van Ginkel Paul R, Gee Ricardo L, Shearer Rebecca L, Subramanian Lalita, Walker Teresa M, Albert Daniel M, Meisner Lorraine F, Varnum Brian C, Polans Arthur S
Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison, Wisconsin 53792, USA.
Cancer Res. 2004 Jan 1;64(1):128-34. doi: 10.1158/0008-5472.can-03-0245.
Metastatic tumor cells originating from cancers of a variety of tissues such as breast, skin, and prostate may remain dormant for long periods of time. In the case of uveal melanoma, the principal malignancy of the eye, complete removal of the primary tumor by enucleation can nonetheless be followed by metastatic tumor growth in distant organs months, years, or even decades later. This suggests that tumor cells have already spread to secondary sites at the time of treatment and remain dormant as micrometastases. Identifying factors that govern long-lived survival of metastatic tumor cells is therefore key to decreasing mortality associated with this and other diseases. While investigating factors differentially expressed in melanoma cells and normal melanocytes, we identified the receptor tyrosine kinase Axl and found up-regulation of Axl in uveal melanomas and melanoma cell lines by RNase protection, Western analysis, and immunohistochemistry. Axl has been shown to mediate cell growth and survival through its ligand Gas6 in non-transformed cells. To test whether stimulation of Axl can enhance survival of uveal melanoma cells, we assessed the degree of mitogenesis and cell survival by bromodeoxyuridine incorporation and trypan blue exclusion, respectively, upon stimulation of Mel 290 uveal melanoma cells with Gas6 in vitro. We show that Gas6 mediates mitogenesis and cell survival in Mel 290 cells. We further demonstrate that these effects occur specifically through the Axl receptor by modulating the expression of Axl with an antisense construct. cDNA microarray analysis of 12,687 genes then revealed that Gas6 stimulation of Axl in Mel 290 cells results primarily in the down-regulation of Cyr61, a member of the CCN protein family involved in tumor progression. These data show that the Axl pathway mediates increased survival of uveal melanoma cells, potentially advantageous during cancer dormancy, and that Axl may function in part through regulation of Cyr61.
源自多种组织（如乳腺、皮肤和前列腺）癌症的转移性肿瘤细胞可能会长期处于休眠状态。就葡萄膜黑色素瘤（眼睛的主要恶性肿瘤）而言，通过眼球摘除术完全切除原发性肿瘤后，数月、数年甚至数十年后仍可能在远处器官出现转移性肿瘤生长。这表明肿瘤细胞在治疗时就已扩散至次要部位，并以微转移灶的形式保持休眠状态。因此，确定控制转移性肿瘤细胞长期存活的因素是降低与这种疾病及其他疾病相关死亡率的关键。在研究黑色素瘤细胞和正常黑素细胞中差异表达的因素时,我们鉴定出受体酪氨酸激酶Axl，并通过核糖核酸酶保护法、蛋白质免疫印迹分析和免疫组织化学发现葡萄膜黑色素瘤和黑色素瘤细胞系中Axl表达上调。在未转化细胞中，Axl已被证明可通过其配体Gas6介导细胞生长和存活。为了测试Axl的激活是否能提高葡萄膜黑色素瘤细胞的存活率，我们在体外使用Gas6刺激Mel 290葡萄膜黑色素瘤细胞后，分别通过溴脱氧尿苷掺入法和台盼蓝拒染法评估了有丝分裂程度和细胞存活率。我们发现Gas6在Mel 290细胞中介导有丝分裂和细胞存活。我们进一步证明，通过用反义构建体调节Axl的表达，这些效应是通过Axl受体特异性发生的。随后对12687个基因进行的cDNA微阵列分析表明，Gas6对Mel 290细胞中Axl的刺激主要导致Cyr61（一种参与肿瘤进展的CCN蛋白家族成员）表达下调。这些数据表明，Axl信号通路介导葡萄膜黑色素瘤细胞存活率增加，这在癌症休眠期间可能具有潜在优势，并且Axl可能部分通过调节Cyr61发挥作用。
