Investigating HER2/EGFR Signaling Dynamics and EV-Mediated Communication in Cancer Cells

HER2 and EGFR are both receptors that help mediate and control cell growth. Both HER2 and EGFR play crucial roles in cancer signaling. In HER2-overexpressing cancers, HER2/EGFR heterodimers form constitutively active complexes that evade recycling, leading to persistent oncogenic signaling. This study explored the internalization dynamics of these receptors and their potential use as extracellular vesicle (EV)-based cancer markers. We used two cell lines: human epithelial-like meningioma (IOMM-LEE) and BT474 (human breast HER2-positive ductal carcinoma) which overexpressed HER2. We tracked the internalization of equimolar HER2/EGFR in meningioma cells and overexpressed HER2 in BT474 cells by using anti-phospho-HER2 and anti-phospho-EGFR antibodies. To investigate EV-mediated signaling, cells were treated with EGF and EVs were collected at 30, 60, and 120 min post-treatment. We explored two possible mechanisms through which receptor dimers drive cancer progression. 1) Activation of cancer gene expression: During endosomal trafficking, instead of recycling to the plasma membrane, receptor dimers bind to importin alpha and translocate to the nucleus, thereby directly influencing gene expression, and 2) EV-mediated signaling: EVs containing phosphorylated receptors facilitate intercellular communication, triggering signaling cascades in recipient cells. This study provides insights into the complex relationship between HER2/EGFR signaling, receptor trafficking, and EV-mediated communication in cancer cells. Our findings suggest that phosphorylated HER2 in EVs may serve as a potential biomarker for diagnostic and therapeutic strategies for HER2-positive cancers.