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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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 9

Expression of estradiol and/or hypoxia target genes and modulation of the transcriptional profile of PCa cells.

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Expression of estradiol and/or hypoxia target genes and modulation of th...
(A) VEGF, hKDR, and GLUT1 mRNA levels were assessed by quantitative RT-PCR in C27IM (G1) and C38IM (G2) cells after 6 hours treatment with E2 (10–7 M), hypoxia (1% O2), or both. Data represent mean ± SEM of 3 experiments. (B and C) C38IM cells transfected with constitutively active HIF-1α or eNOS in basal conditions, treated with DETA-NO for 4 and 12 hours, or under E2, hypoxia, or both. Data, plotted as fold induction in B, represent mean ± SEM of 3 experiments. (B) †P = 0.0003, ††P = 0.003 versus empty vector; †††P = 0.0013, ‡‡P < 10–4, ‡‡P = 0.0006 versus basal condition. (C) ‡P = 0.001 versus empty vector; P = 0.002 versus empty vector plus E2; ¶¶P = 0.013 versus hypoxia empty vector; ¶¶¶P = 0.001 versus E2 plus hypoxia empty vector. (D) G1 cells (C27IM and C19IM) treated with the NOS inhibitors 7-nitroindazole (7N) or N(G)-nitro-l-arginine methyl ester (l-NAME). Data, plotted as percentage, represent mean ± SEM of 2 experiments. *P < 10–4 and **P < 10–4 versus none for C27IM, °P = 0.01 and °°P < 10–4 versus none for C19IM; ^P < 10–4 and ^^P < 10–4 versus none for C27IM; §P < 10–4 and §§P = 0.04 versus none for C19IM; #P < 10–4 and ##P < 10–4 versus none for C27IM; ΥP = 0.05 and ΥΥP = 0.01 versus none for C19IM. (E) Western blot of AKT, phosphorylated AKT, and eNOS in G1 cells (C27IM, C19IM, and C11IM) in basal condition and of HIF-1α and HIF-2α in C27IM treated with N(G)-nitro-l-arginine methyl ester or 7-nitroindazole or transfected with dominant-negative eNOS (S1177A). Hsp70 was the loading control.