Endothelial NOS, estrogen receptor β, and HIFs cooperate in the activation of a prognostic transcriptional pattern in aggressive human prostate cancer
Simona Nanni, … , Massimo Loda, Antonella Farsetti
Simona Nanni, … , Massimo Loda, Antonella Farsetti
Published April 13, 2009
Citation Information: J Clin Invest. 2009;119(5):1093-1108. https://doi.org/10.1172/JCI35079.
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Research Article Oncology

Endothelial NOS, estrogen receptor β, and HIFs cooperate in the activation of a prognostic transcriptional pattern in aggressive human prostate cancer

Abstract

The identification of biomarkers that distinguish between aggressive and indolent forms of prostate cancer (PCa) is crucial for diagnosis and treatment. In this study, we used cultured cells derived from prostate tissue from patients with PCa to define a molecular mechanism underlying the most aggressive form of PCa that involves the functional activation of eNOS and HIFs in association with estrogen receptor β (ERβ). Cells from patients with poor prognosis exhibited a constitutively hypoxic phenotype and increased NO production. Upon estrogen treatment, formation of ERβ/eNOS, ERβ/HIF-1α, or ERβ/HIF-2α combinatorial complexes led to chromatin remodeling and transcriptional induction of prognostic genes. Tissue microarray analysis, using an independent cohort of patients, established a hierarchical predictive power for these proteins, with expression of eNOS plus ERβ and nuclear eNOS plus HIF-2α being the most relevant indicators of adverse clinical outcome. Genetic or pharmacologic modulation of eNOS expression and activity resulted in reciprocal conversion of the transcriptional signature in cells from patients with bad or good outcome, respectively, highlighting the relevance of eNOS in PCa progression. Our work has considerable clinical relevance, since it may enable the earlier diagnosis of aggressive PCa through routine biopsy assessment of eNOS, ERβ, and HIF-2α expression. Furthermore, proposing eNOS as a therapeutic target fosters innovative therapies for PCa with NO inhibitors, which are employed in preclinical trials in non-oncological diseases.

Authors

Simona Nanni, Valentina Benvenuti, Annalisa Grasselli, Carmen Priolo, Aurora Aiello, Stefania Mattiussi, Claudia Colussi, Vittoria Lirangi, Barbara Illi, Manuela D’Eletto, Anna Maria Cianciulli, Michele Gallucci, Piero De Carli, Steno Sentinelli, Marcella Mottolese, Paolo Carlini, Lidia Strigari, Stephen Finn, Elke Mueller, Giorgio Arcangeli, Carlo Gaetano, Maurizio C. Capogrossi, Raffaele Perrone Donnorso, Silvia Bacchetti, Ada Sacchi, Alfredo Pontecorvi, Massimo Loda, Antonella Farsetti

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Figure 1

Generation, characterization, and maintenance of the prognostic signature in immortalized human prostate epithelial cell lines.

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Generation, characterization, and maintenance of the prognostic signatur...
(A) Growth (population doublings) of cell populations derived from tumoral prostate before (C27 and C38) and after retrovirus-mediated transduction of the hTERT and SV40 large T antigen genes (C27IM and C38IM, respectively). Cells were infected at early passage and cultured as described in Supplemental Methods. (B) Steroid receptor expression (AR) by immunocytochemistry. Original magnification is ×20 or ×40 as indicated. (C) ERβ and ERα detected by Western Blot in total or nuclear extracts, respectively, of PCa-derived cells (C19IM, C27IM, and C38IM). HeLa cells were negative control. HeLa cells transiently transfected with ERβ (HeLa + ERβ485aa) and MCF-7 cells were positive control. Tubulin and nuclear protein Sp1 were loading control. (D) Analysis by quantitative RT-PCR (qRT-PCR) of genes from the prognostic expression signature in immortalized PCa cells (PCa-IM) (n = 25) from the poor (G1) or good (G2) prognosis groups. Data are presented as box plot and whisker (see Supplemental Methods). Boxes show medians and upper and lower quartiles and whiskers indicate minimum and maximum values. (E) Western blot of HIF-1α, HIF-2α, and HIF-1β in PCa-IM cells from the G1 (C27IM) or G2 (C38IM) groups in normoxia or hypoxia (1% O2 for 3 hours). PCa cells transfected with an HIF-1α–expression vector (C14IM + HIF-1α) and von Hippel–Lindau–deficient carcinoma 786-O cells were positive controls. Sp1 was the loading control. White lines indicate extracts that were run in noncontiguous lanes.