Cyclin E overexpression confers resistance to trastuzumab through noncanonical phosphorylation of SMAD3 in HER2+breast cancer
Cyclin E overexpression confers resistance to trastuzumab through noncanonical phosphorylation of SMAD3 in HER2+breast cancer
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DOI：
10.1080/15384047.2020.1818518
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发表时间：
2020-10-12
影响因子：
3.6
通讯作者：
Jeruss, Jacqueline S.
中科院分区：
文献类型：
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作者：
Decker, Joseph T.;Kandagatla, Pridvi;Jeruss, Jacqueline S.
曲妥珠单抗是一种治疗 HER2+ 乳腺癌的药物，其疗效可能会因耐药性的产生而受到限制。细胞周期蛋白 E (CCNE) 过度表达与曲妥珠单抗耐药有关。我们试图揭示这种曲妥珠单抗耐药性的潜在机制，并重点关注过表达 HER2+ 乳腺癌的 CCNE 模型和 TGF-β 信号蛋白 SMAD3 的非经典磷酸化。对 HER2+、CCNE 过表达、曲妥珠单抗耐药细胞系 (BT474R2) 转录活性的网络分析发现，SMAD3 活性降低与治疗耐药相关。免疫印迹显示 BT474R2 细胞中 SMAD3 表达显着下调 (p< .01)，并且这些 CCNE 过表达细胞中 SMAD3 的非经典磷酸化增加。此外，针对 CDK2 抑制，还发现了与恢复的经典 SMAD3 信号传导相关的表达模式，包括 cMyc 减少和细胞周期蛋白依赖性抑制剂 p15 增加。 BT474R2 细胞系通过过表达 SMAD3 (BT474R2-SMAD3) 进行修饰，SMAD3 是一种对 CCNE 介导的 SMAD3 非常规磷酸化 (BT474R2-5M) 具有抗性的突变构建体，以及对照 (BT474R2-Blank)。 检查对曲妥珠单抗反应的体外研究显示，对曲妥珠单抗治疗的敏感性增加 BT474R2-5M 电池。然后，这些发现在接种 BT474R2-5M 细胞或 BT474R2 对照细胞的 NSG 小鼠中得到验证。用曲妥珠单抗治疗后，与接种 BT474R2 细胞的小鼠相比，接种 BT474R2-5M 细胞的 NSG 小鼠的肿瘤体积显着降低 (p< .001)。总而言之，这些结果表明，对于 HER2+ 乳腺癌患者，通过非经典 SMAD3 磷酸化调节的 CCNE 介导的曲妥珠单抗耐药机制可以通过 CDK2 抑制进行治疗，以帮助增强曲妥珠单抗治疗的疗效。
The efficacy of trastuzumab, a treatment for HER2+ breast cancer, can be limited by the development of resistance. Cyclin E (CCNE) overexpression has been implicated in trastuzumab resistance. We sought to uncover a potential mechanism for this trastuzumab resistance and focused on a model of CCNE overexpressing HER2+ breast cancer and noncanonical phosphorylation of the TGF-beta signaling protein, SMAD3. Network analysis of transcriptional activity in a HER2+, CCNE overexpressing, trastuzumab-resistant cell line (BT474R2) identified decreased SMAD3 activity was associated with treatment resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p< .01), and noncanonical phosphorylation of SMAD3 was increased in these CCNE-overexpressing cells. Also, in response to CDK2 inhibition, expression patterns linked to restored canonical SMAD3 signaling, including decreased cMyc and increased cyclin-dependent inhibitor, p15, were identified. The BT474R2 cell line was modified through overexpression of SMAD3 (BT474R2-SMAD3), a mutant construct resistant to CCNE-mediated noncanonical phosphorylation of SMAD3 (BT474R2-5M), and a control (BT474R2-Blank).In vitrostudies examining the response to trastuzumab showed increased sensitivity to treatment for BT474R2-5M cells. These findings were then validated in NSG mice inoculated with BT474R2-5M cells or BT474R2 control cells. After treatment with trastuzumab, the NSG mice inoculated with BT474R2-5M cells developed significantly lower tumor volumes (p< .001), when compared to mice inoculated with BT474R2 cells. Taken together, these results indicate that for patients with HER2+ breast cancer, a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, could be treated with CDK2 inhibition to help enhance the efficacy of trastuzumab therapy.