Teramoto Hidemi, Castellone Maria Domenica, Malek Renae L, Letwin Noah, Frank Bryan, Gutkind J Silvio, Lee Norman H
Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892-4330, USA.
Oncogene. 2005 Jan 13;24(3):489-501. doi: 10.1038/sj.onc.1208209.
Activated forms of Ras family members are prevalent in many cancers where Ras mutants transduce signals essential for transformation, angiogenesis, invasion and metastasis. As a cancer progression model, we used NIH3T3 cells to explore the mechanism of Ras-induced tumorigenesis. Ras family mutants H-RasV12 and Rit79L strongly induced foci formation, while Rho family mutants RhoA-QL, Rac1-QL and Cdc42-QL were less effective. A comparison of downstream transcriptional targets of Ras and Rho family members using a 26 383 element cDNA microarray revealed that the osteopontin (OPN) gene exhibited the best correlation between magnitude of gene expression change and level of foci formation (r=0.96, P<0.001). In association with H-RasV12- and Rit79L-mediated transformation, foci secreted OPN protein and upregulated the OPN receptor CD44, suggesting the novel initiation of an aberrant OPN-CD44-Rac autocrine pathway. In support of this were the following observations. First, RGD-deficient OPN protein-binding activity was present in H-RasV12-transformed cells but not in control cells, and binding activity was inhibited by the CD44 blocking antibody. Second, foci formation, cell invasion and Rac activity were induced by H-RasV12 and inhibited by the CD44 blocking antibody. Third, foci formation by H-RasV12 was substantially reduced by a short interfering RNA (siRNA) specifically targeting OPN expression for knockdown. Fourth, H-RasV12-mediated transformation was not blocked by the GRGDS peptide, suggesting that OPN effects were not mediated by the integrins. Lastly, OPN knockdown affected the downstream expression of 160 '2nd tier' genes, and at least a subset of these genes appears to be involved in transformation. Indeed, four genes were selected for knockdown, each resulting in a disruption of foci formation and/or invasion. These results underscore the role of aberrant autocrine signaling and transcriptional networking during tumorigenesis.
Ras家族成员的激活形式在许多癌症中普遍存在，其中Ras突变体转导对细胞转化、血管生成、侵袭和转移至关重要的信号。作为一种癌症进展模型，我们使用NIH3T3细胞来探索Ras诱导肿瘤发生的机制。Ras家族突变体H-RasV12和Rit79L强烈诱导灶形成，而Rho家族突变体RhoA-QL、Rac1-QL和Cdc42-QL效果较差。使用26383元件cDNA微阵列比较Ras和Rho家族成员的下游转录靶点，发现骨桥蛋白（OPN）基因在基因表达变化幅度与灶形成水平之间表现出最佳相关性（r = 0.96，P < 0.001）。与H-RasV12和Rit79L介导的转化相关，灶分泌OPN蛋白并上调OPN受体CD44，提示异常的OPN-CD44-Rac自分泌途径的新启动。支持这一点的有以下观察结果。首先，H-RasV12转化细胞中存在RGD缺陷型OPN蛋白结合活性，而对照细胞中不存在，且结合活性被CD44阻断抗体抑制。其次，H-RasV12诱导灶形成、细胞侵袭和Rac活性，而CD44阻断抗体抑制这些作用。第三，通过特异性靶向OPN表达进行敲低的短干扰RNA（siRNA）可使H-RasV12诱导的灶形成显著减少。第四，GRGDS肽未阻断H-RasV12介导的转化，表明OPN的作用不是由整合素介导的。最后，OPN敲低影响160个“二级”基因的下游表达，并且这些基因中的至少一部分似乎参与了转化。实际上，选择了四个基因进行敲低，每个基因都导致灶形成和/或侵袭的破坏。这些结果强调了异常自分泌信号传导和转录网络在肿瘤发生过程中的作用。
