Medical Research Council Human Reproductive Sciences Unit, The Queen's Medical Research Institute, Little France Crescent, Old Dalkeith Road, Edinburgh EH16 4TJ, UK.
BMC Cancer. 2011 Nov 3;11:476. doi: 10.1186/1471-2407-11-476.
Gonadotrophin releasing hormone (GnRH) analogs lower estrogen levels in pre-menopausal breast cancer patients. GnRH receptor (GnRH-R) activation also directly inhibits the growth of certain cells. The applicability of GnRH anti-proliferation to breast cancer was therefore analyzed.
GnRH-R expression in 298 primary breast cancer samples was measured by quantitative immunofluorescence. Levels of functional GnRH-R in breast-derived cell lines were assessed using 125I-ligand binding and stimulation of 3H-inositol phosphate production. Elevated levels of GnRH-R were stably expressed in cells by transfection. Effects of receptor activation on in vitro cell growth were investigated in comparison with IGF-I and EGF receptor inhibition, and correlated with intracellular signaling using western blotting.
GnRH-R immunoscoring was highest in hormone receptor (triple) negative and grade 3 breast tumors. However prior to transfection, functional endogenous GnRH-R were undetectable in four commonly studied breast cancer cell lines (MCF-7, ZR-75-1, T47D and MDA-MB-231). After transfection with GnRH-R, high levels of cell surface GnRH-R were detected in SVCT and MDA-MB-231 clones while low-moderate levels of GnRH-R occurred in MCF-7 clones and ZR-75-1 clones. MCF-7 sub-clones with high levels of GnRH-R were isolated following hygromycin phosphotransferase transfection. High level cell surface GnRH-R enabled induction of high levels of 3H-inositol phosphate and modest growth-inhibition in SVCT cells. In contrast, growth of MCF-7, ZR-75-1 or MDA-MB-231 clones was unaffected by GnRH-R activation. Cell growth was inhibited by IGF-I or EGF receptor inhibitors. IGF-I receptor inhibitor lowered levels of p-ERK1/2 in MCF-7 clones. Washout of IGF-I receptor inhibitor resulted in transient hyper-elevation of p-ERK1/2, but co-addition of GnRH-R agonist did not alter the dynamics of ERK1/2 re-phosphorylation.
Breast cancers exhibit a range of GnRH-R immunostaining, with higher levels of expression found in triple-negative and grade 3 cancers. However, functional cell surface receptors are rare in cultured cells. Intense GnRH-R signaling in transfected breast cancer cells did not markedly inhibit growth, in contrast to transfected HEK 293 cells indicating the importance of intracellular context. GnRH-R signaling could not counteract IGF-I receptor-tyrosine kinase addiction in MCF-7 cells. These results suggest that combinatorial strategies with growth factor inhibitors will be needed to enhance GnRH anti-proliferative effects in breast cancer.
促性腺激素释放激素（GnRH）类似物可降低绝经前乳腺癌患者的雌激素水平。GnRH 受体（GnRH-R）的激活也可直接抑制某些细胞的生长。因此，分析了 GnRH 抗增殖作用在乳腺癌中的适用性。
通过定量免疫荧光测量 298 例原发性乳腺癌样本中的 GnRH-R 表达。使用 125I-配体结合和 3H-肌醇磷酸盐产生的刺激来评估乳腺衍生细胞系中功能性 GnRH-R 的水平。通过转染稳定表达高 GnRH-R 水平。通过与 IGF-I 和 EGF 受体抑制的比较，研究受体激活对体外细胞生长的影响，并使用 Western 印迹法与细胞内信号相关联。
在激素受体（三重）阴性和 3 级乳腺癌中，GnRH-R 免疫评分最高。然而，在转染之前，在四种常用的乳腺癌细胞系（MCF-7、ZR-75-1、T47D 和 MDA-MB-231）中未检测到功能性内源性 GnRH-R。用 GnRH-R 转染后，SVCT 和 MDA-MB-231 克隆中检测到高水平的细胞表面 GnRH-R，而 MCF-7 克隆和 ZR-75-1 克隆中检测到中低水平的 GnRH-R。在 hygromycin 磷酸转移酶转染后分离出具有高水平 GnRH-R 的 MCF-7 亚克隆。高水平的细胞表面 GnRH-R 可诱导 SVCT 细胞中高水平的 3H-肌醇磷酸盐和适度的生长抑制。相比之下，GnRH-R 激活对 MCF-7、ZR-75-1 或 MDA-MB-231 克隆的生长没有影响。IGF-I 或 EGF 受体抑制剂抑制细胞生长。IGF-I 受体抑制剂降低 MCF-7 克隆中 p-ERK1/2 的水平。IGF-I 受体抑制剂洗脱后，p-ERK1/2 短暂升高，但共添加 GnRH-R 激动剂不会改变 ERK1/2 再磷酸化的动力学。
乳腺癌表现出一系列 GnRH-R 免疫染色，三重阴性和 3 级癌症中表达水平更高。然而，在培养细胞中功能性细胞表面受体很少。转染的乳腺癌细胞中强烈的 GnRH-R 信号并未显著抑制生长，与转染的 HEK 293 细胞相反，这表明细胞内环境的重要性。在 MCF-7 细胞中，GnRH-R 信号不能抵消 IGF-I 受体酪氨酸激酶成瘾。这些结果表明，需要与生长因子抑制剂联合使用组合策略来增强 GnRH 在乳腺癌中的抗增殖作用。
