DNMT3B7 Expression Research Articles

E-Cadherin, NFATC3, and PLP2 Are Differentially Methylated in Multiple Cancers.

It is well documented that cancer cells have abnormal methylation patterns often caused by faulty methylating machinery. Specifically, E-cadherin, NFATC3, and PLP2 are 3 genes known to be aberrantly methylated in cancer cells. These genes are well documented for their role in signaling pathways involved with cell proliferation, adhesion, migration, and other signs of tumor progression. Therefore, changes in gene expression of CDH1, NFATC3, and PLP2 due to aberrant methylation can lead to profound changes in cellular function and tumor formation. In order to ensure that previous in vitro and in vivo methylation studies match what is observed in the clinic, we utilized a bioinformatics approach to complete an extensive analysis of methylation patterns of these 3 genes, analyzing over 5000 patient samples, across all cancers for which both normal and tumor tissues were available. Specifically, we analyzed overall and site-specific methylation patterns, at CpG islands and shores, of all 3 genes across 14 cancer types. Furthermore, we compared these methylation levels in normal and tumor samples of both matched and unmatched patient samples in order to determine any differences between groups. Finally, we examined whether an aberrant DNA methyltransferase, DNMT3B7, known to be expressed in cancer cells and to alter methylation patterns in vitro correlated with altered overall and site-specific methylation of CDH1, NFATC3, and PLP2 in these patient samples. Our results indicate that methylation patterns of CDH1 and NFATC3 were unexpectedly varied across tumors, contrary to previous studies performed in vitro, while PLP2 showed the expected hypomethylation pattern in tumor tissues. We also observed some correlation between DNMT3B7 expression and methylation patterns of these genes, but patterns were inconsistent. Taken together, these results emphasize the necessity for in vivo and patient studies rather than a complete reliance on in vitro data and provide multiple areas of future research.

Aberrant DNMT3B7 expression correlates to tissue type, stage, and survival across cancers

Cancer cells are known for aberrant methylation patterns leading to altered gene expression and tumor progression. DNA methyltransferases (DNMTs) are responsible for regulating DNA methylation in normal cells. However, many aberrant versions of DNMTs have been identified to date and their role in cancer continues to be elucidated. It has been previously shown that an aberrant version of a de novo methylase, DNMT3B7, is expressed in many cancer cell lines and has a functional role in the progression of breast cancer, neuroblastoma, and lymphoma. It is clear that DNMT3B7 is important to tumor development in vitro and in vivo, but it is unknown if expression of the transcript in all of these cell lines translates to relevant clinical results. In this study, a bioinformatics approach was utilized to test the hypothesis that DNMT3B7 expression corresponds to tumor progression in patient samples across cancer types. Gene expression and clinical data were obtained from the Genomic Data Commons for the 33 cancer types available and analyzed for DNMT3B7 expression with relation to tissue type in matched and unmatched samples, staging of tumors, and patient survival. Here we present the results of this analysis indicating a role for DNMT3B7 in tumor progression of many additional cancer types. Based on these data, future in vitro and in vivo studies can be prioritized to examine DNMT3B7 in cancer and, hopefully, develop novel therapeutics to target this aberrant transcript across multiple tumor types.

Aberrant DNMT3B7 Expression Correlates to Age, Race, and Hormone Receptor Status in Breast Cancer Patients

Invasive breast cancer caused almost 40,000 deaths last year alone. Deciphering a way to better understand aggressive phenotypes of breast cancer could potentially provide a novel approach to increase the efficacy of breast cancer treatments. The aberrant gene DNA methyltransferase 3B7, DNMT3B7, has been observed in virtually all cancer types and has been shown to affect multiple facets of breast cancer progression including changes in cell adhesion, cellular proliferation, and anchorage-independent growth. A bioinformatics approach was taken to attempt to determine which clinical parameters are potentially altered by DNMT3B7 expression in breast cancer patients. Clinical parameters including age at initial diagnosis, menopausal status, race, and the status of three hormone receptors — estrogen, progesterone, and human epidermal growth factor 2 (ER, PR, HER2, respectively) — were collected from The Cancer Genome Atlas (TCGA) and analyzed using T-tests and ANOVA. Our results show increased expression of the aberrant gene, DNMT3B7, is correlated with age at initial diagnosis, race, and hormone receptor status (ER negative, PR negative, and HER2 positive) in breast cancer patients, but not with menopausal status. Taken together, these data indicate that DNMT3B7 expression may be an important marker in tumor progression. KEYWORDS: DNMT3B7, DNA Methylation; Bioinformatics; Breast Cancer; Tumor Progression; Estrogen Receptor; Progesterone Receptor: Human Epidermal Growth Factor Receptor 2

DNMT3B7 expression promotes tumor progression to a more aggressive phenotype in breast cancer cells.

