 on their 3’ ends, are preferred targets of DNA methylation linked gene silencing.

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals or cadmium. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. The agglomerative DNA methylation changes induced by arsenicals appear to be common and clinically relevant events, since they occur in other human cancer cell lines and models of malignant transformation, as well as clinical cancer specimens. Aberrant DNA methylation in general occurred more often within histone H3 lysine-27 trimethylation stem cell domains. We found a striking association between enrichment of histone H3 lysine-9 trimethylation stem cell domains and toxicant-induced agglomerative DNA methylation, suggesting these epigenetic modifications may become aberrantly linked during malignant transformation. In summary, we found an association between toxicant-induced malignant transformation and agglomerative DNA methylation, which lends further support to the hypothesis that epigenetic dysfunction plays an important role in toxicant-induced malignant transformation.