Background: Canine mammary tumor (CMT) has long been considered as a good animal model for human breast cancer (HBC) due to their pathological and biological similarities. However, only a few aspects of the epigenome have been explored in both HBC and CMT. Moreover, DNA methylation studies have mainly been limited to the promoter regions of genes. Results: Genome-wide methylation analysis was performed in CMT and adjacent normal tissues and focused on the intron regions as potential targets for epigenetic regulation. As expected, many tumor suppressors and oncogenes were identified. Of note, most cancer-associated biological processes were enriched in differentially methylated genes (DMGs) that included intron DMRs (differentially methylated regions). Interestingly, two PAX motifs, PAX5 (tumor suppressive) and PAX6 (oncogenic), were frequently found in hyper-and hypomethylated intron DMRs, respectively. Hypermethylation at the PAX5 motifs in the intron regions of CDH5 and LRIG1 genes were found to be anti-correlated with gene expression, while CDH2 and ADAM19 genes harboring hypomethylated PAX6 motifs in their intron region were upregulated. These results were validated from the specimens originally MBD-sequenced as well as additional clinical samples. We also comparatively investigated the intron methylation and downstream gene expression of these genes using human breast invasive carcinoma (BRCA) datasets in TCGA (The Cancer Genome Atlas) public database. Regional alteration of methylation was conserved in the corresponding intron regions and, consequently, gene expression was also altered in HBC. Conclusions: This study provides good evidence for the conservation of epigenetic regulation in CMT and HBC, and suggests that intronic methylation can be an important factor in better understanding gene regulation in both CMT and HBC.

Canine mammary tumors (CMT) constitute the most common tumor types found in female dogs. Understanding this cancer through extensive research is important not only for clinical veterinary applications, but also in the scope of comparative oncology. The use of DNA methylation as a biomarker has been noted for numerous cancers in the form of both tissue and liquid biopsies, yet the study of methylation in CMT has been limited. By analyzing our canine methyl-binding domain sequencing (MBD-seq) data, we identified intron regions of canine ANK2 and EPAS1 as differentially methylated regions (DMGs) in CMT. Subsequently, we established quantitative methylation specific PCR (qMSP) of ANK2 and EPAS1 to validate the target hypermethylation in CMT tissue, as well as cell free DNA (cfDNA) from CMT plasma. Both ANK2 and EPAS1 were hypermethylated in CMT and highlighted as potential tissue biomarkers in CMT. ANK2 additionally showed significant hypermethylation in the plasma cfDNA of CMT, indicating that it could be a potential liquid biopsy biomarker as well. A similar trend towards hypermethylation was indicated in HBC at a specific CpG of the ANK2 target on the orthologous human region, which validates the comparative approach using aberrant methylation in CMT.

Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells.

Transcription is regulated through a variety of different mechanisms. One facet of transcriptional regulation is epigenetics, which is the regulation of gene expression without altering the nucleotide sequence of the genome, but rather reversible modifications, e.g. CpG methylation. The CpG islands in tumor suppressor gene promoters are often hypermethylated, thus down regulating their expression. The p73 gene is a member of the p53 tumor suppressor family of proteins, and like p53, has a bifurcated promoter. The p73 P1 promoter transcribes a full-length mRNA, which then translates into the transcriptionally active p73. The p73 P2 promoter transcribes a truncated mRNA, which translated into transcriptionally inactive ΔNp73. The ΔNp73 product lacks the N-terminal trans-activation domain that TAp73 possesses. This study sought to analyze the extent of DNA methylation in the CpG islands in the p73 P1 promoter of three prostate cancer (PCa) cell lines (LNCaP, PC-3, and DU145), and primary prostate cells. We hypothesized that the p73 P1 promoter would be hypermethylated in prostate cancer cell lines and hypomethylated in primary prostate cells. The methylation status of the p73 P1 promoter in all four prostate cell lines was analyzed by using bisulfite sequencing PCR (BSP). Our analyses revealed that a CpG island in the p73 P1 was hypermethylated in LNCaP, PC-3, and DU145 cells compared to primary prostate cells. Furthermore, among the three PCa cell lines evaluated, the p73 P1 promoter was the most methylated in DU145 cells, and the least methylated in LNCaP cells. The P1 region sequenced contained 28 CpG islands, and among them, only five were methylated in the primary prostate cells (18% methylated), 23 were methylated in LNCaP cells (82% methylated), 26 in PC-3 cells (93% methylated), and 28 in DU145 cells (100% methylated). Furthermore, of the 28 CpG sites, 14 of them were differentially methylated in the primary prostate cell line versus the three PCa cell lines evaluated in this study. These data are consistent with the PCa tumor-derived cell lines (LNCaP, PC-3, and DU145) having hypermethylated CpG sites in the p73 P1 promoter compared to the primary prostate cell line. Citation Format: Nicholas E. Braganca, L. Michael Carastro, Johannes J. Schabort, Jong Y. Park. Bisulfite DNA sequencing analyses to detect methylation patterns in the p73 gene promoter in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4363. doi:10.1158/1538-7445.AM2017-4363

