Abstract LB-016: Angiogenin contributes to bladder cancer tumorigenesis by facilitating p53 mediated activation of MMP2
Authors: Peres Rafael , Furuya Hideki , Hokutan Kanani , Shimizu Yoshiko , Chan Owen T. , Pagano Ian , Dyrskjøt Lars , Jensen Jørgen B. , Malmström Per-Uno , Segersten Ulrika , Ng Ho Leung , Janku Filip , Rosser Charles J. 
Published: July 2017

Abstract
Angiogenin (ANG) has been reported to be upregulated and associated with poor prognosis in some human cancers. Previous studies revealed ANG expression stimulated matrix metallopeptidase-2 (MMP2) expression through a) phosphorylation of ERK1/2 (ANG-ERK1/2-MMP2 pathway), which led to a pronounced tumorigenesis, and b) hypomethylation of the MMP2 gene by negatively regulating DNA methyltransferase 3b (DNMT3b) expression (ANG-DNMT3b-MMP2 pathway). However, since ANG-DNMT3b-MMP2 pathway is independent of ANG-ERK1/2-MMP2, the exact mechanism through which ANG-induced MMP2 expression remains unclear. Here we show that ANG-induced p53 expression is regulated by ERK1/2 and is a missing link between ERK1/2 and MMP2 in ANG-ERK1/2-MMP2 axis. The analysis of the serial deletion mutant MMP2 promoters demonstrated that the p53-binding site is critical for ANG to induce MMP2 promoter activity. We also found that ANG increased the DNA binding activity of p53, which was suppressed by knockdown of p53 leading to the reduction in MMP2 expression. Remarkably, we revealed that mutated p53 isoforms were able to induce MMP2 expression, illustrating that in this setting, the primary driver of MMP2 expression is the amount of p53 protein (wild-type and mutants) available to bind to the MMP2 promoter. ChIP-qPCR and promoter assay using MMP2 promoter constructs confirmed that both wild-type and p53 mutants (V143A, R175H, R248W, R249S and R273H) increased MMP2 promoter transcriptional activities in BCa cell lines. We also performed computational modeling to support energetic and structural feasibility of our wild-type and mutant p53 observations. We predicted that p53 mutants can bind to p53 binding sites on the MMP2 promoter with binding energy comparable to wild type. The mutants may have induced, however, changes on the expression of other p53 targets. In addition, we present data that the p53 mutants are able to induce cell growth in bladder cells, in opposition to the wild type p53. Finally, the results from human bladder tumors a) confirmed the strong correlation between ANG, p53 and MMP2 and b) reduced survival when ANG, p53 and/or MMP2 were overexpressed in human bladder tumors. We provide multiple lines of evidence indicting ANG oncogenic potential. Thus, we suggest that targeting ANG and associated factors could provide a novel strategy to inhibit tumor angiogenesis and growth.
Citation Format: Rafael Peres, Hideki Furuya, Kanani Hokutan, Yoshiko Shimizu, Owen T. Chan, Ian Pagano, Lars Dyrskjøt, Jørgen B. Jensen, Per-Uno Malmström, Ulrika Segersten, Ho Leung Ng, Filip Janku, Charles J. Rosser. Angiogenin contributes to bladder cancer tumorigenesis by facilitating p53 mediated activation of MMP2 [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 LB-016. doi:10.1158/1538-7445.AM2017-LB-016