Epigenetic changes, such as DNA methylation, have been shown to promote breast cancer progression. However, the mechanism by which cancer cells acquire and maintain abnormal DNA methylation is not well understood. We have previously identified an aberrant splice form of a DNA methyltransferase, DNMT3B7, expressed in virtually all cancer cell lines but at very low levels in normal cells. Furthermore, aggressive MDA-MB-231 breast cancer cells have been shown to express increased levels of DNMT3B7 compared to poorly invasive MCF-7 cells, indicating that DNMT3B7 may have a role in promoting a more invasive phenotype. Using data gathered from The Cancer Genome Atlas, we show that DNMT3B7 expression is increased in breast cancer patient tissues compared to normal tissue. To determine the mechanism by which DNMT3B7 was functioning in breast cancer cells, two poorly invasive breast cancer cell lines, MCF-7 and T-47D, were stably transfected with a DNMT3B7 expression construct. Expression of DNMT3B7 led to hypermethylation and down-regulation of E-cadherin, altered localization of β-catenin, as well as increased adhesion turnover, cell proliferation, and anchorage-independent growth. The novel results presented in this study suggest a role for DNMT3B7 in the progression of breast cancer to a more aggressive state and the potential for future development of novel therapeutics.

DNMT3B7 expression related to MENT expression and its promoter methylation in human lymphomas

DNA methyltransferase (DNMT) 3B7 is the most expressed DNMT3B splice variant. It was reported that the loss of DNMT3B function led to overexpression of the MEthylated in Normal Thymocyes (MENT) and accelerated mouse lymphomagenesis. We investigated the DNMT3B7 expression and its relationship to MENT expression and promoter methylation in human lymphomas. DNMT3B7 and MENT expression were significantly (p<0.0001, p<0.01) higher in lymphomas than in non-malignant. Expression of DNMT3B7 and MENT were associated with MENT promoter hypomethylation. DNMT3B7 overexpression might interfere with the normal DNA methylation mechanism required for silencing the MENT proto-oncogene, and may accelerate human lymphomagenesis.

Truncated DNMT3B Isoform DNMT3B7 Suppresses Growth, Induces Differentiation, and Alters DNA Methylation in Human Neuroblastoma

Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7, which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tumors. Higher levels of DNMT3B7 were detected in differentiated ganglioneuroblastomas compared to undifferentiated neuroblastomas, suggesting that expression of DNMT3B7 may induce a less aggressive clinical phenotype. To test this hypothesis, we investigated the effects of enforced DNMT3B7 expression in neuroblastoma cells, finding a significant inhibition of cell proliferation in vitro and angiogenesis and tumor growth in vivo. DNMT3B7-positive cells had higher levels of total genomic methylation and a dramatic decrease in expression of the FOS and JUN family members that comprise AP1 transcription factors. Consistent with an established antagonistic relationship between AP1 expression and retinoic acid receptor activity, increased differentiation was seen in the DNMT3B7-expressing neuroblastoma cells following treatment with all-trans retinoic acid (ATRA) compared to controls. Our results indicate that DNMT3B7 modifies the epigenome in neuroblastoma cells to induce changes in gene expression, inhibit tumor growth, and increase sensitivity to ATRA.

Abstract 1040: A truncated DNMT3B protein (DNMT3B7) drives DNA methylation and suppresses growth in human neuroblastoma

