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

LINE-1 hypomethylation of cell-free DNA has been described as an epigenetic biomarker of human aging. However, in the past, insufficient differentiation between cellular and cell-free DNA may have confounded analyses of genome-wide methylation levels in aging cells. Here we present a new methodological strategy to properly and unambiguously extract DNA methylation patterns of repetitive, as well as single genetic loci from pure cell-free DNA from peripheral blood. Since this nucleic acid fraction originates mainly in apoptotic, senescent and cancerous cells, this approach allows efficient analysis of aged and cancerous cell-specific DNA methylation patterns for diagnostic and prognostic purposes. Using this methodology, we observe a significant age-associated erosion of LINE-1 methylation in cfDNA suggesting that the threshold of hypomethylation sufficient for relevant LINE-1 activation and consequential harmful retrotransposition might be reached at higher age. We speculate that this process might contribute to making aging the main risk factor for many cancers.

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