Basic Hallmarks of Urothelial Cancer Unleashed in Primary Uroepithelium by Interference with the Epigenetic Master Regulator ODC1

Abstract

Urothelial carcinoma (UC) is a common disease causing significant morbidity and mortality as well as considerable costs for health systems. extensive aberrant methylation of DnA is broadly documented in early Uc, contributing to genetic instability, altered gene expression and tumor progression. However the triggers initiating aberrant methylation are unknown. Recently we discovered that several genes encoding key enzymes of methyl group and polyamine metabolism, including Ornithine Decarboxylase 1 (ODC1), are affected by DNA methylation in early stage UC. In this study, we investigated the hypothesis that these epigenetic alterations act in a feed-forward fashion to promote aberrant DnA methylation in Uc. We demonstrate that siRnA-mediated knockdown of ODC1 expression elicits genome-wide LINE-1 demethylation, induction of LINE-1 transcripts and doublestrand DnA breaks and decreases viability in primary cultured uroepithelial cells. Similarly, following siRnA-mediated knockdown of ODC1, Uc cells undergo double-strand DnA breaks and apoptosis. Collectively, our findings provide evidence that ODC1 gene hypermethylation could be a starting point for the onset of genome-wide epigenetic aberrations in urothelial carcinogenesis. Furthermore, LINE-1 induction enabled by ODC1 interference provides a new experimental model to study mechanisms and consequences of LINE-1 activation in the etiology and progression of UC as well as presumably other cancers. Urothelial carcinoma (UC), the most common cancer of the urinary bladder, is a frequent disease with yearly 549,393 new cases and 199,922 deaths worldwide 1. Relapse rates of 30-70% and rates of progression at 10-30% for high-grade tumors present a serious health care challenge and impose enormous financial burdens on the health care systems 2. In particular, UC that have invaded the muscle-layers of the bladder are highly lethal. Tobacco smoking is thought to contribute to 50% of all UC cases as a risk factor 3. Aromatic amines such as 2-naphthylamin and polycyclic aromatic hydrocarbons in cigarette smoke cause mutations in key cancer-related genes by forming DNA adducts 4. Accordingly, exposure to tobacco smoke alone is sufficient to induce

RETRACTED ARTICLE: Aging-associated distinctive DNA methylation changes of LINE-1 retrotransposons in pure cell-free DNA from human blood

“…that DNA methylation represses repetitive DNA elements including LINE-1 retrotransposons to ensure genomic integrity, its stability and to prevent cancer. LINE-1 hypomethylation seems to be a sufficient criterion for LINE-1 transcription, as demonstrated by us and others 15 . However, additional potent mechanisms in healthy somatic cells inhibit LINE-1 protein expression.…”

“…LINE-1 hypomethylation occurs in a broad variety of different cancers, often at early stages, e.g. in urothelial cancer 15 , but also appears to contribute to cancer progression, especially once the additional cellular defense mechanisms preventing LINE-1 retrotransposition have been overcome. Therefore, we expect that a degree of LINE-1 hypomethylation in cfDNA that is markedly higher than the average value of healthy individuals at a specific age could be indicative of a higher risk of developing or harboring cancer.…”

“…DNA served as template for amplification of methylated LINE-1 promotor sequences in a normalized real-time MS-PCR for genetically imbalanced DNA specimens as described 15,17,22,44 . Quantification was done using Ste-pOne Plus Real Time PCR System (Applied Biosystems, Foster City, United States of America) with the following primers for sL1met: 5′-GCG CGA GTC GAA GTA GGG C-3′; asL1met:5′-CTC CGA CCA AAT ATA AAA TAT AAT CTCG-3′.…”

“…Amplification of an adjacent region without CpGs was used to normalize the amount of LINE-1 methylation. The primers used for reference amplification were for sLcontrol: 5′-AGG TTT TAT TTT TGG GGG TAG GGT ATA-3′; asL1control:5′-CCC CTA CTA AAA AAT ACC TCC CAA TTA AAC -3′ 15 .…”

“…For instance, we have recently shown that interference with methyl group availability by downregulation of ornithine decarboxylase, a key enzyme of polyamine biosynthesis, can induce LINE-1 hypomethylation in primary uroepithelial cells, leading to increased LINE-1 transcript levels, double-strand DNA breaks and decreased viability 15 .…”