Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.
Oncogene. 2012 Oct 11;31(41):4434-46. doi: 10.1038/onc.2011.617. Epub 2012 Jan 16.
Trastuzumab (Herceptin) resistance is a major obstacle in the treatment of patients with HER2-positive breast cancers. We recently reported that the transcription factor Y-box binding protein-1 (YB-1) leads to acquisition of resistance to trastuzumab in a phosphorylation-dependent manner that relies on p90 ribosomal S6 kinase (RSK). To explore how this may occur we compared YB-1 target genes between trastuzumab-sensitive cells (BT474) and those with acquired resistance (HR5 and HR6) using genome-wide chromatin immunoprecipitation sequencing (ChIP-sequencing), which identified 1391 genes uniquely bound by YB-1 in the resistant cell lines. We then examined differences in protein expression and phosphorylation between these cell lines using the Kinexus Kinex antibody microarrays. Cross-referencing these two data sets identified the mitogen-activated protein kinase-interacting kinase (MNK) family as potentially being involved in acquired resistance downstream from YB-1. MNK1 and MNK2 were subsequently shown to be overexpressed in the resistant cell lines; however, only the former was a YB-1 target based on ChIP-PCR and small interfering RNA (siRNA) studies. Importantly, loss of MNK1 expression using siRNA enhanced sensitivity to trastuzumab. Further, MNK1 overexpression was sufficient to confer resistance to trastuzumab in cells that were previously sensitive. We then developed a de novo model of acquired resistance by exposing BT474 cells to trastuzumab for 60 days (BT474LT). Similar to the HR5/HR6 cells, the BT474LT cells had elevated MNK1 levels and were dependent on it for survival. In addition, we demonstrated that RSK phosphorylated MNK1, and that this phosphorylation was required for ability of MNK1 to mediate resistance to trastuzumab. Furthermore, inhibition of RSK with the small molecule BI-D1870 repressed the MNK1-mediated trastuzumab resistance. In conclusion, this unbiased integrated approach identified MNK1 as a player in mediating trastuzumab resistance as a consequence of YB-1 activation, and demonstrated RSK inhibition as a means to overcome recalcitrance to trastuzumab.
曲妥珠单抗（赫赛汀）耐药是治疗 HER2 阳性乳腺癌患者的主要障碍。我们最近报道，转录因子 Y 盒结合蛋白-1（YB-1）通过依赖于 p90 核糖体 S6 激酶（RSK）的磷酸化依赖性方式导致对曲妥珠单抗的耐药性。为了探索这种情况如何发生，我们使用全基因组染色质免疫沉淀测序（ChIP-seq）比较了曲妥珠单抗敏感细胞（BT474）和获得耐药性的细胞（HR5 和 HR6）之间的 YB-1 靶基因，该方法鉴定了在耐药细胞系中由 YB-1 特异性结合的 1391 个基因。然后，我们使用 Kinexus Kinex 抗体微阵列检查了这些细胞系之间的蛋白表达和磷酸化差异。将这两个数据集交叉引用确定丝裂原激活的蛋白激酶相互作用激酶（MNK）家族可能是 YB-1 下游获得性耐药的参与者。MNK1 和 MNK2 在耐药细胞系中表达上调；然而，只有前者是基于 ChIP-PCR 和小干扰 RNA（siRNA）研究的 YB-1 靶标。重要的是，使用 siRNA 敲低 MNK1 表达可增强曲妥珠单抗的敏感性。此外，MNK1 过表达足以使先前敏感的细胞对曲妥珠单抗产生耐药性。然后，我们通过将 BT474 细胞暴露于曲妥珠单抗中 60 天（BT474LT）来开发获得性耐药的从头模型。与 HR5/HR6 细胞类似，BT474LT 细胞具有升高的 MNK1 水平，并且依赖于它来生存。此外，我们证明 RSK 磷酸化 MNK1，并且这种磷酸化对于 MNK1 介导曲妥珠单抗耐药性是必需的。此外，用小分子 BI-D1870 抑制 RSK 抑制了 MNK1 介导的曲妥珠单抗耐药性。总之，这种无偏置的综合方法确定 MNK1 是 YB-1 激活导致曲妥珠单抗耐药的中介物，并证明 RSK 抑制是克服曲妥珠单抗抗性的一种手段。
