In the clinical management of prostate cancer (PCa) an early discrimination between aggressive and indolent forms of the disease is of paramount importance. We have recently contributed to this goal by revealing a novel and pivotal function of endothelial nitric oxide synthase (eNOS) in the acquisition of an aggressive PCa phenotype (1). Specifically, we demonstrated that activation of eNOS is crucial for tumor progression in the prostate microenvironment, highly sensitive to abnormal estrogen level and hypoxia. Nitric oxide (NO), the product of eNOS, is a free radical involved in a variety of biological processes. It is well established that NO plays a key role in angiogenesis and is a powerful modulator of the function of endothelial cells and their progenitors. However, only recently it has been shown that activated eNOS can translocate into the nucleus where it regulates gene transcription (2). We (1, 3) have provided insights on the mechanism underlying this eNOS nuclear function in two experimental models: primary cultures of human endothelium and PCa-derived cell lines. We demonstrated that: i. the NO/eNOS nuclear signalling is a key pathway in the maintenance of vascular homeostasis and in the progression of PCa; and ii. formation of eNOS/ER? combinatorial complexes determines localized remodelling of chromatin leading to transcriptional regulation of previously identified prognostic genes (e.g. hTERT, MSH2, Cyclin B1 and PS2), all extremely sensitive to estrogen stimulation and/or variations in the intracellular levels of oxygen and NO (1). We are currently investigating whether eNOS as "co-factor" of ERs plays a role also in the transcriptional silencing of prognostic genes, focusing on the Glutathione S-Transferase P1 (GSTP1) (4). First, in TMAs from a retrospective cohort we were able to correlate loss of GSTP1 expression with decreased disease-specific survival. Further, in our ex-vivo model of tumor cell lines from PCa patients, we recapitulated loss of GSTP1 mRNA and protein expression and of biochemical activity, particularly in cells from patients with worse outcome. Silencing of GSTP1, however, did not appear to involve methylation of its promoter as described (5), unlike the case with LNCaP cells. Rather ChIP and re-ChIP assays have provided evidence in favor of an active repression mechanism, apparently mediated by dynamic recruitment of the combinatorial complex eNOS/ER??along a 5-kb region of the GSTP1 regulatory sequences. In support of this hypothesis, a synthetic antagonist of ER and/or overexpression of a dominant negative eNOS appeared capable to relieve repression of GSTP1 mRNA and, conversely, a NO donor repressed mRNA level in cells still expressing GSTP1. Overall our findings strongly support a model where eNOS functions as a co-factor of ERs in gene transcription, and formation of eNOS/ERs complexes induces local production of NO with major effects on the acquisition of a malignant phenotype by human prostate epithelial cells. 1. Nanni et al., 2009 2. Goetz et al., 1999; Feng et al., 1999; Klinz et al., 2005 ; Gobeil et al., 2006 3. Grasselli et al., 2008 4. Nanni et al., 2006 5. Lee et al., 1994 ; Esteller et al., 1998, Lin et al., 2001 ; Harden et al., 2003; Nakayama et al., 2004
FUNCTIONAL ROLE OF THE eNOS/ERbeta COMBINATORIAL COMPLEX IN THE PROGRESSION OF PROSTATE CANCER: TRANSCRIPTIONAL REGULATION OF PROGNOSTIC GENES.
Farsetti A
2009
Abstract
In the clinical management of prostate cancer (PCa) an early discrimination between aggressive and indolent forms of the disease is of paramount importance. We have recently contributed to this goal by revealing a novel and pivotal function of endothelial nitric oxide synthase (eNOS) in the acquisition of an aggressive PCa phenotype (1). Specifically, we demonstrated that activation of eNOS is crucial for tumor progression in the prostate microenvironment, highly sensitive to abnormal estrogen level and hypoxia. Nitric oxide (NO), the product of eNOS, is a free radical involved in a variety of biological processes. It is well established that NO plays a key role in angiogenesis and is a powerful modulator of the function of endothelial cells and their progenitors. However, only recently it has been shown that activated eNOS can translocate into the nucleus where it regulates gene transcription (2). We (1, 3) have provided insights on the mechanism underlying this eNOS nuclear function in two experimental models: primary cultures of human endothelium and PCa-derived cell lines. We demonstrated that: i. the NO/eNOS nuclear signalling is a key pathway in the maintenance of vascular homeostasis and in the progression of PCa; and ii. formation of eNOS/ER? combinatorial complexes determines localized remodelling of chromatin leading to transcriptional regulation of previously identified prognostic genes (e.g. hTERT, MSH2, Cyclin B1 and PS2), all extremely sensitive to estrogen stimulation and/or variations in the intracellular levels of oxygen and NO (1). We are currently investigating whether eNOS as "co-factor" of ERs plays a role also in the transcriptional silencing of prognostic genes, focusing on the Glutathione S-Transferase P1 (GSTP1) (4). First, in TMAs from a retrospective cohort we were able to correlate loss of GSTP1 expression with decreased disease-specific survival. Further, in our ex-vivo model of tumor cell lines from PCa patients, we recapitulated loss of GSTP1 mRNA and protein expression and of biochemical activity, particularly in cells from patients with worse outcome. Silencing of GSTP1, however, did not appear to involve methylation of its promoter as described (5), unlike the case with LNCaP cells. Rather ChIP and re-ChIP assays have provided evidence in favor of an active repression mechanism, apparently mediated by dynamic recruitment of the combinatorial complex eNOS/ER??along a 5-kb region of the GSTP1 regulatory sequences. In support of this hypothesis, a synthetic antagonist of ER and/or overexpression of a dominant negative eNOS appeared capable to relieve repression of GSTP1 mRNA and, conversely, a NO donor repressed mRNA level in cells still expressing GSTP1. Overall our findings strongly support a model where eNOS functions as a co-factor of ERs in gene transcription, and formation of eNOS/ERs complexes induces local production of NO with major effects on the acquisition of a malignant phenotype by human prostate epithelial cells. 1. Nanni et al., 2009 2. Goetz et al., 1999; Feng et al., 1999; Klinz et al., 2005 ; Gobeil et al., 2006 3. Grasselli et al., 2008 4. Nanni et al., 2006 5. Lee et al., 1994 ; Esteller et al., 1998, Lin et al., 2001 ; Harden et al., 2003; Nakayama et al., 2004I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