Abstract Cancer cells have an altered distribution of DNA methylation relative to normal cells. We have shown previously that human cancer cells express aberrant DNMT3B transcripts via abnormal gene splicing. Many of these aberrant transcripts encode truncated proteins, including DNMT3B7, one of the most commonly expressed aberrant isoforms expressed in cancer. In vitro, DNMT3B7 expression modifies the pattern of DNA methylation and gene expression. Neuroblastoma is a pediatric tumor of the neural crest cell lineage. A clinically aggressive disease and poor outcome is associated with CpG island methylator phenotype in neuroblastoma. We found that the expression of four or more DNMT3B transcripts, in primary neuroblastic tumors correlated with high-risk and stage IV disease. Further, the expression of ΔDNMT3B5 also correlated to high-risk and stage IV tumors. Additionally, higher levels of DNMT3B7 expression were detected in more differentiated neuroblastic tumors (ganglioneuroblastomas) compared to undifferentiated neuroblastomas. To determine if DNMT3B7 expression might alter neuroblastoma phenotype, we induced exogenous expression of DNMT3B7 in SMS-KCNR and LA1-55n cells, both aggressive neuroblastoma cell lines. Forced DNMT3B7 expression significantly inhibited neuroblastoma tumor growth in both cell lines. Histologic evaluation of the DNMT3B7-positive xenografts revealed decreased neuroblastoma cell proliferation, increased apoptosis, and inhibited angiogenesis. RNA-Sequencing of the DNMT3B7-expressing LA1-55n cells revealed dramatic decreased expression of FOS and JUN family members (FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, JUND), which encode the components of the AP1 complex. In addition to acting as a transcription factor, AP1 can also antagonize the activity of retinoic acid receptors. We therefore hypothesized that with reduced levels of AP1, retinoic acid receptors would be more active and may drive the DNMT3B7-expressing cells toward differentiation. Among 24 genes that have been used as markers for differentiation in neuroblastoma, 19 have expression changes in DNMT3B7-expressing LA1-55n cells relative to control cells that correlate with a more differentiated phenotype. We also found higher levels of total genomic methylation in neuroblastoma cells with forced DNMT3B7 expression compared to controls in addition to gene promoter methylation changes that correlated to gene expression data. Our results demonstrate that high levels of DNMT3B7 increases total DNA methylation and inhibits neuroblastoma cell proliferation, angiogenesis, and tumor growth. Further knowledge regarding mechanisms by which DNMT3B7 regulates gene methylation may lead ultimately to the development of therapeutic strategies that reverse the epigenetic aberrations that drive neuroblastoma pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1040. doi:1538-7445.AM2012-1040

Abstract 1053: DNMT3B7 alters E-cadherin expression and cell proliferation in breast cancer cells

Abstract Breast cancer is the second-leading cause of cancer death among women in the United States and the progression from non-invasive to invasive/metastatic disease correlates with an increased risk of death. Changes in DNA methylation have been shown to be important in breast cancers. However, the mechanism by which cancer cells acquire and maintain abnormal methylation is not well understood. DNA methyltransferases (DNMTs) are responsible for monitoring and altering DNA methylation patterns in cells. Interestingly, we have identified many aberrant DNMT3B transcripts in cancer cells compared to normal cells. Therefore, we hypothesize that aberrant DNMT3B isoforms alter gene and protein expression to promote tumor progression in breast cancer cells. Our studies indicate that the breast cancer cell lines MCF-7 and MDA-MB-231 express many aberrant DNMT3B isoforms including DNMT3B7 and ΔDNMT3B6. Interestingly, the invasive MDA-MB-231 cells express increased levels of DNMT3B7 and ΔDNMT3B6 compared to the poorly invasive MCF-7 cells. To further our studies, poorly invasive MCF-7 cells were stably transfected with a DNMT3B7 expression construct to determine the role of this aberrant transcript in tumor progression. Preliminary data indicate that the increased expression of DNMT3B7 in these cells correlates with a significant increase in growth. Specifically, cells expressing more DNMT3B7 grow faster than those with lower levels of the aberrant protein. Furthermore, E-cadherin expression is inversely correlated with DNMT3B7 expression indicating that the expression of this aberrant protein may lead to a more aggressive phenotype. Finally, soft agar assays have been performed to determine the functional significance of these findings. The novel results presented in this study suggest the need for additional studies to determine the role of aberrant DNMT3B transcripts in cancer. Ultimately, this work may lead to a better understanding of breast cancer progression and the development of novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1053. doi:1538-7445.AM2012-1053

Myc-Mediated Lymphomagenesis Is Driven by DNA Methylation Changes Induced by DNMT3B7 Expression and Dnmt3b Heterozygosity

