Mol Endocrinol. 2008 Feb;22(2):273-86. Epub 2007 Nov 1.

Interplay of nuclear factor-kappaB and B-myb in the negative regulation of androgen receptor expression by tumor necrosis factor alpha.

Ko S, Shi L, Kim S, Song CS, Chatterjee B.

Source

Department of Molecular Medicine/Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245, USA.

Abstract

Increased androgen receptor (AR) levels are associated with prostate cancer progression to androgen independence and therapy resistance. Evidence has suggested that chronic inflammation is closely linked to various cancers including prostate cancer. Herein we show that the proinflammatory cytokine TNFalpha negatively regulates AR mRNA and protein expression and reduces androgen sensitivity in androgen-dependent LNCaP human prostate cancer cells. Decreased AR expression results from transcription repression involving essential in cis interaction of nuclear factor-kappaB (NF-kappaB) with the B-myb transcription factor at a composite genomic element in the 5'-untranslated region of AR. The negative regulation was abrogated when NF-kappaB activity was inhibited by a superrepressor of the inhibitory kappaB protein. In contrast, androgen-independent C4-2 (LNCaP-derived) cells fail to show AR down-regulation by TNFalpha, despite expression of B-myb and TNFalpha-induced NF-kappaB activity similar to that in LNCaP cells. The negatively regulated AR gene chromatin region showed TNFalpha-dependent enrichment of B-myb and the NF-kappaB proteins p65 and p50. In parallel, the histone deacetylase 1, corepressor silencing mediator of retinoid and thyroid hormone receptor and the corepressor-associated scaffold protein mSin3A were recruited to the inhibitory site. In C4-2 cells, neither NF-kappaB and B-myb, nor any of the corepressor components, were detected at the negative site in response to TNFalpha. Apoptosis was induced in TNFalpha-treated LNCaP cells, likely in part due to the down-regulation of AR. The androgen-independent, AR-expressing C4-2 and C4-2B (derived from C4-2) cells were resistant to TNFalpha-induced apoptosis. The results linking androgen dependence to the NF-kappaB and AR pathways may be insightful in identifying novel treatment targets for prostate cancer.

PMID:: 17975021; [PubMed - indexed for MEDLINE]
PMCID:: PMC2234585

Free PMC Article

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Figure 4

AR Promoter Repression by NF-κB-p65/p50 in Androgen-Dependent LNCaP and RWPE-1 Prostate Cells and Lack of the Repression in Androgen-Independent C4-2 Cells
A, Luciferase reporter activity is shown for each construct in p65 and p50 cotransfected LNCaP cells relative to pcDNA3.1-transfected cells. Reporter expression was directed by the human AR promoters (−110/+246, −311/+560, −726/+560) or from the pGL3b vector itself. Data are averages with sd from three independent transfections. B, Repression of the −110 to +246 AR promoter by TNFα in LNCaP cells, and lack of the negative regulation in the presence of IκB-SR. The AR promoter-reporter was transfected into LNCaP (solid bar) or LNCaP-SR-IκB cells (open bars), and 24 h later the cells were treated with vehicle or TNFα (20 ng/ml) for an additional 24 h before the cells were analyzed for luciferase. Luciferase values are presented relative to the 100% value set for LNCaP-IκB-SR cells in the absence of TNFα. C, Repression of the AR promoter (−110 to +246) in RWPE-1 (AR-positive, nontumorigenic) cells treated with TNFα. Transfection conditions were same as in panel B. D, Absence of the TNFα effect on the AR promoter (−110 to +246) in C4-2 cells. It should be noted that the luciferase values were comparable in LNCaP and C4-2 cells in the absence of TNFα treatment. In contrast to LNCaP cells, C4-2 cells showed no reduction of luciferase activity after TNFα treatment. E, Comparable NF-κB activation by TNFα in LNCaP and C4-2 cells. Transactivation of a canonical NF-κB element was assayed in transfected LNCaP and C4-2 cells with or without TNFα treatment. F, NF-κB DNA-binding activity in TNFα-treated LNCaP and C4-2 cells. The NF-κB element from the IL-6 gene was used as the EMSA probe. TNFα treatments in panels B–F were for 24 h at 20 ng/ml.

Interplay of Nuclear Factor-κB and B-myb in the Negative Regulation of Androgen Receptor Expression by Tumor Necrosis Factor α

Mol Endocrinol. Mol Endocrinol;22(2):273-286.

Figure 1

TNFα Suppressed AR Expression and Activity in LNCaP Human Prostate Cancer Cells
A, Reduced AR protein level. Cells were treated with TNFα at indicated doses for 12 h, and AR and β-actin proteins were assayed by Western blot. B, Reduced AR mRNAs in cells treated with TNFα for 6 h (at 20 ng/ml) or 12 h (at 20 ng/ml and 50 ng/ml). AR mRNAs were assayed by real-time RT-qPCR, normalized to β-actin mRNAs. Minus TNFα control cells were treated with vehicle (PBS). C, Reduced androgen sensitivity indicated by decreased androgen-induced PSA mRNA expression. Cells were incubated with 10 nm R1881 (or 0.001% ethanol as vehicle) for 24 h. Thereafter, TNFα (20 ng/ml) was added to the culture medium. Control cells (minus TNFα) received PBS. Incubation continued for 12 additional hours, after which cells were harvested for total RNA preparation. In one indicated case, TNFα incubation continued for additional 12 h (total TNFα treatment time: 24 h), thus extending R1881 incubation period to 48 h in this case. PSA mRNAs relative to the invariant β-actin mRNAs were assayed by real-time RT-qPCR. Error bars are averages of three experiments ± sd. PCR primers for AR, PSA, and β-actin mRNAs are described in Materials and Methods. Error bars are not shown in cases where assays were repeated twice, each in duplicate. Data in these cases are presented as average values. D, IκB-SR blocked TNFα-induced AR down-regulation. LNCaP cells were infected with IκB-SR-expressing adenovirus (at 20 moi) or the control GFP-expressing adenovirus (at 20 moi), and 48 h later cells were treated with TNFα (20 ng/ml) or PBS (vehicle) for an additional 24 h. Cell lysates were immunoblotted with anti-AR or anti-β-actin. E, Reduced AR expression in UV-C-irradiated LNCaP cells. UV-C (<290 nm) exposure was at 40 J/m² (1 min). AR and ER-β protein levels were analyzed by Western blot. ER-β, Estrogen receptor-β; veh, vehicle.

Interplay of Nuclear Factor-κB and B-myb in the Negative Regulation of Androgen Receptor Expression by Tumor Necrosis Factor α

Mol Endocrinol. Mol Endocrinol;22(2):273-286.