Department of Pathology, University of Colorado Denver Health Science Center, Aurora, Colorado, USA.
BMC Cancer. 2010 Jan 14;10:16. doi: 10.1186/1471-2407-10-16.
Dysregulated CD44 expression characterizes most human cancers, including prostate cancer (PCa). PCa loses expression of CD44 standard (CD44s) that is present in benign epithelium, and overexpresses the novel splice variant isoform, CD44v7-10.
Using retroviral gene delivery to PC-3M PCa cells, we expressed luciferase-only, enforced CD44s re-expression as a fusion protein with luciferase at its C-terminus or as a protein separate from luciferase, or knocked down CD44v7-10 by RNAi. Invasion, migration, proliferation, soft agar colony formation, adhesion, Docetaxel sensitivity, and xenograft growth assays were carried out. Expression responses of merlin, a CD44 binding partner, and growth-permissive phospho-merlin, were assessed by western blot.
Compared to luciferase-only PC-3M cells, all three treatments reduced invasion and migration. Growth and soft agar colony formation were reduced only by re-expression of CD44s as a separate or fusion protein but not CD44v7-10 RNAi. Hyaluronan and osteopontin binding were greatly strengthened by CD44s expression as a separate protein, but not a fusion protein. CD44v7-10 RNAi in PC-3M cells caused marked sensitization to Docetaxel; the two CD44s re-expression approaches caused minimal sensitization. In limited numbers of mouse subcutaneous xenografts, all three alterations produced only nonsignificant trends toward slower growth compared with luciferase-only controls. The expression of CD44s as a separate protein, but not a fusion protein, caused emergence of a strongly-expressed, hypophosphorylated species of phospho-merlin.
Stable re-expression of CD44s reduces PCa growth and invasion in vitro, and possibly in vivo, suggesting CD44 alterations have potential as gene therapy. When the C-terminus of CD44s is fused to another protein, most phenotypic effects are lessened, particularly hyaluronan adhesion. Finally, CD44v7-10, although it was not functionally significant for growth, may be a target for chemosensitization.
CD44 表达失调是大多数人类癌症的特征，包括前列腺癌 (PCa)。PCa 失去了良性上皮中存在的 CD44 标准 (CD44s) 的表达，并过度表达新型剪接变体同工型 CD44v7-10。
我们使用逆转录病毒基因传递将荧光素酶仅表达、强制 CD44s 再表达为与荧光素酶 C 端融合的蛋白或与荧光素酶分离的蛋白，或通过 RNAi 敲低 CD44v7-10。进行侵袭、迁移、增殖、软琼脂集落形成、粘附、多西紫杉醇敏感性和异种移植生长测定。通过 Western blot 评估 Merlin 的表达反应，Merlin 是 CD44 的结合伴侣，也是生长允许的磷酸化 Merlin。
与仅荧光素酶的 PC-3M PCa 细胞相比，所有三种处理均降低了侵袭和迁移。只有 CD44s 的再表达作为单独的或融合蛋白，而不是 CD44v7-10 RNAi 降低了生长和软琼脂集落形成。CD44s 作为单独的蛋白表达大大增强了透明质酸和骨桥蛋白的结合，但融合蛋白则不然。CD44v7-10 RNAi 在 PC-3M 细胞中导致对多西紫杉醇的显著敏感性；两种 CD44s 再表达方法仅引起最小的敏感性。在有限数量的小鼠皮下异种移植中，与仅荧光素酶对照相比，所有三种改变仅导致生长速度的非显著趋势较慢。CD44s 作为单独的蛋白表达，但不是融合蛋白，导致出现强表达、低磷酸化的磷酸化 Merlin 种。
CD44s 的稳定再表达可降低 PCa 的体外生长和侵袭，并且可能在体内降低生长，表明 CD44 改变具有作为基因治疗的潜力。当 CD44s 的 C 端与另一种蛋白融合时，大多数表型效应减弱，特别是透明质酸粘附。最后，CD44v7-10 虽然对生长没有功能意义，但可能是化学增敏的靶点。
