Iczkowski Kenneth A
Department of Pathology, University of Colorado Health Science Center Aurora, CO, USA.
Am J Transl Res. 2010 Sep 12;3(1):1-7.
CD44 is a cell adhesion glycoprotein that also governs cell signaling. 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 (v) isoform, CD44v7-10. We studied CD44 in PCa for more than a decade, and in a series of papers, established its functional significance. Using retrovi-ral 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. Compared to luciferase-only PC-3M cells, all 3 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 2 CD44s re-expression approaches caused minimal sensitization. In limited numbers of mouse subcutaneous xeno-grafts, all 3 alterations produced only nonsignificant trends toward slower growth compared with luciferase-only controls. In further work, we tested the effects of the anti-growth compound silibinin, a milk thistle derivative. Using a luciferase promoter construct to test for CD44 promoter activity, silibinin significantly and dose-dependently inhibited promoter activity at physiologic doses. Total CD44 RNA and CD44v7-10 RNA were significantly decreased; both were also decreased at the protein level. Phenyl-methylene hydantoins (PMH), guanidine alkaloids derived from Red Sea sponges, have the ability to increase cell-cell adhesion in prostate cancer cells and reduce invasion. Expression of CD44 total mRNA and CD44v7-10 were markedly decreased by PMH and its S-ethyl derivative. The oncogenic mi-croRNAs, miR-373 and miR-520c, which interact with CD44, were studied in prostate cancer cells and human tissues. We found that they bound the 3' untranslated region of the CD44 RNA, and suppressed CD44 in prostate cancer, by preventing the translation of CD44 RNA, rather than by degrading the RNA. Thus, stable re-expression of CD44s reduces PCa growth and invasion in vitro, and possibly in vivo, suggesting CD44's potential as gene therapy. Finally, CD44v7-10 may be a target for chemosensitization, and plays a role in nutraceutical abrogation of tumor development. In vivo effects of CD44 alteration still need to be investigated by use of orthotopic or renal capsule xenografts, which confer a different stromal microenvironment than that of the subcutaneous grafts.
CD44是一种细胞黏附糖蛋白，也参与细胞信号传导。CD44表达失调是大多数人类癌症的特征，包括前列腺癌（PCa）。PCa会失去良性上皮细胞中存在的CD44标准型（CD44s）的表达，并过度表达新型剪接变体（v）亚型CD44v7-10。我们对PCa中的CD44进行了十多年的研究，并在一系列论文中确立了其功能意义。通过逆转录病毒基因传递至PC-3M前列腺癌细胞，我们分别表达了仅含荧光素酶的基因、在其C末端与荧光素酶融合表达CD44s、单独表达CD44s蛋白，或通过RNA干扰敲低CD44v7-10。进行了侵袭、迁移、增殖、软琼脂集落形成、黏附、多西他赛敏感性和异种移植生长试验。与仅含荧光素酶的PC-3M细胞相比，所有这三种处理均降低了侵袭和迁移能力。只有将CD44s作为单独或融合蛋白重新表达时，生长和软琼脂集落形成才会减少，而CD44v7-10 RNA干扰则不会。作为单独蛋白表达的CD44s能大大增强透明质酸和骨桥蛋白的结合，但融合蛋白则不能。PC-3M细胞中的CD44v7-10 RNA干扰导致对多西他赛的敏感性显著增加；而两种CD44s重新表达方法引起的敏感性增加则微乎其微。在数量有限的小鼠皮下异种移植中，与仅含荧光素酶的对照组相比，所有这三种改变仅产生了生长缓慢的不显著趋势。在进一步的研究中，我们测试了抗生长化合物水飞蓟宾（一种水飞蓟衍生物）的作用。使用荧光素酶启动子构建体检测CD44启动子活性，水飞蓟宾在生理剂量下能显著且剂量依赖性地抑制启动子活性。总CD44 RNA和CD44v7-10 RNA显著降低；蛋白水平也降低。源自红海海绵的胍生物碱苯亚甲基乙内酰脲（PMH）具有增强前列腺癌细胞间黏附并减少侵袭的能力。PMH及其S-乙基衍生物可显著降低CD44总mRNA和CD44v7-10的表达。在前列腺癌细胞和人体组织中研究了与CD44相互作用的致癌性微小RNA miR-373和miR-520c。我们发现它们与CD44 RNA的3'非翻译区结合，并通过阻止CD44 RNA的翻译而非降解RNA来抑制前列腺癌中的CD44。因此，稳定重新表达CD44s可在体外（可能也在体内）降低PCa的生长和侵袭，提示CD44具有作为基因治疗的潜力。最后，CD44v7-10可能是化学增敏的靶点，并在营养保健品消除肿瘤发展中发挥作用。CD44改变的体内效应仍需通过原位或肾包膜异种移植进行研究，因为它们提供的基质微环境与皮下移植不同。
