Our previous studies (Misiti et al., 2000 and Nanni et al. JCI 2002) assigned a crucial role to ER signaling in terms of telomerase activation in human ovary epithelial cells as well as in a traditionally androgen-dependent tumor, the Prostate cancer (PCa) and identified nuclearized endothelial NOS (eNOS) as partner of both Estrogen Receptors, ER?/ER? and Hypoxia Inducible Factors in PCa (Grasselli et al., 2008; Nanni et al., 2009; Re et al., 2011). Protein conformation at target promoters (e.g. hTERT) is affected substantially by formation of these complexes in response to estrogen and hypoxia stimuli that, in turn, regulate expression of genes associated with adverse prognosis in PCa. To better define the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells derived from a primary tumor associated with poor outcome and from metastatic LNCaP cells, before and after treatment with 17?-estradiol (E2). By this approach, we have documented the existence, on a genome-wide scale, of a considerable number of eNOS-DNA associations that define transcriptional active regions modulated by estrogen (Nanni et al., 2013). In addition, numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets, including small non coding RNAs (miRs). In particular, we found a cluster of miRs, among which miR-34a, strongly silenced in PCa cells associated with poor outcome, suggesting a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. Specifically, we revealed a feedback loop involving transcriptional downregulation of pri-miR34a by the eNOS/SIRT1 complex in an estrogen-dependent fashion. As a consequence we observed induction of the miR-34a target SIRT1 that sequentially activates eNOS itself by post-translational modification. These findings revealed novel functions of eNOS and of the eNOS/SIRT1 interplay, fine-tuned by E2-activated ER signaling, favouring the concept of eNOS as critical molecular determinant in aggressive PCa. Recent studies have placed long non coding RNAs (lncRNAs) at the leading edge of cancer research and suggested that they may serve as master drivers of carcinogenesis. However, their identity, function and deregulation in cancer are only beginning to be understood. In line with the above, we have contributed to the identification, by ChIP-Sequencing, of a consistent number of eNOS-bound complexes in the genome regions of many lncRNAs strictly associated with cancer, including PCa (Prensner JR and Chinnaiyan AM, 2011). Intriguingly, in the genomic regions overlapping with, or in the proximity of these transcripts the number of eNOS peaks significantly increases upon estrogen treatment, with a specific pattern for the primary tumor as compared to the metastatic cell line. These data are being validated by ChIP/re-ChIP and RIP assays with proteins of interest (eNOS, ER?, ER? and androgen receptor, AR) and lncRNAs emerging from the ChIP-Seq analysis. We currently aim at expanding such findings to a different microenvironment, breast cancer, by analysing, in this context, the occupancy by the eNOS complexes of genome regions of specific cancer-associated LncRNAs. Although arising in organs that are different for anatomy and physiological function, both breast and prostate cancers are typically hormone-dependent and have remarkable biological similarities. They are both driven by the sex steroid hormones, estrogen and androgen, that activate their respective nuclear receptors, ER?/ER? or AR. These nuclear receptors interact with a plethora of other transcription factors and co-factors, like eNOS, which aid ER and AR in the activation of a pro-tumorigenic gene expression program. To this end the expression of sex steroid hormone receptors (ER?, ER? or AR) and eNOS has been evaluated and compared in a large population of breast and prostate cancer cell lines, and 4 sub-groups among them have been identified on the basis of their different expression pattern. Levels of the most relevant cancer-associated lncRNAs (specifically HOTAIR, GAS5, H19, and CDKN2B AS/ANRIL and MALAT1) were quantified by qRT-PCR in each cell line, before and after estrogens or hypoxia or upon interfering with the NO signaling by genetic or pharmacological approaches or upon combined treatments. Preliminary data indicate that: i. lncRNAs HOTAIR and H19 basal expression in breast cancer cell lines appears highly correlated with presence of ER?, being undetectable in the ER?- cells; ii. lncRNAs HOTAIR, MALAT1 and GAS5 have higher expression levels in metastatic prostate cancer cell lines than in primary tumors-derived PCa cells, suggesting that their induction correlates with poor patient outcome and cancer metastasis; iii. lncRNAs H19, HOTAIR and CDKN2B-AS/ANRIL expression significantly decreases after eNOS inhibition exclusively in ER?+ breast cancer cell lines, strongly suggesting that eNOS and ER? are strictly required for the transcriptional control of selected cancer-associated lncRNAs in hormone-driven cancers.
