Fig 2: 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 3: Integrated genomic and proteomic analysis of gallbladder cancer. (a) The heat map represents somatic mutation landscape in gallbladder cancer patients (n = 17) and primary tumor derived cancer cell lines (n = 5) using whole exome sequencing. Clinicopathological features such as gender, gallstones, tumor location and liver involvement are shown. The gray solid boxes denote females, presence of gallstone, tumor location (neck) and positive for liver involvement. The white box denotes males, absence of gallstones, tumor location (body) and negative for liver involvement. The genes are arranged in decreasing order of their frequency. Black solid box indicates the presence of mutation in the heatmap. Mutation frequencies of the genes mentioned are shown in our study, COSMIC-GBC and Li et al. study. The transition to transversion ratio is shown in percentage for each patient indicated by different shades (Black denotes transversion and gray denotes transition). Somatic mutation rate/30 Mb is derived from whole exome sequencing data is indicated by white line. Overall copy number changes derived from whole exome sequencing data. The horizontal-axis is represented by a score of segment gain or segment loss (SGOL score) while the vertical-axis represents the chromosomal positions. Copy number gain is indicated by red with positive SGOL score while copy number loss is indicated by blue with a negative SGOL score. Representative cancer-associated genes are annotated in their respective amplified/deleted regions. (b) Schematic representation of ERBB family mutation validation by Sanger sequencing in an additional set of 27 samples. Solid box indicate presence for mutation in the respective samples, white boxes indicates no event. (c) RTK array analysis of gallbladder cancer cells (OCUG1, TGBC2TKB, G415, and NOZ) for 10 min exposure of blot is shown. Each RTK is spotted in duplicate and the pair of dots in each corner of the membrane corresponds to positive and negative control. Tyrosine phosphorylation of EGFR (ERBB1) and ERBB2 were observed consistently, indicated by arrow. D) Immunohistochemistry was performed for ERBB2 expression in tumor samples (n = 25). Representative images of IHC stained photomicrographs from tumor and normal samples are shown. Brown color indicates positive expression. The corresponding H/E images are indicated in the upper panel. Below table indicates the quantification of ERBB2 immunostaining data. [Color figure can be viewed at http://wileyonlinelibrary.com]

Fig 4: Loss of stromal PTEN increases fibroblast EGF ligand expression and associated ErbB signaling activity in neighboring epithelium. a Areg, Btc, Egf, Ereg, Hbegf, Nrg1, Nrg4 and Tgfa mRNA in immortalized control (Ptenfl/fl) and PTEN-null (Fsp-cre;Ptenfl/fl) MMFs. Bars represent mean expression of technical replicates relative to Gapdh ± s.e.m. Nrg2 and Nrg3 were not detectable (N.D.). b Western blot for PTEN, TGF-a and GAPDH in the immortalized control (Ptenfl/fl) and PTEN-null (Fsp-cre;Ptenfl/fl) MMFs. c Representative phospho-ErbB2 (Y1221/22) immunostaining and quantification (mean ± s.e.m.) of mammary epithelium in Ptenfl/fl and Fsp-cre;Ptenfl/fl mice. P value determined by two-tailed Mann–Whitney (Ptenfl/fl n = 61 fields/4 mice, Fsp-cre;Ptenfl/fl n = 71 fields/4 mice). Scale bar = 20 µm. d Representative phospho-EGFR (Y1068) immunostaining and quantification (mean ± s.e.m.) of mammary epithelium in Ptenfl/fl and Fsp-cre;Ptenfl/fl mice. P value determined by Welch’s t-test (Ptenfl/fl n = 48 fields/3 mice, Fsp-cre;Ptenfl/fl n = 48 fields/3 mice). Scale bar = 20 µm

Fig 5: Blocking HER1/HER4, ER and ERK1/2 signaling inhibits the proliferation of ER+ cells with HER3T355I(A) T47D cells (HER3WT and HER3T355I) were plated and treated with vehicle (DMSO), lapatinib (1 µM), fulvestrant (1 µM), SCH772984 (1 µM), or indicated combinations. Media and inhibitors were replenished every second day and stained with crystal violet. The value is represented as mean ± SEM (n = 3 independent experiments performed in triplicate). *P < 0.05 versus WT, **P < 0.05 versus treated groups as indicated. (B) MCF-7 cells (HER3WT and HER3T355I) were plated and treated with vehicle (DMSO), lapatinib (1 µM), fulvestrant (1 µM), SCH772984 (1 µM) or indicated combinations. Intensities were analyzed and represented as mean ± SEM (n = 3 independent experiments performed in triplicate). *P < 0.05 versus WT, **P < 0.05 versus respective treatment groups as shown. (C–D) ER+ T47D and MCF-7 cells with HER3WT and HER3T355I expression were seeded on a basement membrane of matrigel ± vehicle (DMSO), lapatinib (1 µM), fulvestrant (1 µM), SCH772984 (1 µM) or indicated combinations. The average size of each cellular structure was quantified using ImageJ and expressed relative to respective control. The value is represented as mean of areas ± SEM (n = 3 independent experiments performed in triplicate). *P < 0.05 versus WT, **P < 0.05 versus respective treatment groups as indicated.