Department of Pharmacology, Emory University, Atlanta, GA 30322, USA.
Biochem Pharmacol. 2011 Nov 1;82(9):1090-9. doi: 10.1016/j.bcp.2011.07.082. Epub 2011 Jul 23.
Resistance to the anti-HER2 monoclonal antibody trastuzumab is a major problem in the treatment of HER2-overexpressing metastatic breast cancer. Growth differentiation factor 15 (GDF15), which is structurally similar to TGF beta, has been reported to stimulate phosphorylation of HER2. We tested the hypothesis that GDF15-mediated phosphorylation of HER2 reduces the sensitivity of HER2-overexpressing breast cancer cell lines to trastuzumab. Gene microarray analysis, real-time PCR, and ELISA were used to assess GDF15 expression. Growth inhibition and proliferation assays in response to pharmacologic inhibitors of HER2, TGF beta receptor, or Src were performed on cells stimulated with recombinant human GDF15 or stable GDF15 transfectants. Western blotting was performed to determine effects of GDF15 on HER2 signaling. Cells were infected with lentiviral GDF15 shRNA plasmid to determine effects of GDF15 knockdown on cell survival in response to trastuzumab. Cells with acquired or primary trastuzumab resistance showed increased GDF15 expression. Exposure of trastuzumab-sensitive cells to recombinant human GDF15 or stable transfection of a GDF15 expression plasmid inhibited trastuzumab-mediated growth inhibition. HER2 tyrosine kinase inhibition abrogated GDF15-mediated Akt and Erk1/2 phosphorylation and blocked GDF15-mediated trastuzumab resistance. Pharmacologic inhibition of TGF beta receptor blocked GDF15-mediated phosphorylation of Src. Further, TGF beta receptor inhibition or Src inhibition blocked GDF15-mediated trastuzumab resistance. Finally, lentiviral GDF15 shRNA increased trastuzumab sensitivity in cells with acquired or primary trastuzumab resistance. These results support GDF15-mediated activation of TGF beta receptor-Src-HER2 signaling crosstalk as a novel mechanism of trastuzumab resistance.
曲妥珠单抗耐药是治疗 HER2 过表达转移性乳腺癌的主要问题。结构上与 TGFβ相似的生长分化因子 15（GDF15）已被报道可刺激 HER2 的磷酸化。我们检验了这样一个假设，即 GDF15 介导的 HER2 磷酸化降低了 HER2 过表达乳腺癌细胞系对曲妥珠单抗的敏感性。采用基因微阵列分析、实时 PCR 和 ELISA 来评估 GDF15 的表达。用重组人 GDF15 或稳定转染的 GDF15 转染细胞进行针对 HER2、TGFβ受体或Src 的药物抑制剂的生长抑制和增殖测定。Western blot 用于确定 GDF15 对 HER2 信号的影响。用慢病毒 GDF15 shRNA 质粒感染细胞，以确定 GDF15 敲低对曲妥珠单抗治疗时细胞存活的影响。获得或原发性曲妥珠单抗耐药的细胞显示出 GDF15 表达增加。曲妥珠单抗敏感细胞暴露于重组人 GDF15 或稳定转染 GDF15 表达质粒可抑制曲妥珠单抗介导的生长抑制。HER2 酪氨酸激酶抑制可消除 GDF15 介导的 Akt 和 Erk1/2 磷酸化，并阻断 GDF15 介导的曲妥珠单抗耐药。TGFβ受体的药理学抑制阻断了 GDF15 介导的Src 磷酸化。此外，TGFβ受体抑制或Src 抑制阻断了 GDF15 介导的曲妥珠单抗耐药。最后，慢病毒 GDF15 shRNA 增加了获得性或原发性曲妥珠单抗耐药细胞中曲妥珠单抗的敏感性。这些结果支持 GDF15 介导的 TGFβ受体-Src-HER2 信号转导串扰作为曲妥珠单抗耐药的一种新机制。