Abstract 225Cancer cells are characterized by abnormal DNA methylation, including overall genomic hypomethylation with concurrent region-specific hypo- and hyper-methylation, causing aberrant activation of some genes and the silencing of others. Three DNA methyltransferase (DNMT) enzymes catalyze DNA methylation in eukaryotic cells, DNMT1, DNMT3A, and DNMT3B. We discovered previously that cancer cells exhibit aberrant splicing of the DNMT3B gene, which produces transcripts containing premature stop codons that encode truncated proteins lacking the catalytic domain. When we bred transgenic mice expressing DNMT3B7, one of the aberrantly spliced DNMT3B isoforms found most commonly in cancer cells, with the Eμ-Myc mice, a mouse model for B cell lymphomas, we observed an acceleration of mediastinal lymphomagenesis along with changes in the expression of several genes involved in oncogenesis. The acceleration in tumorigenesis was associated with global DNA hypermethylation, and further analyses showed that these changes in DNA methylation were heterogeneous in tumors derived from Eμ-Myc/DNMT3B7 mice, a phenomenon reminiscent of human tumors. We hypothesized that DNMT3B7 altered DNA methylation by functioning as a dominant negative isoform of full-length endogenous mouse Dnmt3b, and therefore tested a second mouse model that has defects in DNA methylation.The introduction of Dnmt3b heterozygosity (Dnmt3b+/−) into the Eμ-Myc background accelerated mediastinal lymphomagenesis to an even greater extent, with more than 90% of the Eμ-Myc/Dnmt3b+/− mice developing mediastinal lymphomas within the first 120 days. This was also associated with an increase in global DNA methylation as measured by liquid chromatography-mass spectrometry, to a larger extent than in the Eμ-Myc/DNMT3B7 mice. Interestingly, the tumors from Eμ-Myc mice themselves showed global hypermethylation when compared to non-transformed cells from Eμ-Myc mice, suggesting that the transformation of cells that express Myc is a key aspect in the induction of global DNA hypermethylation.These observations led us to the hypothesis that Myc-mediated tumorigenesis is particularly sensitive to changes in DNA methylation. Brenner et al. demonstrated that Myc binds to Dnmt3a/b and recruits the methyltransferases to promoter regions of Myc targets, leading to DNA hypermethylation in these regions. We have also found previously that DNNMT3B7 binds with full-length DNMT3B, by co-immunoprecipitation studies. We hypothesize that either in the presence of DNMT3B7 or with Dnmt3b heterozygosity, Myc-Dnmt3a/b binding at promoters is enhanced, which leads to hypermethylation and repression of gene expression. Using Mycbp, a gene that was repressed in Eμ-Myc/DNMT3B7 tumors, we demonstrated that its promoter region was hypermethylated in both Eμ-Myc/DNMT3B7 and Eμ-Myc/Dnmt3b+/− tumors. The E-box, a conserved sequence located ∼100bp upstream of the transcription start site that Myc binds specifically, was hypomethylated in the Eμ-Myc/DNMT3B7 tumors, suggesting that there was an enrichment of Myc binding at this region. Chromatin immunoprecipitation analyses confirmed increased binding of Myc at the E-box of Mycbp in the Eμ-Myc/DNMT3B7 tumors. Furthermore, we also demonstrated that Myc expression induced all the three DNA methyltransferases, suggesting that Myc-mediated lymphomagenesis may occur using a feedback loop which enhances expression of the DNA methyltransferases to regulate particular genes involved in tumorigenesis. This study offers an insight into the mechanism behind Myc-mediated tumorigenesis and provides evidence for the central role played by changes in DNA methylation patterns in this process. Disclosures:No relevant conflicts of interest to declare.

DNMT3B7, a Truncated DNMT3B Isoform Expressed in Human Tumors, Disrupts Embryonic Development and Accelerates Lymphomagenesis

Epigenetic changes are among the most common alterations observed in cancer cells, yet the mechanism by which cancer cells acquire and maintain abnormal DNA methylation patterns is not understood. Cancer cells have an altered distribution of DNA methylation and express aberrant DNA methyltransferase 3B transcripts, which encode truncated proteins, some of which lack the COOH-terminal catalytic domain. To test if a truncated DNMT3B isoform disrupts DNA methylation in vivo, we constructed two lines of transgenic mice expressing DNMT3B7, a truncated DNMT3B isoform commonly found in cancer cells. DNMT3B7 transgenic mice exhibit altered embryonic development, including lymphopenia, craniofacial abnormalities, and cardiac defects, similar to Dnmt3b-deficient animals, but rarely develop cancer. However, when DNMT3B7 transgenic mice are bred with Emicro-Myc transgenic mice, which model aggressive B-cell lymphoma, DNMT3B7 expression increases the frequency of mediastinal lymphomas in Emicro-Myc animals. Emicro-Myc/DNMT3B7 mediastinal lymphomas have more chromosomal rearrangements, increased global DNA methylation levels, and more locus-specific perturbations in DNA methylation patterns compared with Emicro-Myc lymphomas. These data represent the first in vivo modeling of cancer-associated DNA methylation changes and suggest that truncated DNMT3B isoforms contribute to the redistribution of DNA methylation characterizing virtually every human tumor.