Figure 5. Therapeutic inhibition of SFKs can block nEGFR translocation in TNBC cell lines and tumor models.

(A) Dasatinib can inhibit nEGFR translocation and enhance non-nuclear EGFR levels. Cells were treated with vehicle or dasatinib (25 nM) for 24 and 72 hr prior to harvesting whole cell, non-nuclear and nuclear proteins. (B) Dasatinib can block nEGFR translocation measured by Nuance imaging analysis. Cells were treated with vehicle or dasatinib (50 nM) for 24 and 48 hr prior to staining for EGFR, E-Cadherin, and DAPI. Nuclear EGFR fluorescence detected from dasatinib treated cells was normalized to nEGFR fluorescence detected from vehicle treated cells using InForm software (n=2). (C) Dasatinib can enhance plasma membrane-bound EGFR levels measured by flow cytometry. Cells were treated with dasatinib (25 nM) for 24 hr prior to EGFR surface level analysis. Surface level EGFR expression of dasatinib treated cells was normalized to vehicle treated cells (n=3). Shaded histogram= vehicle treated cells, Non-shaded histograms= dasatinib treated cells. IgG treated cells are used as a control (dotted line). (D, E) Dasatinib can block nEGFR translocation in MDAMB468 xenograft tumors. Mice with established MDAMB468 tumors were treated with 50 mg/kg of dasatinib or vehicle once a day for 4 days. Tumors were analyzed by confocal IF (D) and IHC (E) for EGFR expression. IF: merged images were magnified to depict nEGFR (arrows) and non-nEGFR (triangle). Magnification 600X for IF and 400X for IHC. Four tumors from vehicle (tumor # 1–4) or dasatinib treated mice (tumor # 5–8) were harvested for protein and analyzed for the indicated proteins. Data points are represented as mean±s.e.m. **p<0.01.