Abstract. Ribosomal RNA (rRNA) expression, one of the most important factors regulating ribosome production, is primarily controlled by a CG-rich 45S rDNA promoter. However, the DNA methylation state of the 45S rDNA promoter, as well as its effect on rRNA gene expression in types of human cancers is controversial. In the present study we analyzed the methylation status of the rDNA promoter (-380 to +53 bp) as well as associated rRNA expression levels in breast cancer cell lines and breast tumor-normal tissue pairs. We found that the aforementioned regulatory region was extensively methylated (74-96%) in all cell lines and in 68% (13/19 tumor-normal pairs) of the tumors. Expression levels of rRNA transcripts 18S, 28S, 5.8S and 45S external transcribed spacer (45S ETS) greatly varied in the breast cancer cell lines regardless of their methylation status. Analyses of rRNA transcript expression levels in the breast tumor and normal matched tissues showed no significant difference when normalized with TBP. On the other hand, using the geometric mean of the rRNA expression values (GM-rRNA) as reference enabled us to identify significant changes in the relative expression of rRNAs in the tissue samples. We propose GM-rRNA normalization as a novel strategy to analyze expression differences between rRNA transcripts. Accordingly, the 18S rRNA/GM-rRNA ratio was significantly higher whereas the 5.8S rRNA/GM-rRNA ratio was significantly lower in breast tumor samples than this ratio in the matched normal samples. Moreover, the 18S rRNA/GM-rRNA ratio was negatively correlated with the 45S rDNA promoter methylation level in the normal breast tissue samples, yet not in the breast tumors. Significant correlations observed between the expression levels of rRNA transcripts in the normal samples were lost in the tumor samples. We showed that the expression of rRNA transcripts may not be based solely on promoter methylation. Carcinogenesis may cause dysregulation of the correlation between spliced rRNA expression levels, possibly due to changes in rRNA processing, which requires further investigation. IntroductionBreast cancer, the most common type of cancer among women, was also the primary and secondary cause of cancer-related deaths among women living in less developed (14.3% of all cancer-related deaths) and more developed regions (15.4% after lung cancer) in 2012, respectively (1). Familial or somatic mutations of BRCA1, BRCA2 and TP53 (alias p53) are wellknown high risk factors for breast cancer formation while others (PALB2, BRIP1, ATM, CHEK2, PTEN and CDH1) have been estimated to have moderate or weak effects (2).Contrary to mutations that modify the DNA sequence itself, epigenetic alterations affect gene expression via DNA methylation, histone modifications and chromatin remodeling. DNA methylation, the frequently studied epigenetic modification in the context of embryogenesis, X chromosome inactivation and imprinting (3-5), is also important for the protection of genome integrity and hence cancer. Global hypomethylation of ...