Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation - PubMed (original) (raw)

. 2012 Mar 8;31(10):1254-63.

doi: 10.1038/onc.2011.319. Epub 2011 Aug 8.

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Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation

M Uemura et al. Oncogene. 2012.

Free PMC article

Abstract

Alterations in nucleoli, including increased numbers, increased size, altered architecture and increased function are hallmarks of prostate cancer cells. The mechanisms that result in increased nucleolar size, number and function in prostate cancer have not been fully elucidated. The nucleolus is formed around repeats of a transcriptional unit encoding a 45S ribosomal RNA (rRNA) precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. Although it has been generally accepted that tumor cells overexpress rRNA species, this has not been examined in clinical prostate cancer. We find that indeed levels of the 45S rRNA, 28S, 18S and 5.8S are overexpressed in the majority of human primary prostate cancer specimens as compared with matched benign tissues. One mechanism that can alter nucleolar function and structure in cancer cells is hypomethylation of CpG dinucleotides of the upstream rDNA promoter region. However, this mechanism has not been examined in prostate cancer. To determine whether rRNA overexpression could be explained by hypomethylation of these CpG sites, we also evaluated the DNA methylation status of the rDNA promoter in prostate cancer cell lines and the clinical specimens. Bisulfite sequencing of genomic DNA revealed two roughly equal populations of loci in cell lines consisting of those that contained densely methylated deoxycytidine residues within CpGs and those that were largely unmethylated. All clinical specimens also contained two populations with no marked changes in methylation of this region in cancer as compared with normal. We recently reported that MYC can regulate rRNA levels in human prostate cancer; here we show that MYC mRNA levels are correlated with 45S, 18S and 5.8S rRNA levels. Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels. We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells. Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

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Figures

Figure 1

Figure 1

The human rDNA promoter region. (a) Genotyping of rDNA promoter region. The sequence of the rDNA promoter as shown was identical for all prostate cancer tissues and normal tissues, as well as all the cell lines. In this map, primer sequences for genotyping are indicated by the underlined regions and the primer sequences for bisulfite sequencing are indicated as dotted underlines. CpG dinucleotides are boxed with the location marked relative to the transcription start site (arrow). Discrepancies with the published DNA sequence (U13369) are marked. Nucleotides that show base substitutions as compared with the reference U13369 seqeunce are illustrated with dots above the DNA sequence. Deletions (generally a single base), relative to the reference sequence, are marked by a ‘v ‘between nucleotides. (b) Schematic representation of a single human rDNA repeat. The approximate positions relative to the transcription start site are indicated (DNA base number in kb). (c) Vertical lines indicate locations of CpG dinucleotides on the 45S rRNA promoter relative to the transcription start site, indicated with the dashed curved arrow, with primer pairs used for bisulfite mapping marked by inward pointing arrows.

Figure 2

Figure 2

rRNA levels are increased in human prostate cancer tissues. Box plots show the relative levels of the indicated rRNA species in tumor compared with non-tumor (normal) tissue. The sign test was used to test the equality of the matched pairs of observation (_P_-values are indicated).

Figure 3

Figure 3

Methylation status of the rRNA promoter in the parental colorectal carcinoma cells HCT116 and its derivative knockout cell lines. The methylation status of each CpG dinucleotide spanning −158 to +29 bp of human rRNA promoter in HCT116 colorectal carcinoma cells and derivative lines with targeted disruption of DNMT1, DNMT3b or both genes. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 16–19 randomly selected clones from each sample were subjected to automated sequencing. Each row represents the sequence of an individual clone, whereas each column depicts the position of the CpG. The filled and open circles denote methylated and unmethylated CpGs, respectively. The top row shows a heat map of methylated CpGs representing the frequency of methylation in each of the individual clones shown below. The degree of hypermethylation (NIM) at three CpG islands was assessed using quantitative real-time methylation-specific PCR and was normalized to the amount of bisulfite-converted input as described in ‘Materials and methods.'

Figure 4

Figure 4

The methylation status of the rRNA promoter in prostate cancer cell lines and normal prostate epithelial cells. The methylation status for each cytosine within each CpG dinucleotide spanning from −158 to +29 bp of the human rRNA promoter in prostate cancer cell lines and normal prostate epithelial cells was determined. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 10–18 randomly selected clones from each sample were subjected to automated sequencing.

Figure 5

Figure 5

The methylation status of the rRNA promoter in clinical prostate cancer relative to matched normal tissues. (a) The methylation status of each cytosine within each CpG dinucleotide spanning −158 to +29 bp of the human rRNA promoter in 20 human prostate cancers and matched normal tissues. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 10–18 randomly selected clones from each sample were subjected to automated sequencing. (b) Quantitative analysis of methylation density at each CpG and all CpGs with respect to the +1 site of the rRNA promoter in individual tumors and matched normal prostate tissues is shown. The percentage of clones methylated and unmethylated at each position among clones is represented in this bar diagram. T and N denote prostate cancer and normal tissue, whereas Unmet and Met indicate unmethylated and methylated CpGs, respectively. The average percentage of methylation for all CpG sites at each CpG site, for prostate cancer (_N_=20; left-side bars) and matched normal tissues (_N_=20; right-side bars). Data are expressed as *P<0.05; ** P<0.01, measured by _χ_2-test.

Figure 5

Figure 5

The methylation status of the rRNA promoter in clinical prostate cancer relative to matched normal tissues. (a) The methylation status of each cytosine within each CpG dinucleotide spanning −158 to +29 bp of the human rRNA promoter in 20 human prostate cancers and matched normal tissues. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 10–18 randomly selected clones from each sample were subjected to automated sequencing. (b) Quantitative analysis of methylation density at each CpG and all CpGs with respect to the +1 site of the rRNA promoter in individual tumors and matched normal prostate tissues is shown. The percentage of clones methylated and unmethylated at each position among clones is represented in this bar diagram. T and N denote prostate cancer and normal tissue, whereas Unmet and Met indicate unmethylated and methylated CpGs, respectively. The average percentage of methylation for all CpG sites at each CpG site, for prostate cancer (_N_=20; left-side bars) and matched normal tissues (_N_=20; right-side bars). Data are expressed as *P<0.05; ** P<0.01, measured by _χ_2-test.

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