<i>DNMT3B7</i> 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.</p

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

<i>DNMT3B7</i> expression in breast cancer.

<p><i>DNMT3B7</i> reads per kilobase per million (RPKM) values of A) normal tissue versus primary tumors in unmatched patient samples or B) matched normal tissue versus primary tumor. C) <i>DNMT3B7</i> expression in all primary tumors segregated by respective tumor stages. D) <i>DNMT3B7</i> expression in all primary tumors segregated by respective molecular subtype: luminal A (ER and/or PR positive and HER2 negative), luminal B (ER and/or PR positive and HER2 positive), triple negative/basal-like (ER negative, PR negative, HER2 negative), and HER2 type (ER negative, PR negative, HER2 positive). Statistical significance is indicated on the graphs. Immunoblot analysis of nuclear lysates of (E) MCF-7 and (F) T-47D cells stably transfected with a DNMT3B7-expression construct or an empty control vector. Topoisomerase was utilized as a nuclear lysate loading control.</p

<i>DNMT3B7</i> regulates cell adhesion, proliferation, and growth in soft agar.

<p>A-C) MCF-7 or (D-F) T-47D cells stably expressing DNMT3B7 or a control vector were measured for changes in adhesion, proliferation, and growth in soft agar. A, D) Adhesion was measured as the number of cells that could adhere to a 6-well dish in 1 hour normalized to the control (100%). B, E) Cells were plated on a dish and counted twice a week for 3 weeks to measure proliferative ability. C, F) Anchorage-independent growth was determined after cells were grown in soft agar for 2 weeks, stained with crystal violet to visualize colonies, counted, and normalized to the control vector. Statistical significance is indicated on the graphs. * represents statistical significance, <i>p</i><0.05.</p

Hypermethylation and down-regulation of E-cadherin in the presence of <i>DNMT3B7</i>.

<p>Genomic DNA from stably transfected DNMT3B7-expressing cell lines and vector controls was collected, bisulfite treated, and subjected to methylation-specific PCR analysis. Primers were designed to incorporate 15 CpGs within an island encompassing the promoter region, exon 1, and the translation start site (TSS, indicated by the arrow between CpG 12 and 13). Dark shading in pie charts indicates percent of methylated C:T ratio at 15 CpG sites in (A) MCF-7 and (B) T-47D cells expressing DNMT3B7 or a control vector. Immunoblot analysis of E-cadherin expression compared to an actin loading control in (C) MCF-7 and (D) T-47D cells correlates with methylation data. Quantitative analysis using densitometry of representative blots is shown for (E) MCF-7 and (F) T-47D cells. Statistical significance is indicated on the graphs.</p

Putative model of <i>DNMT3B7</i> action in poorly invasive breast cancer cells.

<p>MCF-7 and T-47D cell lines normally have low levels of DNMT3B7 and high expression of E-cadherin. Upon stable transfection of <i>DNMT3B7</i> we observe inhibition of E-cadherin, localization of β-catenin to the nucleus, and tumor progression to a more aggressive phenotype. Future studies will examine the potential for cadherin switching in the presence of DNMT3B7 as well as changes in E-cadherin binding partners, such as catenins, and downstream signaling pathways which may promote tumor progression. Studies examining the role of other aberrant <i>DNMT</i>s will also be imperative to fully understand the role of aberrant <i>DNMT</i> transcripts in breast cancer progression.</p

Expression of <i>DNMT3B7</i> alters β-catenin localization in breast cancer cells.

<p>Immunofluorescence of β-catenin in MCF-7 (left panel) and T-47D (right panel) cells shows membrane staining in vector controls (A-B) as well as in DNMT3B7-expressing cells (C-F). Nuclear localization of β-catenin is only observed in cells expressing DNMT3B7 (G-H).</p