Cancer Proteomics Laboratory, EGA Institute for Women's Health, University College London, Cruciform Building, Gower Street, London WC1E 6BT, UK.
BMC Cancer. 2010 Sep 14;10:490. doi: 10.1186/1471-2407-10-490.
Members of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression.
Gene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression.
775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signalling and proliferation were enhanced in ErbB2-overexpressing cells, whilst loss of ErbB2 expression by siRNA silencing reduced IGF1 signalling. Furthermore, IGFBP3 knockdown resulted in basal ERK and Akt activation in luminal epithelial cells and increased invasiveness and anchorage-independent colony formation in SKBR3 cells.
These data show IGFBP3 as a negative regulator of transformation and that its down-regulation enhances IGF1-dependent signalling. They also show that ErbB2 can up-regulate IGF1-dependent signalling, possibly via the regulated expression of IGFBP3.
表皮生长因子受体家族成员与上皮细胞生物学、发育和肿瘤发生密切相关；然而，其下游信号转导的机制尚不清楚。事实上，目前尚不清楚如何实现信号特异性，以及每个受体对特定基因表达的相对贡献。
使用 cDNA 微阵列和常见的 RNA 参考方法，对过表达 ErbB2 的人乳腺腔上皮细胞模型进行基因表达谱分析，以检查在 ErbB2 过表达的情况下，EGF 和 heregulin beta1 处理的长期重叠和差异反应。使用定量实时 PCR 和/或免疫印迹验证改变的基因表达。针对一个感兴趣的基因进行了进一步的表征，其中检查了 siRNA 介导的沉默对 IGF1 依赖性信号和细胞表型的影响，并将其与 ErbB2 表达缺失的影响进行了比较。
775 个基因根据其生长因子反应性差异表达并聚类。除了确定未被表征的基因作为 ErbB2 依赖性信号的新靶点外，ErbB2 过表达还增强了多个参与增殖（如 MYC、MAP2K1、MAP2K3）、自分泌生长因子信号（VEGF、PDGF）和粘附/细胞骨架调节（ZYX、THBS1、VCL、CNN3、ITGA2、ITGA3、NEDD9、TAGLN）的基因的诱导，将它们与 ErbB2 过表达细胞的过度增殖和改变的粘附表型联系起来。我们还报告了 ErbB2 依赖性下调多个干扰素刺激基因，这可能使 ErbB2 过表达细胞能够抵抗干扰素的抗增殖作用。最后，IGFBP3 的调节模式是独特的，我们进一步研究了 IGFBP3 下调在 ErbB2 依赖性转化中的可能作用，通过抑制 IGF1 信号。我们表明，IGF1 依赖性信号和增殖在 ErbB2 过表达细胞中增强，而 siRNA 沉默导致 ErbB2 表达缺失则降低了 IGF1 信号。此外，IGFBP3 的敲低导致腔上皮细胞中基础 ERK 和 Akt 的激活，并增加了 SKBR3 细胞的侵袭性和无锚定集落形成。
这些数据表明 IGFBP3 是转化的负调节剂，其下调增强了 IGF1 依赖性信号。它们还表明，ErbB2 可以上调 IGF1 依赖性信号，可能是通过调节 IGFBP3 的表达。