Estrogen/ERs, Hypoxia/HIFs and NO/eNOS signaling in hormone-driven cancers
Antonella Farsetti
2014
Abstract
Our previous studies (Misiti et al., 2000 and Nanni et al. JCI 2002) assigned a crucial role to ER signaling in terms of telomerase activation in human ovary epithelial cells as well as in a traditionally androgen-dependent tumor, the Prostate cancer (PCa) and identified nuclearized endothelial NOS (eNOS) as partner of both Estrogen Receptors, ER?/ER? and Hypoxia Inducible Factors in PCa (Grasselli et al., 2008; Nanni et al., 2009; Re et al., 2011). Protein conformation at target promoters (e.g. hTERT) is affected substantially by formation of these complexes in response to estrogen and hypoxia stimuli that, in turn, regulate expression of genes associated with adverse prognosis in PCa. To better define the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells derived from a primary tumor associated with poor outcome and from metastatic LNCaP cells, before and after treatment with 17?-estradiol (E2). By this approach, we have documented the existence, on a genome-wide scale, of a considerable number of eNOS-DNA associations that define transcriptional active regions modulated by estrogen (Nanni et al., 2013). In addition, numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets, including small non coding RNAs (miRs). In particular, we found a cluster of miRs, among which miR-34a, strongly silenced in PCa cells associated with poor outcome, suggesting a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. Specifically, we revealed a feedback loop involving transcriptional downregulation of pri-miR34a by the eNOS/SIRT1 complex in an estrogen-dependent fashion. As a consequence we observed induction of the miR-34a target SIRT1 that sequentially activates eNOS itself by post-translational modification. These findings revealed novel functions of eNOS and of the eNOS/SIRT1 interplay, fine-tuned by E2-activated ER signaling, favouring the concept of eNOS as critical molecular determinant in aggressive PCa. Recent studies have placed long non coding RNAs (lncRNAs) at the leading edge of cancer research and suggested that they may serve as master drivers of carcinogenesis. However, their identity, function and deregulation in cancer are only beginning to be understood. In line with the above, we have contributed to the identification, by ChIP-Sequencing, of a consistent number of eNOS-bound complexes in the genome regions of many lncRNAs strictly associated with cancer, including PCa (Prensner JR and Chinnaiyan AM, 2011). Intriguingly, in the genomic regions overlapping with, or in the proximity of these transcripts the number of eNOS peaks significantly increases upon estrogen treatment, with a specific pattern for the primary tumor as compared to the metastatic cell line. These data are being validated by ChIP/re-ChIP and RIP assays with proteins of interest (eNOS, ER?, ER? and androgen receptor, AR) and lncRNAs emerging from the ChIP-Seq analysis. We currently aim at expanding such findings to a different microenvironment, breast cancer, by analysing, in this context, the occupancy by the eNOS complexes of genome regions of specific cancer-associated LncRNAs. Although arising in organs that are different for anatomy and physiological function, both breast and prostate cancers are typically hormone-dependent and have remarkable biological similarities. They are both driven by the sex steroid hormones, estrogen and androgen, that activate their respective nuclear receptors, ER?/ER? or AR. These nuclear receptors interact with a plethora of other transcription factors and co-factors, like eNOS, which aid ER and AR in the activation of a pro-tumorigenic gene expression program. To this end the expression of sex steroid hormone receptors (ER?, ER? or AR) and eNOS has been evaluated and compared in a large population of breast and prostate cancer cell lines, and 4 sub-groups among them have been identified on the basis of their different expression pattern. Levels of the most relevant cancer-associated lncRNAs (specifically HOTAIR, GAS5, H19, and CDKN2B AS/ANRIL and MALAT1) were quantified by qRT-PCR in each cell line, before and after estrogens or hypoxia or upon interfering with the NO signaling by genetic or pharmacological approaches or upon combined treatments. Preliminary data indicate that: i. lncRNAs HOTAIR and H19 basal expression in breast cancer cell lines appears highly correlated with presence of ER?, being undetectable in the ER?- cells; ii. lncRNAs HOTAIR, MALAT1 and GAS5 have higher expression levels in metastatic prostate cancer cell lines than in primary tumors-derived PCa cells, suggesting that their induction correlates with poor patient outcome and cancer metastasis; iii. lncRNAs H19, HOTAIR and CDKN2B-AS/ANRIL expression significantly decreases after eNOS inhibition exclusively in ER?+ breast cancer cell lines, strongly suggesting that eNOS and ER? are strictly required for the transcriptional control of selected cancer-associated lncRNAs in hormone-driven cancers.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
