Molecular Oncology Unit, Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, 75, M. Asias Street, 11527, Athens, Greece.
First Department of Internal Medicine, 'Laiko' Hospital, Medical School, National and Kapodistrian University of Athens, 11527, Athens, Greece.
Breast Cancer Res. 2019 Dec 3;21(1):132. doi: 10.1186/s13058-019-1226-9.
ERBB-2 is overexpressed in about 20% of breast cancers (BCs), indicating poor prognosis. The receptor activator of nuclear factor-κB (RANK) pathway is implicated in ERBB-2 (+) BC. The purpose of this study was to elucidate the underlying molecular mechanism of this interaction and the beneficial impact of dual targeting of RANK and ERBB-2 pathways.
We used SKBR3, MCF7, MDA-MB-453, and BT-474 human BC cell lines. We examined RANK and RANKL expression using RT-PCR, Western blot, and immunofluorescence. The evaluation of RANK expression in a cohort of BC patients was performed using immunohistochemistry. The interaction between RANK and ERBB family members was detected using proximity ligation assay (PLA), which enables the visualization of interacting proteins. We used inhibitors of both pathways [trastuzumab (T), pertuzumab (P), denosumab (D)]. NF-κB pathway activation was studied using Western blot. Cell growth and viability was evaluated using XTT, flow cytometry, and clonogenic assay. For cell migration evaluation, scratch assay was performed. Data were analyzed by one-way ANOVA.
Cell lines express RANK and RANKL. RANK immunostaining was also detected in human BC tissue samples. RANK receptor dimerizes with ERBB family members. RANK/ERBB-2 dimer number seems to be associated with ERBB-2 expression (SKBR3, 5.4; BT-474, 8.2; MCF7, 0.7; MDA-MB-453, 0.3). RANK/ERBB-2 dimers were decreased in the presence of the inhibitors D, T, and P, while they were increased after RANKL (R) treatment in SKBR3 (m, 5.4; D, 1.2; T, 1.9; DT, 0.6; TP, 1; DTP, 0.4; R, 11.8) and BT-474 (m, 8.2; D, 3.1; T, 4.3; DT, 0.7; TP, 3.4; DTP, 3.2; R, 11.6). Combination targeting of SKBR3 further decreased NF-κB pathway activation compared to single targeting. In SKBR3, RANKL and ERBB-2 blockage resulted in reduced cell proliferation, increased apoptosis, and lower metastatic potential compared to mock cells (m) and reversed values in RANKL presence. The combination treatment of SKBR3 with D, T, and P had an advantage in functional traits compared to single targeting. Denosumab suppressed NF-κB signaling and diminished proliferation rate in MDA-MB-453 cells. MCF7 did not correspond to inhibitors.
The results indicate a novel physical and molecular association between ERBB-2 and RANK pathways that affects ERBB-2 (+) BC growth. We also present data suggesting that the combination of anti-ERBB-2 agents and RANKL inhibitors have a potential direct anti-tumor effect and should be further tested in certain BC patients.
大约 20%的乳腺癌（BC）中存在 ERBB-2 过表达，提示预后不良。核因子-κB（NF-κB）受体激活剂（RANK）通路与 ERBB-2（+）BC 有关。本研究旨在阐明这种相互作用的潜在分子机制以及双重靶向 RANK 和 ERBB-2 通路的有益影响。
我们使用了 SKBR3、MCF7、MDA-MB-453 和 BT-474 人 BC 细胞系。我们使用 RT-PCR、Western blot 和免疫荧光检测 RANK 和 RANKL 的表达。使用免疫组织化学检测 BC 患者队列中 RANK 的表达。使用接近连接测定（PLA）检测 RANK 与 ERBB 家族成员之间的相互作用，该测定可可视化相互作用的蛋白。我们使用了两种通路的抑制剂[曲妥珠单抗（T）、帕妥珠单抗（P）、地舒单抗（D）]。使用 Western blot 研究 NF-κB 通路的激活。使用 XTT、流式细胞术和集落形成测定评估细胞生长和活力。为了评估细胞迁移，进行了划痕实验。通过单因素方差分析对数据进行分析。
细胞系表达 RANK 和 RANKL。还在人 BC 组织样本中检测到 RANK 免疫染色。RANK 受体与 ERBB 家族成员形成二聚体。RANK/ERBB-2 二聚体的数量似乎与 ERBB-2 表达相关（SKBR3，5.4；BT-474，8.2；MCF7，0.7；MDA-MB-453，0.3）。在存在抑制剂 D、T 和 P 的情况下，RANK/ERBB-2 二聚体减少，而在用 RANKL（R）处理 SKBR3 后（m，5.4；D，1.2；T，1.9；DT，0.6；TP，1；DTP，0.4；R，11.8）和 BT-474 后（m，8.2；D，3.1；T，4.3；DT，0.7；TP，3.4；DTP，3.2；R，11.6）增加。与单独靶向相比，SKBR3 的联合靶向进一步降低了 NF-κB 通路的激活。与模拟细胞（m）相比，RANKL 和 ERBB-2 阻断导致 SKBR3 中的细胞增殖减少、凋亡增加和转移潜力降低，而在 RANKL 存在下则逆转了这些值。与单独靶向相比，SKBR3 的联合治疗在功能特征方面具有优势。地舒单抗抑制 NF-κB 信号并降低 MDA-MB-453 细胞的增殖率。MCF7 与抑制剂不对应。
结果表明 ERBB-2 和 RANK 通路之间存在新的物理和分子关联，这影响 ERBB-2（+）BC 的生长。我们还提供的数据表明，抗 ERBB-2 药物和 RANKL 抑制剂的联合具有潜在的直接抗肿瘤作用，应在某些 BC 患者中进一步测试。
