Background: EGF receptor (EGFR) signaling is regulated by endocytosis. Results: The intracellular localization of the EGFR affects its signaling. Conclusion: EGFRs on the limiting membrane of endosomes, but not from the intraluminal vesicles, can induce apoptosis. Significance: EGFR signaling is spatially regulated at multiple steps of the endocytic pathway.

Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared (NIR) EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe prior to MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a non-specific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at six hours post-injection, demonstrating specific intra-tumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of EGF receptor.

Both constitutive and ligand-mediated membrane trafficking regulate Epidermal Growth Factor Receptor (EGFR) signaling. The constitutive endocytosis and recycling of the unliganded EGFR is a critical determinant of cell surface EGFR expression and the cell’s sensitivity to ligands. We report that two proteins with established roles in trafficking the EGF:EGFR complex to the lysosome also regulate the recycling of the unliganded EGFR. Knock down of either Tumor suppressor gene 101 (TSG101) or RAB7 causes the endosomal accumulation of the inactive, unliganded receptor in morphologically and biochemically distinct organelles. Knock down of TSG101 causes the EGFR to accumulate in low density endosomes whereas RAB7 knock down results in EGFR accumulation in high density endosomes. Knock down of either protein caused the receptor to co-localize primarily with LAMP-1, but not EEA1. These two proteins regulate EGFR slow, perinuclear recycling, via distinct mechanism and are new molecular targets that regulate cell surface EGFR expression.

There are 13 known endogenous ligands for the epidermal growth factor receptor (EGFR) and its closely related ErbB receptor family members. We previously reported that betacellulin (BTC) is more efficacious than epidermal growth factor (EGF) in mediating corneal wound healing, although the molecular basis for this difference was unknown. For the most part, differences between ligands can be attributed to variability in binding properties, such as the unique rate of association and dissociation, pH sensitivity, and selective binding to individual ErbB family members of each ligand. However, this was not the case for BTC. Despite being better at promoting wound healing via enhanced cell migration, BTC has reduced receptor affinity and weaker induction of EGFR phosphorylation. These data indicate that the response of BTC is not due to enhanced affinity or kinase activity. Receptor phosphorylation and proximity ligation assays indicate that BTC treatment significantly increases ErbB3 phosphorylation and EGFR-ErbB3 heterodimers when compared with EGF treatment. We observed that EGFR-ErbB3 heterodimers contribute to cell migration, because the addition of an ErbB3 antagonist (MM-121) or RNA interferencemediated knockdown of ErbB3 attenuates BTC-stimulated cell migration compared with EGF. Thus, we demonstrate that, despite both ligands binding to the EGFR, BTC biases the EGFR to dimerize with ErbB3 to regulate the biologic response.

Manipulating the duration of EGFR activity can enhance the rate of restoration of the corneal epithelial layer. Based on our findings, c-Cbl is a new therapeutic target to enhance EGFR-mediated corneal epithelial homeostasis that bypasses the limitations of previous approaches.