Around 15–20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

中文翻译：

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抗 HER2 治疗后 RANK 信号增加导致 HER2 阳性乳腺癌出现耐药性

大约 15-20% 的原发性乳腺癌以 HER2 蛋白过度表达和/或 HER2 基因扩增为特征。尽管成功开发了抗 HER2 药物，但内在和获得性耐药性仍是一个主要障碍。本研究旨在分析 RANK 通路在 HER2 阳性乳腺癌和抗 HER2 治疗耐药中的作用。RANK 和 RANKL 蛋白表达在来自对抗 HER2 治疗有抗性的 HER2 阳性乳腺癌患者和未接受治疗的患者的样本中进行评估。在来自接受 SOLTI-1114 PAMELA 试验的新辅助双重 HER2 阻断剂（拉帕替尼和曲妥珠单抗）治疗的患者的配对样本中分析了 RANK 和 RANKL 基因表达。此外，HER2 阳性乳腺癌细胞系用于调节 RANK 表达，并在体外分析 RANK 信号对抗 HER2 抗性和下游信号的贡献。RANK 和 RANKL 蛋白在对抗 HER2 疗法获得耐药性的 HER2 阳性肿瘤中比在未接受治疗的肿瘤中更频繁地检测到。在 SOLTI-1114 PAMELA 试验的队列中，在双重抗 HER2 新辅助治疗后，RANK（但不是 RANKL）基因表达增加。HER2 阳性乳腺癌细胞系的结果概括了临床观察结果，在用 HER2 抑制剂拉帕替尼或双重抗 HER2 疗法短期治疗后以及在拉帕替尼耐药细胞中观察到 RANK 表达增加。在 RANKL 刺激后，拉帕替尼抗性细胞与其敏感细胞相比显示出更高的 NF-κB 活化，证实了 RANK 通路在抗 HER2 耐药乳腺癌中的增强功能。RANK 信号通路的过度激活增强了 ERK 和 NF-κB 信号传导并增加了不同 HER2 阳性乳腺癌细胞系中拉帕替尼的耐药性，而 RANK 丢失会使拉帕替尼耐药细胞对药物敏感。我们的结果表明，在几种 HER2 阳性细胞系中，RANK/RANKL 驱动的 ERK 激活需要 ErbB 信号传导。相比之下，拉帕替尼不能抵消 RANK 过表达细胞中 RANKL 处理后引发的 NF-κB 激活。最后，我们表明 RANK 与乳腺癌细胞中的 HER2 结合，并且增强的 RANK 通路激活改变了 HER2 磷酸化状态。我们的数据支持乳腺癌中 RANK 和 HER2 信号传导之间的物理和功能联系，并证明 RANK 信号传导增加可能通过 NF-κB 激活促进拉帕替尼耐药的发展。具有肿瘤 RANK 表达的 HER2 阳性乳腺癌患者是否可能受益于 HER2 和 RANK 双重抑制治疗仍有待阐明。