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Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer.

Nanni S, Aiello A, Re A, Guffanti A, Benvenuti V, Colussi C, Castro-Vega LJ, Felsani A, Londono-Vallejo A, Capogrossi MC, Bacchetti S, Gaetano C, Pontecorvi A, Farsetti A - PLoS ONE (2013)

Bottom Line: To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells.The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS.E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, National Cancer Institute Regina Elena, Rome, Italy.

ABSTRACT
In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5' domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

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Prognostic miRNA signature.A) Supervised cluster analysis of microRNAs profiling (Exiqon Array) for the two groups of patients defined by recurrence status (Good or Bad prognosis). B) Validation by quantitative real time PCR of differential miRNAs levels in the G1 (Bad prognosis n = 7) and G2 (Good prognosis n = 8) groups, *p<0,05 G1 vs G2. C) Differential level of primary transcripts (pri-miR) in G1 (n = 7) and G2 (n = 8), *p<0,05 G1 vs G2. Data are represented as box plot on a logarithm scale.
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pone-0062522-g002: Prognostic miRNA signature.A) Supervised cluster analysis of microRNAs profiling (Exiqon Array) for the two groups of patients defined by recurrence status (Good or Bad prognosis). B) Validation by quantitative real time PCR of differential miRNAs levels in the G1 (Bad prognosis n = 7) and G2 (Good prognosis n = 8) groups, *p<0,05 G1 vs G2. C) Differential level of primary transcripts (pri-miR) in G1 (n = 7) and G2 (n = 8), *p<0,05 G1 vs G2. Data are represented as box plot on a logarithm scale.

Mentions: Lastly, we validated the eNOS ChIP-Seq data enrichment observed upon estradiol treatment. The E2 effect was monitored in C27IM cells, untreated (NT) or E2-treated, using anti-eNOS antibody and ChIP-qPCR on eight gene promoters previously identified or derived from microRNA profile analysis (Figure 1E and Figure 2A below) [1]. Our results reveal a significant correlation between the presence of eNOS-peaks, as emerging from ChIP-Seq data set upon estrogen treatment (Figure S4 and data not shown), and the estrogen-induced recruitment of eNOS onto the same genomic regions as assessed by traditional ChIP-qPCR. Enrichments were normalized to the absence of antibody (noAb), or an unrelated antibody (Ab IgG). Specificity of the ChIp-Seq was ensured using primers amplifying a genomic region within chromosome 5 lacking eNOS peaks and simultaneously showing the absence of eNOS recruitment by classical ChIP-qPCR.


Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer.

Nanni S, Aiello A, Re A, Guffanti A, Benvenuti V, Colussi C, Castro-Vega LJ, Felsani A, Londono-Vallejo A, Capogrossi MC, Bacchetti S, Gaetano C, Pontecorvi A, Farsetti A - PLoS ONE (2013)

Prognostic miRNA signature.A) Supervised cluster analysis of microRNAs profiling (Exiqon Array) for the two groups of patients defined by recurrence status (Good or Bad prognosis). B) Validation by quantitative real time PCR of differential miRNAs levels in the G1 (Bad prognosis n = 7) and G2 (Good prognosis n = 8) groups, *p<0,05 G1 vs G2. C) Differential level of primary transcripts (pri-miR) in G1 (n = 7) and G2 (n = 8), *p<0,05 G1 vs G2. Data are represented as box plot on a logarithm scale.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643940&req=5

pone-0062522-g002: Prognostic miRNA signature.A) Supervised cluster analysis of microRNAs profiling (Exiqon Array) for the two groups of patients defined by recurrence status (Good or Bad prognosis). B) Validation by quantitative real time PCR of differential miRNAs levels in the G1 (Bad prognosis n = 7) and G2 (Good prognosis n = 8) groups, *p<0,05 G1 vs G2. C) Differential level of primary transcripts (pri-miR) in G1 (n = 7) and G2 (n = 8), *p<0,05 G1 vs G2. Data are represented as box plot on a logarithm scale.
Mentions: Lastly, we validated the eNOS ChIP-Seq data enrichment observed upon estradiol treatment. The E2 effect was monitored in C27IM cells, untreated (NT) or E2-treated, using anti-eNOS antibody and ChIP-qPCR on eight gene promoters previously identified or derived from microRNA profile analysis (Figure 1E and Figure 2A below) [1]. Our results reveal a significant correlation between the presence of eNOS-peaks, as emerging from ChIP-Seq data set upon estrogen treatment (Figure S4 and data not shown), and the estrogen-induced recruitment of eNOS onto the same genomic regions as assessed by traditional ChIP-qPCR. Enrichments were normalized to the absence of antibody (noAb), or an unrelated antibody (Ab IgG). Specificity of the ChIp-Seq was ensured using primers amplifying a genomic region within chromosome 5 lacking eNOS peaks and simultaneously showing the absence of eNOS recruitment by classical ChIP-qPCR.

Bottom Line: To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells.The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS.E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, National Cancer Institute Regina Elena, Rome, Italy.

ABSTRACT
In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5' domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

Show MeSH
Related in: MedlinePlus