Moore Lakisha D, Isayeva Tatyana, Siegal Gene P, Ponnazhagan Selvarangan
Department of Pathology, The University of Alabama at Birmingham, Birmingham, Alabama 35294-0007, USA.
Clin Cancer Res. 2008 Aug 1;14(15):4961-70. doi: 10.1158/1078-0432.CCR-07-4604.
Overexpression of transforming growth factor (TGF)-beta has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-beta1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management.
Small interfering RNA constructs targeting TGF-beta1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis.
Inhibition of TGF-beta1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-beta signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-beta receptor expression showed a decrease in both receptor I and II expression in TGF-beta1 silenced cells. These results suggest that inhibition of TGF-beta1 ligand may act as a negative feedback loop to disrupt the function of all TGF-beta isoforms.
Therapies targeting the TGF-beta signaling pathway may be more effective in late-stage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-beta levels.
转化生长因子（TGF）-β的过表达与多种癌症（包括乳腺癌）中促进免疫抑制、肿瘤血管生成、肿瘤细胞迁移和侵袭有关。因此，在原位乳腺癌中靶向下调TGF-β1表达并确定其影响将为疾病管理提供新的治疗方法。
验证了靶向TGF-β1的小干扰RNA构建体，并用于开发转移性乳腺癌细胞系MDA-MB-435的克隆衍生物。这些细胞用于多种体外分析，包括迁移、侵袭、3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐、凋亡和信号转导分析。采用伤口愈合试验来确定培养物中细胞的迁移情况，采用Boyden小室Transwell试验来检测侵袭情况。此外，将这些克隆用于体内小鼠模型，以研究肿瘤生长动力学以及转移后原发部位和肺部的基因表达情况。
MDA-MB-435细胞中TGF-β1表达的抑制在体外使迁移减少35%，侵袭减少55%，增殖增加50%，且对凋亡无影响。体内分析表明，出现肉眼可见肺转移的小鼠数量减少了90%；然而，与亲本MDA-MB-435细胞相比，原发性肿瘤的生长动力学没有任何差异。对克隆衍生物中TGF-β信号通路的分析表明，Smad2激活减少，AKT和细胞外信号调节激酶激活增加。有趣的是，对TGF-β受体表达的分析表明，在TGF-β1沉默的细胞中，受体I和受体II的表达均降低。这些结果表明，抑制TGF-β1配体可能作为负反馈环来破坏所有TGF-β亚型的功能。
靶向TGF-β信号通路的疗法在晚期疾病中可能更有效地预防器官转移，但对原发性肿瘤形成无效，并且可能与其他通常受循环TGF-β水平升高限制的肿瘤靶向疗法联合使用。
