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. 2009 Jun 15;69(9):949-55.
doi: 10.1002/pros.20944.

Soluble factors derived from stroma activated androgen receptor phosphorylation in human prostate LNCaP cells: roles of ERK/MAP kinase

Katsumi Shigemura  1 , Shuji Isotani, Ruoxiang Wang, Masato Fujisawa, Akinobu Gotoh, Fray F Marshall, Haiyen E Zhau, Leland W K Chung
Affiliations

Soluble factors derived from stroma activated androgen receptor phosphorylation in human prostate LNCaP cells: roles of ERK/MAP kinase

Katsumi Shigemura et al. Prostate. .

Abstract

Background: Accumulated evidence suggests stromal-epithelial interactions are critical to the progression of prostate cancer. In this study, we characterized AR phosphorylation in LNCaP cells co-cultured with the conditioned medium (CM) from human prostate stromal fibroblasts.

Methods: CM harvested from prostate stromal fibroblasts was added to LNCaP cells, and both anchorage-dependent and -independent growth was determined. Status of AR phosphorylation at Ser-81 and Ser-213 was assessed by immunoblot analysis. ERK kinase activity was measured using MBP-2 protein as the substrate.

Results: The growth of LNCaP cells on plastic dishes increased by 1.7-fold upon exposure to stromal CM or androgen, and their combination resulted in additive growth (2.4-fold). Anchorage-independent growth of LNCaP cells in soft agar, however, was induced synergistically at 80-fold by both stromal CM and androgen. Stromal CM or androgen alone induced LNCaP cell growth by 10- and 26-fold, respectively. We observed ERK kinase inhibitor, U0126, but not phosphatidylinositol 3-kinase (PI-3K), LY294002, or protein kinase A (PKA) inhibitor, H-89, inhibited stromal CM or androgen-induced PSA promoter luciferase activities, and anchorage-independent growth of LNCaP cells. Our results demonstrated for the first time how stromal CM acts in synergy with androgen by activation of ERK kinase and AR phosphorylation at Ser-81 but not Ser-213, for AR-regulated PSA promoter and anchorage-independent growth of human prostate cancer cells.

Conclusions: A stromal factor-activated ERK pathway mediated by AR phosphorylation at Ser-81 could be responsible for stimulating the growth of human prostate cancer cells.

