ERBB2-target therapies in breast cancer: Investigating the biological mechanisms regulated by the pan-ERBB inhibitor Neratinib

Breast carcinoma (BCa) represents the leading cause of cancer-related death among women worldwide, and the overexpression of the Tyrosine Kinase Receptor (RTK) ERBB2 correlates with aggressive disease and poor prognosis. Thus, ERBB2 became one of the molecules chosen for the development of targeted therapies. The Pan-ERBB inhibitor Neratinib (NE), was approved by Food and Drug Administration (FDA) in 2017, for ERBB2+ BC treatment. Despite its broad clinical applications, its mechanisms of action toward the ERBB2 receptor and the downstream effects that sustain its clinical outcome are currently not fully understood. Here we explored the effects of IC50 low nanomolar doses of NE on ERBB2+ Breast Cancer cell lines regarding: the internalization of ERBB2 receptor, the regulation of intracellular trafficking, the autophagy process, and extracellular vesicle (EV) release. These are mechanisms highly dysregulated in cancer, sustaining cancer progression and resistance to treatments. Combining imaging, biochemical, and molecular approaches, in this study we assessed that low nanomolar IC50 doses of NE transiently reduced the activation of its downstream signaling pathways (Ras/MAPK and PI3K/Akt/mTOR), prompted an increase in the clathrin-mediated endocytosis and expanded the CD63+ MVB compartment without inducing a strong internalization of the ERBB2 receptor. Moreover, NE exerted its antitumoral function on the metastatic cell line SKBR-3 impairing autophagic machinery and processes. Additionally, we observed that cells under treatment increase the EV release while lowering the amount of ERBB2 on EVs, possibly preventing a potential metastatic mechanism. We reasoned that the outcome on autophagy could be the result of the reduced activation of TFEB and TFE3 transcription factors, two master regulators of autophagy and lysosomal exocytosis. Indeed, the expression of TFEB and TFE3 was strikingly increased in the cytosolic compartment upon treatment. Recent findings unveiled that TFEB and TFE3 are responsible for mitochondrial surveillance since they modulate a selective type of autophagy against damaged mitochondria (mitophagy). Therefore, we investigated mitochondrial morphology and functionality upon NE treatment. NE impaired the metabolic functionality of mitochondria that appeared also more fragmented compared to untreated cells, as a possible consequence of TFEB and TFE3 inhibition. Overall, these data extend the understanding of NE effects on ERBB2+ BCa cells, which result in an impairment of autophagy, a diminished ERBB2 potentially transferred to distant sites, and a detriment of mitochondrial metabolism, as its possible antitumoral functions.