One facet of epigenetic regulation is methylation of CpG islands within the promoter region of genes. When CpG islands are hypermethylated, this often down regulates transcription of the gene as transcription factors are hindered from binding. This sometimes occurs in the promoter regions of tumor suppressor genes, such as in cancerous cell lines, e.g., p73. The p73 gene is a member of the p53 tumor suppressor family. The p73 gene promoter is bifurcated, with transcription of a full‐length, wild‐type mRNA, TAp73, from the P1 promoter, while the P2 promoter transcribes a truncated, dominant negative version of the p73 mRNA, deltaNp73. Previously, we reported bisulfite sequencing PCR (BSP) data which identified methylated CpG positions in the p73 gene P1 promoter in primary prostate cells, as well as three prostate cancer (PCa) cell lines: LNCaP, PC‐3, and DU 145. Upon analyzing this BSP methylation data for the P1 promoter region in all four cell lines studied, we reported the three PCa cell lines contained more methylated CpG positions compared to the primary prostate cell line which contained the fewest methylated CpG positions. Furthermore, within the three PCa cell lines, the number of methylated CpG positions was higher in more transformed cell lines, DU145 and PC‐3, compared to the less transformed cell lines, LNCaP. The purpose of this work was to combine our unreported BSP data with previously reported data and determine the overall degree of CpG methylation in the p73 gene P1 promoter region. We hypothesized the overall number of methylation CpG methylation observed in the P1 promoter of the p73 gene would be greater in the three PCa cell lines compared to primary prostate cells. Within the 635‐bp region of the P1 promoter analyzed, there were 230 potential CpG positions which could have been methylated. Of these 230 CpG position, 58 were found to be hypermethylated (>50% frequency) in one or more of these cell lines. Among these 58 confirmed CpG methylation positions, 13 (5.7%) were hypermethylated in primary prostate cells, 40 (17.4%) in LNCaP cells, 52 (22.6%) in PC‐3 cells, and 57 (24.8%) in DU145 cells. This analysis of our data is consistent with the stated hypothesis, in that, within the p73 P1 promoter region analyzed, a higher degree of CpG methylation was detected in the three PCa lines as compared to primary prostate cells. Currently, we are quantifying the extent of methylation, in all four cell lines, at all 58 detected CpG positions. Methylation quantification is being accomplished by subcloning p73 gene P1 promoter BSP amplimers into pET41a vectors, transforming ligation products into DH5‐alpha cells (NEB) from which plasmids are isolated. At least 20 clones from each amplimer ligation reaction are being analyzed by Sanger sequencing and the extent of CpG methylation at each position is being determined quantitatively.Support or Funding InformationThis work was financially supported by funding from the Department of Chemistry, Biochemistry & Physics at the University of Tampa to support student research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.