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Figures

Figure 1
Figure 1. Effects of prostate stromal CM and a synthetic androgen, R-1881, on LNCaP cell proliferation
C1 and C2, two independent isolates of stromal cells, were used as described in Materials and Methods. Effects of stromal CM on growth and PSA promoter reporter activity by LNCaP cells were evaluated after 72 hrs treatment. Effects of stromal CM and R1881 (10 nM) on the proliferation of LNCaP cells were assayed by MTS assay (Materials and Methods). R1881, stromal CM (C1) and stromal CM (C2) induced significantly more cell proliferation than control (p=0.005, 0.0012, and 0.0007, respectively). In addition, combining R1881 with stromal CM (C1) and stromal CM (C2) induced more cell proliferation than R1881 alone (p=0.0493, and 0.0269, respectively). Data are presented as fold of induction relative to control, mean ± SD of a triplicate assay. Asterisks denote statistically significant changes compared with controls.
Figure 2
Figure 2. Effects of prostate stromal CM and a synthetic androgen, R-1881, on AR transcriptional activity of PSA promoter luciferase in LNCaP cells
Panel A, effects of stromal CM and R1881 (10 nM) on AR transcriptional activity measured by 6.1 kb PSA promoter luciferase assay with relative luciferase activity/million cell number (see Materials and Methods). LNCaP cells transiently transfected with a PSA reporter luciferase genewere subjected to androgen starvation before treatment with stromal CM and/or R1881. The specificity of PSA promoter reporter activation by androgen was determined by the simultaneous addition of R1881 (1 nM), an androgen agonist, and an androgen antagonist, bicalutamide (100 nM). Luciferase activity was determined 72 hrs after the initiation of treatment with R1881 (1 nM), MEK inhibitor U0126 (20 μM), PI-3K inhibitor LY294002 (20 μM), and/or PKA inhibitor H89 (10 μM). Data represent fold of induction of relative luciferase activity, mean ± SD of three separate assays. Asterisks denote statistically significant changes compared with controls. Panel B, effects of stromal CM on anchorage-independent LNCaP soft agar colony formation. LNCaP cells suspended in agar were treated with stromal CM for 21 days. Photographic results were analyzed with computer imaging software. Data are presented with bars representing different sizes of the colony stacked to give the total number of colonies. Effects of R1881 (10 nM), antiandrogen, bicalutamide (100 nM), and MEK inhibitor U0126 (10 nM) on LNCaP soft agar formation were assessed. In each treatment, the standard deviation from 3 independent experiments was less than 10% of the mean. The insets show representative colony formation from control and treated groups (×40).
Figure 2
Figure 2. Effects of prostate stromal CM and a synthetic androgen, R-1881, on AR transcriptional activity of PSA promoter luciferase in LNCaP cells
Panel A, effects of stromal CM and R1881 (10 nM) on AR transcriptional activity measured by 6.1 kb PSA promoter luciferase assay with relative luciferase activity/million cell number (see Materials and Methods). LNCaP cells transiently transfected with a PSA reporter luciferase genewere subjected to androgen starvation before treatment with stromal CM and/or R1881. The specificity of PSA promoter reporter activation by androgen was determined by the simultaneous addition of R1881 (1 nM), an androgen agonist, and an androgen antagonist, bicalutamide (100 nM). Luciferase activity was determined 72 hrs after the initiation of treatment with R1881 (1 nM), MEK inhibitor U0126 (20 μM), PI-3K inhibitor LY294002 (20 μM), and/or PKA inhibitor H89 (10 μM). Data represent fold of induction of relative luciferase activity, mean ± SD of three separate assays. Asterisks denote statistically significant changes compared with controls. Panel B, effects of stromal CM on anchorage-independent LNCaP soft agar colony formation. LNCaP cells suspended in agar were treated with stromal CM for 21 days. Photographic results were analyzed with computer imaging software. Data are presented with bars representing different sizes of the colony stacked to give the total number of colonies. Effects of R1881 (10 nM), antiandrogen, bicalutamide (100 nM), and MEK inhibitor U0126 (10 nM) on LNCaP soft agar formation were assessed. In each treatment, the standard deviation from 3 independent experiments was less than 10% of the mean. The insets show representative colony formation from control and treated groups (×40).
Figure 3
Figure 3. Stromal CM induced concomitant ERK activation and AR phosphorylation
