Shigemura Katsumi, Isotani Shuji, Wang Ruoxiang, Fujisawa Masato, Gotoh Akinobu, Marshall Fray F, Zhau Haiyen E, Chung Leland W K
Molecular Urology and Therapeutics Program, Department of Urology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Prostate. 2009 Jun 15;69(9):949-55. doi: 10.1002/pros.20944.
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.
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.
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.
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.
越来越多的证据表明，基质 - 上皮相互作用对前列腺癌的进展至关重要。在本研究中，我们对与来自人前列腺基质成纤维细胞的条件培养基（CM）共培养的LNCaP细胞中的雄激素受体（AR）磷酸化进行了表征。
将从前列腺基质成纤维细胞收获的CM添加到LNCaP细胞中，并测定其贴壁依赖性和非贴壁依赖性生长。通过免疫印迹分析评估AR在Ser-81和Ser-213处的磷酸化状态。使用MBP-2蛋白作为底物测量细胞外信号调节激酶（ERK）活性。
暴露于基质CM或雄激素后，LNCaP细胞在塑料培养皿上的生长增加了1.7倍，两者联合导致生长增加（2.4倍）。然而，基质CM和雄激素协同诱导LNCaP细胞在软琼脂中的非贴壁依赖性生长增加80倍。单独的基质CM或雄激素分别诱导LNCaP细胞生长10倍和26倍。我们观察到ERK激酶抑制剂U0126可抑制基质CM或雄激素诱导的前列腺特异性抗原（PSA）启动子荧光素酶活性以及LNCaP细胞的非贴壁依赖性生长，而磷脂酰肌醇3激酶（PI-3K）抑制剂LY294002或蛋白激酶A（PKA）抑制剂H-89则无此作用。我们的结果首次证明了基质CM如何通过激活ERK激酶以及AR在Ser-81而非Ser-213处的磷酸化与雄激素协同作用，以调节AR调控的PSA启动子以及人前列腺癌细胞的非贴壁依赖性生长。
由AR在Ser-81处磷酸化介导的基质因子激活的ERK途径可能是刺激人前列腺癌细胞生长的原因。
