Relative <i>DNMT3B7</i> expression correlates to clinical staging.

<p><i>DNMT3B7</i> expression was compared to clinical stage and shown to be significantly different in (A) ESCA, <i>p</i> = 0.012; (B) KIRC, <i>p</i> = 0.010; (C) KIRP, <i>p</i> = 0.02; (D) LUSC, <i>p</i> = 0.003; (E) TGCT, <i>p</i><0.001; and (F) LAML, <i>p</i><0.001. For (E) TGCT, there were no patient samples with a stage IV diagnosis. (F) LAML staging was measured using the French-American-British (FAB) classifications.</p

Patients with high levels of <i>DNMT3B7</i> expression have lower survival rates than those with low expression levels.

<p>The median <i>DNMT3B7</i> expression for each individual tumor was determined to divide patients with that tumor into “high” (gray, dotted line) and “low” (black, solid line) expression groups. Kaplan-Meier curves were generated and statistical significance was determined for (A) CESC, <i>p</i> = 0.025; (B) KIRC, <i>p</i> = 0.009; (C) LAML, <i>p</i> = 0.035; (D) MESO, <i>p</i> = 0.013; (E) SARC, <i>p</i> = 0.003; and (F) SKCM, <i>p</i> = 0.003.</p

Expression of <i>DNMT3B7</i> in normal and tumor patient samples.

<p>Representative graphs of 6 different tumor samples showing relative <i>DNMT3B7</i> expression, as measured by reads per kilobase per million (RPKM) or RNA-Seq by Expectation Maximization (RSEM), in unmatched normal and tumor tissues in (A) HNSC, (B) UCEC, and (C) THCA. Expression of <i>DNMT3B7</i> in matched patient samples is shown in (D) LIHC and (E) LUAD. <i>DNMT3B7</i> expression in primary and recurrent tissues in (F) LGG (<i>p</i> = 0.005) was assessed when normal samples were not available. All samples shown here were significant, <i>p</i> < 0.001, unless otherwise stated.</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

<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

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