Panel A, androgen-deprived LNCaP cells were treated with stromal CM (C1). The cells were harvested at different time points for ERK kinase and western blotting analyses. Top panels, phosphorylated ERK (pERK), phosphorylated myelin basic protein (pMBP), and total ERK proteins were detected by specific antibodies. Bottom panels, AR status in these samples was determined with specific antibodies to phosphorylated AR at the site Ser-81(pARS81) and total AR (AR). We used the relative ratio of phosphorylated ARS81/AR, pERK1/ERK1, and pERK2/ERK2 to represent the relative extent of phosphorylation of pARS81, pERK1, and pERK2, respectively. The same amount of protein was loaded in each lane. The value of the ratios of the 1st lane was set as 1.0. Panel B, stromal CM-activated ERK-catalyzed phosphorylation of AR at Ser-81 was assayed. LNCaP cells were treated as indicated and immunoprecipitated with anti-ERK antibodies purified and bound to G-Sepharose (G-S) beads. For endogenous AR protein, androgen-deprived LNCaP cells were used in immunoprecipitation with the anti-AR antibody (PG21) and bound to protein G-S. The ERK-G-S complex was mixed with the substrate AR-G-S complex in the kinase reaction, then fractionated on SDS-PAGE. Specific antibodies to phosphorylated Ser-81 (pARS81) and Ser-213 (pARS213) were used. Total AR and ERK proteins were detected to determine the efficiency of the immunoprecipitation. Synthetic androgen R1881 (10 nM), bicalutamide (100 nM) and MEK inhibitor U0126 (20 μM) were used. The relative ratios of phosphorylated ARS81/AR and phosphorylated ARS213/AR represented the relative intensities of pARS81 and pARS213. The value of these ratios of the 1st lane was set as 1.0. The ERK inhibitor U0126 successfully inhibited the pARS81 expression enhanced by combined treatment with CM (C1) and R1881 even though the anti-androgen bicalutamide did not). The same amount of protein was loaded in each lane. All experiments were conducted independently three times.
Figure 3
Figure 3. Stromal CM induced concomitant ERK activation and AR phosphorylation
Panel A, androgen-deprived LNCaP cells were treated with stromal CM (C1). The cells were harvested at different time points for ERK kinase and western blotting analyses. Top panels, phosphorylated ERK (pERK), phosphorylated myelin basic protein (pMBP), and total ERK proteins were detected by specific antibodies. Bottom panels, AR status in these samples was determined with specific antibodies to phosphorylated AR at the site Ser-81(pARS81) and total AR (AR). We used the relative ratio of phosphorylated ARS81/AR, pERK1/ERK1, and pERK2/ERK2 to represent the relative extent of phosphorylation of pARS81, pERK1, and pERK2, respectively. The same amount of protein was loaded in each lane. The value of the ratios of the 1st lane was set as 1.0. Panel B, stromal CM-activated ERK-catalyzed phosphorylation of AR at Ser-81 was assayed. LNCaP cells were treated as indicated and immunoprecipitated with anti-ERK antibodies purified and bound to G-Sepharose (G-S) beads. For endogenous AR protein, androgen-deprived LNCaP cells were used in immunoprecipitation with the anti-AR antibody (PG21) and bound to protein G-S. The ERK-G-S complex was mixed with the substrate AR-G-S complex in the kinase reaction, then fractionated on SDS-PAGE. Specific antibodies to phosphorylated Ser-81 (pARS81) and Ser-213 (pARS213) were used. Total AR and ERK proteins were detected to determine the efficiency of the immunoprecipitation. Synthetic androgen R1881 (10 nM), bicalutamide (100 nM) and MEK inhibitor U0126 (20 μM) were used. The relative ratios of phosphorylated ARS81/AR and phosphorylated ARS213/AR represented the relative intensities of pARS81 and pARS213. The value of these ratios of the 1st lane was set as 1.0. The ERK inhibitor U0126 successfully inhibited the pARS81 expression enhanced by combined treatment with CM (C1) and R1881 even though the anti-androgen bicalutamide did not). The same amount of protein was loaded in each lane. All experiments were conducted independently three times.
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References

    1. Chung LW. Implications of stromal-epithelial interaction in human prostate cancer growth, progression and differentiation. Semin Cancer Biol. 1993;4(3):183–192. - PubMed
    1. Cunha GR, Shannon JM, Neubauer BL, Sawyer LM, Fujii H, Taguchi O, Chung LW. Mesenchymal-epithelial interactions in sex differentiation. Hum Genet. 1981;58(1):68–77. - PubMed
    1. Djakiew D, Pflug B, Onoda M. Stromal-epithelial paracrine interactions in the neoplastic rat and human prostate. Adv Exp Med Biol. 1993;330:185–202. - PubMed
    1. Sung SY, Chung LW. Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting. Differentiation. 2002;70(9-10):506–521. - PubMed
    1. Fu M, Rao M, Wu K, Wang C, Zhang X, Hessien M, Yeung YG, Gioeli D, Weber MJ, Pestell RG. The androgen receptor acetylation site regulates cAMP and AKT but not ERK-induced activity. J Biol Chem. 2004;279(28):29436–29449. - PubMed

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