Narita Keishi, Staub Julie, Chien Jeremy, Meyer Kristy, Bauer Maret, Friedl Andreas, Ramakrishnan Sundaram, Shridhar Viji
Department of Experimental Pathology, Mayo Clinic Cancer Center, Rochester, Minnesota 55905, USA.
Cancer Res. 2006 Jun 15;66(12):6025-32. doi: 10.1158/0008-5472.CAN-05-3582.
We previously identified HSulf-1 as a down-regulated gene in several tumor types including ovarian, breast, and hepatocellular carcinomas. Loss of HSulf-1, which selectively removes 6-O-sulfate from heparan sulfate, up-regulates heparin-binding growth factor signaling and confers resistance to chemotherapy-induced apoptosis. Here we report that HSulf-1 expression in MDA-MB-468 breast carcinoma clonal lines leads to reduced proliferation in vitro and reduced tumor burden in athymic nude mice in vivo. Additionally, xenografts derived from HSulf-1-expressing stable clones of carcinoma cells showed reduced vessel density, marked necrosis, and apoptosis, indicative of inhibition of angiogenesis. Consistent with this observation, HSulf-1-expressing clonal lines showed reduced staining with the endothelial marker CD31 in Matrigel plug assay, indicating that HSulf-1 expression inhibits angiogenesis. More importantly, HSulf-1 expression in the xenografts was associated with a reduced ability of vascular endothelial cell heparan sulfate to participate in a complex with fibroblast growth factor 2 (FGF-2) and its receptor tyrosine kinase FGF receptor 1c. In vitro, short hairpin RNA-mediated down-regulation of HSulf-1 in human umbilical vein endothelial cells (HUVEC) resulted in an increased proliferation mediated by heparan sulfate-dependent FGF-2, hepatocyte growth factor, and vascular endothelial growth factor 165 (VEGF165) but not by heparan sulfate-independent VEGF121. HSulf-1 down-regulation also enhanced downstream signaling through the extracellular signal-regulated kinase pathway compared with untreated cells. Consistent with the role of heparan sulfate glycosaminoglycan sulfation in VEGF-mediated signaling, treatment of HUVEC cells with chlorate, which inhibits heparan sulfate glycosaminoglycan sulfation and therefore mimics HSulf-1 overexpression, led to an attenuated VEGF-mediated signaling. Collectively, these observations provide the first evidence of a novel mechanism by which HSulf-1 modulates the function of heparan sulfate binding VEGF165 in proliferation and angiogenesis.
我们之前在包括卵巢癌、乳腺癌和肝细胞癌在内的多种肿瘤类型中，将HSulf-1鉴定为下调基因。HSulf-1可选择性地从硫酸乙酰肝素中去除6-O-硫酸酯，其缺失会上调肝素结合生长因子信号传导，并赋予对化疗诱导的细胞凋亡的抗性。在此，我们报告MDA-MB-468乳腺癌克隆系中HSulf-1的表达导致体外增殖减少以及体内无胸腺裸鼠的肿瘤负荷降低。此外，源自表达HSulf-1的癌细胞稳定克隆的异种移植物显示血管密度降低、明显坏死和凋亡，表明血管生成受到抑制。与该观察结果一致，在基质胶栓试验中，表达HSulf-1的克隆系与内皮标志物CD31的染色减少，表明HSulf-1的表达抑制血管生成。更重要的是，异种移植物中HSulf-1的表达与血管内皮细胞硫酸乙酰肝素参与与成纤维细胞生长因子2（FGF-2）及其受体酪氨酸激酶FGF受体1c形成复合物的能力降低有关。在体外，短发夹RNA介导的人脐静脉内皮细胞（HUVEC）中HSulf-1的下调导致由硫酸乙酰肝素依赖性FGF-2、肝细胞生长因子和血管内皮生长因子165（VEGF）介导的增殖增加，但不由硫酸乙酰肝素非依赖性VEGF121介导。与未处理的细胞相比，HSulf-1的下调还增强了通过细胞外信号调节激酶途径的下游信号传导。与硫酸乙酰肝素糖胺聚糖硫酸化在VEGF介导的信号传导中的作用一致，用氯酸盐处理HUVEC细胞，氯酸盐抑制硫酸乙酰肝素糖胺聚糖硫酸化，因此模拟HSulf-1过表达，导致VEGF介导的信号传导减弱。总的来说，这些观察结果提供了首个证据，证明HSulf-1通过一种新机制调节硫酸乙酰肝素结合VEGF165在增殖和血管生成中的功能。
