Hyaluronidase 1 is a vital mediator of brain metastasis in melanoma.
Hyaluronidase 1 is a vital mediator of brain metastasis in melanoma.
复制标题
DOI：
--
复制
发表时间：
2008-05
期刊：
影响因子：
11.2
通讯作者：
D. O'Sullivan;Melissa A. O’Neal;B. Felding-Habermann
中科院分区：
文献类型：
--
作者：
D. O'Sullivan;Melissa A. O’Neal;B. Felding-Habermann
3672黑色素瘤具有向CNS转移的高倾向，脑转移患者预后差。由于黑色素瘤从最初的脑病变颅内转移到整个脑内产生多个肿瘤的倾向，使治疗这些患者的难度加剧。脑内的肿瘤生长和其他病变的种植都需要脑细胞外基质的降解和重塑。大脑独特的细胞外基质的组成尚不完全清楚，但其结构组织是基于长链的非常高分子量的透明质酸，其装饰有各种聚集的蛋白质和蛋白聚糖，形成交联纤维的网络。使用人黑色素瘤细胞系MDA MB 435，我们发现源自SCID小鼠原位植入细胞的肺、骨和脑转移瘤的变异亚系，透明质酸降解酶透明质酸酶1（Hyal 1）显着上调。透明质酸酶谱显示，亲本435细胞系表达几乎检测不到量的Hyal 1，并且在原位植入部位由实验性原发性肿瘤培养的细胞分泌的酶水平也非常低。Hyal 1的显著的转移相关上调表明，这种酶可能是黑色素瘤转移过程的中心介质。我们的实验室特别感兴趣的是开发一个分子概况的事件背后的生存和传播的肿瘤细胞在大脑中。在这项研究中，在组织培养中建立了经酶标记的MDA MB 435细胞（435/脑）的脑转移变体，并用于检查Hyal 1在SCID小鼠脑肿瘤生长过程中的作用。我们使用慢病毒载体中的shRNA在435/Brain细胞中产生了一组稳定的Hyal 1敲除。通过PCR、透明质酸酶谱和通过琼脂糖凝胶电泳分析HA消化产物来证实Hyal 1的敲低。在所有实验中使用与已知人基因序列没有同源性的对照shRNA序列作为基线比较。荧光素酶标记的435/Brain细胞的体内成像用于在立体定向引导植入SCID小鼠的脑纹状体中后可视化对照和Hyal 1敲除的生长和转移。对照和敲低细胞在植入部位形成肿瘤。然而，Hyal 1敲低强烈降低了细胞从初始肿瘤位置迁移的能力，与对照细胞相反，对照细胞在注射动物的大脑和脊柱中的多个位置快速接种转移瘤。这些数据表明，肿瘤衍生的Hyal 1，一种在几种肿瘤类型的转移扩散中很重要的酶，也可以降解脑细胞外基质中发现的透明质酸形式，并介导癌细胞的颅内迁移。
3672 Melanoma has a high propensity to metastasize to the CNS and patients with brain metastases have a poor prognosis. The difficulty of treating these patients is exacerbated by the tendency of melanoma to produce multiple tumors throughout the brain by intracranial metastasis from the initial brain lesion. Both tumor growth within the brain and the seeding of additional lesions requires degradation and remodeling of the brain extracellular matrix. The composition of the brain’s unique extracellular matrix is incompletely understood but its structural organization is based on long strands of very high molecular weight hyaluronic acid decorated with a variety of aggregated proteins and proteoglycans which form networks of crosslinked fibers. Using the human melanoma cell line, MDA MB 435, we found that variant sub-lines derived from lung, bone and brain metastases from orthotopically implanted cells in SCID mice, share a significant upregulation of the hyaluronic acid degrading enzyme, hyaluronidase 1 (Hyal1). Hyaluronic acid zymography showed that the parental 435 cell line expresses barely detectable amounts of Hyal1 and the level of enzyme secreted by cells cultured from the experimental primary tumors at the site of orthotopic implantation is also very low. The striking metastasis-associated up-regulation of Hyal1 suggested that this enzyme might be a central mediator of the metastatic process in melanoma. Our laboratory is especially interested in developing a molecular profile of the events underlying survival and dissemination of tumor cells in the brain. In this study, brain-metastatic variants of luciferase-tagged MDA MB 435 cells (435/Brain) were established in tissue culture and used to examine the role of Hyal1 during tumor growth in the brains of SCID mice. We generated a panel of stable knockdowns of Hyal1 in 435/Brain cells using shRNA in a lentiviral vector. Knockdown of Hyal1 was confirmed by PCR, hyaluronic acid zymography and by analysis of HA digestion products by agarose gel electrophoresis. A control shRNA sequence which has no homology to known human gene sequences was used as a baseline comparison in all experiments. In vivo imaging of luciferase tagged 435/Brain cells was used to visualize growth and metastasis of control and Hyal1 knockdowns after stereotactically guided implantation in the brain striatum of SCID mice. Control and knockdown cells formed tumors at the site of implantation. However, Hyal 1 knockdown strongly diminished the capacity of the cells to migrate from the initial tumor location, in contrast to control cells which rapidly seeded metastases in multiple locations in the brains and spines of the injected animals. These data imply that tumor-derived Hyal1, an enzyme which has been shown to be important in the metastatic spread of several tumor types, can also degrade the form of hyaluronic acid found in the brain extracellular matrix and mediate intra-cranial migration of cancer cells.