Angiogenin Contributes To Bladder Cancer Tumorigenesis By Facilitating P53/Dnmt3b-Mediated Activation Of Mmp2

Epigenetic-mediated gene activation/silencing plays a crucial role in human tumorigenesis. Eliciting the underlying mechanism behind certain epigenetic changes is essential for understanding tumor biology. An important epigenetic process that is implicated in mammalian development is the methylation of cytosine within the context of a simple dinucleotide site, CpG. Deregulated DNA methylation can be a major driver of pathologic conditions such as neurological and autoimmune diseases, as well as cancers Previous studies in human cancers revealed an unrecognized interplay between Angiogenin (ANG)-MAPK/ERK- matrix metalloproteinase-2 (MMP2) leading to pronounced tumorigenesis. Specifically, we showed that forced ANG expression in a benign human bladder UROtsa cell line induced cellular survival, proliferation, endothelial tube formation and xenograft angiogenesis and growth by mediating the MAPK/ERK-MMP2 axis. Here we provide multiple lines of evidence indicating ANG oncogenic potential and how ANG regulates MMP2 expression. Here we have shown that deletion and site-directed mutagenesis analysis of the MMP2 promoter demonstrated that the p53-binding site is critical for ANG-induced MMP2 promoter activity. To further determine whether ANG facilitates the recruitment of p53 to its targets in cells, we used a ChIP assay to examine p53 binding to the promoter region of the MMP2 gene in UROtsaEmpty, UROtsaANG, RT112 Scr and RT112 KD-ANG. Overexpression of ANG greatly enriched p53 association with the MMP2 promoter and ANG depletion decreased enrichment of p53 with MMP2 promoter. To further explore the mechanism by which ANG regulates MMP2 expression, we investigated whether ANG is also functionally linked to DNA methylation-based transcriptional repression. First, we quantitatively assessed the methylation status of the MMP2 promoter. Overexpression of ANG was associated with a reduction in methylation status of the MMP2 promoter, which led to increased gene expression of MMP2. The alteration of genome-wide DNA methylation in these cells was then analyzed using Illumina whole genome methylation array. ANG expression was noted to be associated with a change in global DNA methylation levels compared to control cells. Mechanistically, ANG negatively regulated DNA methyltransferase 3b (DNMT3b) enzymatic activity by down-regulating its expression and inhibiting its recruitment to the MMP2 promoter. To further define the role of ANG, p53 and MMP2 in tumorigenesis, we collected 78 fresh bladder cancer samples from bladder cancer patients and extracted DNA and RNA for analysis. Muscle invasive bladder cancer specimens had increased levels of ANG, p53 and MMP2 compared to non-muscle invasive bladder cancer. More importantly, disease free survival was adversely and significantly effected if ANG, p53 and MMP2 were overexpressed. In this study we demonstrate that ANG is overexpressed in approximately 45% of bladder tumors resulting in overexpression of p53 and MMP2, a reduction in DNMT3b and deep changes in the methylation status genome wide. Such molecular alterations are associated with higher rates of muscle invasive bladder cancers as well as a reduction in disease specific survival, thus attesting to ANG, p53 and MMP2 ability to further risk stratify patients that may require a more aggressive, even personalized, management plan. Therefore, the study provides novel evidence that ANG plays an important role in the promoter activation of the tumor oncogenic gene MMP2, which likely contributes to ANG oncogenic activity. Citation Format: Rafael Peres, Hideki Furuya, Ian Pagano, Yoshiko Shimizu, Kanani Hokutan, Joanna Gawecka, Charles Joel Rosser. Angiogenin contributes to bladder cancer tumorigenesis by facilitating p53/DNMT3b-mediated activation of MMP2. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A05.