HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

中文翻译：

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在 PIK3CA 中携带激酶和螺旋结构域突变的 HER2 扩增乳腺癌细胞对靶向治疗的敏感性不同

HER2 扩增的乳腺癌是临床定义的乳腺癌亚型，有多种可行的靶向治疗。对这些靶向治疗的耐药性是一个常见问题，但耐药性发生的机制仍未完全确定。已提出的一种机制是通过 PI3 激酶途径中的基因突变。来自 HER2 通路的细胞内信号可通过 PI3 激酶发生，并且已知编码基因 PIK3CA 的突变是致癌的。在约 20% 的 HER2 扩增亚型病例中，PIK3CA 突变与 HER2 扩增同时发生。我们使用腺相关病毒介导的基因打靶，在 HER2 扩增的乳腺癌细胞中生成了每个 PIK3CA 热点突变的等基因敲入突变体。使用组合药物筛选分析等基因克隆以确定对 HER2 靶向治疗的不同反应。蛋白质印迹分析和免疫荧光揭示了对 HER2 靶向治疗具有抗性的细胞中独特的细胞内信号动力学。随后的组合药物筛选用于探索神经调节蛋白 1 介导的对 HER2 靶向治疗的耐药性。最后，体外实验的结果被外推到公开可用的数据集。HER2 靶向治疗表明激酶结构域 (H1047R) 而非螺旋结构域 (E545K) 的突变增加了对拉帕替尼的耐药性。从机制上讲，持续的 AKT 信号在具有激酶结构域突变的细胞中驱动拉帕替尼耐药性，如通过 PI3 激酶的细胞内产物 PIP3 染色所证明的。这种耐药性可以通过与下游激酶 AKT 抑制剂共同处理来克服。此外，敲除 PIP3 磷酸酶 PTEN 可以表型复制该结果。我们还表明，neuregulin-1 是 HER 家族受体的配体，它赋予对携带任一热点突变的细胞的抵抗力，并调节对组合疗法的反应。最后，我们通过对 TCGA 和 METABRIC 数据队列的分析，展示了热点突变具有与治疗耐药性相关的不同表达谱的临床证据。我们的结果证明了独特的细胞内信号差异，这取决于细胞携带的 PIK3CA 突变。只有激酶结构域中的突变才能完全激活 PI3 激酶信号通路，并在 HER2 抑制的情况下维持下游信号传导。而且，