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

Uemura M, Zheng Q, Koh CM, Nelson WG, Yegnasubramanian S, De Marzo AM - Oncogene (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

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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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 χ2-test.
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fig5: 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 χ2-test.

Mentions: We next isolated genomic DNA from the same samples used above for RNA isolation (20 of the 21 cases contained enough tissue for DNA isolation). Figure 5a shows bisulfite-sequencing results from these human prostate cancer specimens as compared with matched normal specimens. As in the cell lines, all cases showed two populations of alleles. And, there was no clear difference in tumor samples compared with normal samples. A comparison of the methylation pattern of the 20 individual prostates revealed variations among them, which is likely the result of germ line driven polymorphisms in this epigenetic patterning as the differences between individuals were generally present in both tumor and normal tissues (Figure 5b). For example, when comparing tumor with normal samples the fraction of methylated to total CpGs across the whole region correlated relatively strongly with each other (r=0.7663, P=0.0001, Pearson's moment correlation). It is presently unknown whether this pattern of individual variation of rDNA promoter methylation status in humans is also found in other tissues in the body. When comparing tumor vs normal at individual CpG sites, there were some relatively subtle differences uncovered such that there was hypomethylation at −30 and −25 in the core promoter region and hypermethylation at +8 in prostate cancer compared with normal tissues (Figure 5c). Despite these minor changes, it is clear that demethylation of rDNA promoter regions does not appear to explain why tumor cell nucleoli are so enlarged, nor why these cells tend to have increased numbers of nucleoli.


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

Uemura M, Zheng Q, Koh CM, Nelson WG, Yegnasubramanian S, De Marzo AM - Oncogene (2011)

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 χ2-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298623&req=5

fig5: 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 χ2-test.
Mentions: We next isolated genomic DNA from the same samples used above for RNA isolation (20 of the 21 cases contained enough tissue for DNA isolation). Figure 5a shows bisulfite-sequencing results from these human prostate cancer specimens as compared with matched normal specimens. As in the cell lines, all cases showed two populations of alleles. And, there was no clear difference in tumor samples compared with normal samples. A comparison of the methylation pattern of the 20 individual prostates revealed variations among them, which is likely the result of germ line driven polymorphisms in this epigenetic patterning as the differences between individuals were generally present in both tumor and normal tissues (Figure 5b). For example, when comparing tumor with normal samples the fraction of methylated to total CpGs across the whole region correlated relatively strongly with each other (r=0.7663, P=0.0001, Pearson's moment correlation). It is presently unknown whether this pattern of individual variation of rDNA promoter methylation status in humans is also found in other tissues in the body. When comparing tumor vs normal at individual CpG sites, there were some relatively subtle differences uncovered such that there was hypomethylation at −30 and −25 in the core promoter region and hypermethylation at +8 in prostate cancer compared with normal tissues (Figure 5c). Despite these minor changes, it is clear that demethylation of rDNA promoter regions does not appear to explain why tumor cell nucleoli are so enlarged, nor why these cells tend to have increased numbers of nucleoli.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

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.

Show MeSH
Related in: MedlinePlus