Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration–approved pan-ERBB inhibitor employed for the treatment of ERBB2⁺ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30–100 nm) ERBB2⁻ EVs and large (>100 nm) ERBB2⁺ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2⁺ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs:

ERBB2 扩增的乳腺癌 (BCa) 表现出肿瘤生长迅速、疾病进展加快和生存率较低。细胞内运输失调和细胞外囊泡 (EV) 释放是支持癌症进展和治疗耐药的机制。 Neratinib (NE) 是一种经美国食品和药物管理局批准的泛 ERBB 抑制剂，用于治疗 ERBB2 ⁺ BCa，阻断信号传导并导致生存抑制。然而，NE 对 ERBB2 内化、其向多泡体 (MVB) 的运输以及源自这些细胞器的 EV 释放的影响仍然很少研究。通过共聚焦和电子显微镜，我们观察到低纳摩尔剂量的 NE 诱导适度的 ERBB2 内化，同时网格蛋白介导的内吞作用和 SKBR-3 细胞中 CD63+ MVB 区室的增加。此外，我们根据大小和 ERBB2 阳性，在培养物上清液中显示了两个不同的 EV 子集：小 (30-100 nm) ERBB2 ^- EV 和大 (>100 nm) ERBB2 ⁺ EV。特别是，我们发现 NE 增加了 EV 的总体释放，与对照组相比，其表现出 ERBB2 阳性率降低。总而言之，这些结果为 NE 对 ERBB2 ⁺ BCa 细胞的影响提供了新的见解，这可能导致通过 EV 转移到远处靶细胞的 ERBB2 减少：