Fig 2: Abrogation of HER1/HER4 and ERK1/2 signaling inhibits cyclinD1 expression in ER+ cells with HER3T355I(A–B) Serum starved T47D cells expressing HER3WT and HER3T355I were treated with vehicle (DMSO), lapatinib (1 µM), fulvestrant (1 µM), SCH772984 (1 µM) or indicated combinations for 4 hr and cell lysates were collected and immunoblotted using p-HER4, p-ERK1/2, ERa, p-AKT and cyclinD1 antibodies. Actin served as loading control. (C–D) Serum starved MCF-7 cells (HER3WT and HER3T355I) were treated with vehicle (DMSO), lapatinib (1 µM), fulvestrant (1 µM), SCH772984 (1 µM) or indicated combinations for 4 hr and cell lysates were collected and immunoblotted using indicated antibodies. (E) T47D cells with HER3WT and HER3T355I expression were transfected with an HER3-specific siRNA or control (siCon) for 48 hr and analyzed using specific antibodies. Actin was used as loading control.

Fig 3: Structural analysis of HER3T355I mutation(A-B) The locations of the conserved T355 residue mapped onto the structure of the inactive HER3 extracellular domain (ECD) monomer (PDB ID: 1M6B) (A) and the active EGFR ECD homodimer (PDB ID: 3NJP) (B) illustrating the conformational changes associated with ligand dependent receptor activation. In EGFR, domains I and II rotate around the domain II/III hinge as a single rigid body to form an extended, activated conformation poised to interact with dimerization partners. For clarity, one monomer of the EGFR ECD homodimer is shown using the same surface representation as for the inactive HER3 ECD monomer. The second monomer is shown as grey ribbons. (C) Cartoon representation of interactions between T355 and residues of the hinge region pocket in the inactive HER3 ECD. (D) Cartoon representation of the modelled isoleucine residue at position 355 (I355) in the HER3 ECD reveals that the bulkier hydrophobic residue produces a steric clash with the polar pocket. (E) Schematic representation of HER4/HER1-activated ERK1/2-dependent and cyclinD1-mediated signaling in ER+ T47D and MCF-7 cells expressing HER3T355I mutant.

Fig 4: DDA activation of UPR is separable from effects on HER1-3 levels and Akt phosphorylation(A) MDA-MB-468 and BT474 cells were treated for 24 hours with 20 µM RBF3, 100 µM 2-Aminoethoxydiphenyl Borate (2-APB), or 4 mM 2-deoxyglucose (2-DOG) in the indicated combinations and cell extracts were analyzed by immunoblot. (B) MDA-MB-468 cells were treated for 24 hours with 20 µM RBF3, 400 nM thapsigargin, 500 ng/ml tunicamycin or dithiothreitol (DTT) at the indicated concentrations and cell extracts were analyzed by immunoblot. The effects of these same treatments on the splicing of the mRNA coding for XBP1s was assessed by reverse transcription of mRNA followed by DNA amplification (RT-PCR). (C) BT474 cells were treated for 24 hours with 20 µM RBF3, 400 nM thapsigargin, 500 ng/ml tunicamycin, or DTT at the indicated concentrations, and cell extracts were analyzed by immunoblot. The effects of these same treatments on the splicing of the mRNA coding for XBP1s was assessed by reverse transcription of mRNA followed by DNA amplification (RT-PCR). (D) Wild type or eIF2a[S51A] double knock-in mutant MEFs were treated for 24 hours with 20 µM RBF3, 400 nM thapsigargin, 500 ng/ml tunicamycin, 5 mM DTT, or vehicle and cell extracts were prepared and analyzed by immunoblot.

Fig 5: TUSC3 inhibits EGFR phosphorylationA., Subcellular localization of ectopic TUSC3 protein at the ER. SW480 cells were transfected with FLAG-TUSC3 plasmid, fixed and stained for immunofluorescence microscopy. Color legend: red = FLAG-TUSC3, green = calnexin (ER marker) or phalloidin (actin), blue = nuclei (DAPI). Overlay of TUSC3 with calnexin or actin appears in yellow. Magnification 630x. B., TUSC3 blocks tyrosine phosphorylation of EGFR/Her1. SW480 cells were transfected with TUSC3 and EV plasmids for 24 h, followed by serum removal (“starvation”) for 16 h and a restimulation with 20 % FCS (“serum shock”) for 0 to 3 h before cell harvest. Cells were then subjected to subcellular fractionation (SCF). Western blot analyses were done with an Ab against the C-terminal cytoplasmic (intracellular) domain containing phospho-tyrosine residue Y1068 important for EGFR activity (P-EGFR). O.D. values from bands in gels were calculated as -fold ± S.E. (*p < 0.05 TUSC3 vs. EV, Two-way ANOVA, n = 3). C., TUSC3 inhibits EGFR phosphorylation without affecting total cellular EGFR protein levels. Cells were transfected and treated as in B and total cell lysate (TCL) was subjected to Western blot using the C-terminal Ab against the phosphorylated (Y1068) (*p < 0.05 TUSC3 vs. EV, Two-way ANOVA, n = 3) and unphosphorylated intracellular domain of the EGFR (n.s.). Data are calculated as in B. Similar results were obtained for the transferrin receptor (TFR/CD71). D., TUSC3 knock-down increases EGFR phosphorylation. HCT116 cells were transfected with siRNAs and analyzed for P-EGFR in TCL as in B (*p < 0.05 TUSC3-siRNA vs. control-siRNA, Two-way ANOVA, n = 4). E., Representative Western blots of the experiments in C-D are shown.