E-peptides inhibit myoblast differentiation.

<p><b>A–C</b>. C2C12 cells were grown to confluency and switched to differentiation media (Day 0). Media was changed every day and synthetic peptides (100 nM) were added to the fresh media. Quantitative RT-PCR was used to measure expression of differentiation markers: MyoD (<i>Myod</i>, A), Myogenin (<i>Myog</i>, B), Embryonic Myosin (<i>Myh3</i>, C). Expression of the markers at Days 1, 2, and 3 were compared to Day 0 to obtain fold change. Bars represent fold change means ± s.e.m. of N = 3 replicates. *, p<0.05 for fold change expression comparisons via 2-way ANOVA followed by a Bonferroni post-test. <b>D</b>. Synthetic EA and EB peptides were incubated with growth media and aliquots were taken at times indicated for immunoblotting analysis. <b>E–F</b>. Quantification of (D) and analysis of peptide half-life. Bars represent percent of original intensity ± s.e.m. of N = 3 replicates.</p

E-peptides augment IGF-IR activation and cell surface localization.

<p><b>A</b>. P6 cells overexpressing IGF-IR were treated with synthetic E-peptides with and without recombinant IGF-I for 15 minutes, and cell lysates were utilized for KIRA assays. Level of absorbance indicates the extent of IGF-IR phosphorylation. Bars represent means ± s.e.m. of N = 6 wells. <b>B</b>. OD 450 from A were compared to No Peptide for each IGF-I concentration, and the % change is graphed. <b>C</b>. P6 cells were treated as in A for a localization assay for times indicated, and biotin labeled before lysis. The optimal concentrations of E-peptides and IGF-I from the A were used (E-peptides 100 nM, IGF-I 10 nM). Surface IGF-IR was normalized to Total IGF-IR and compared to NoTx at t0 to get % IGF-IR on cell surface. Bars represent means ± s.e.m. of N = 6 wells. Samples were compared to no peptide (A and B,*), NoTx (C,*) or IGF-I (C,†) via 2-way ANOVA followed by a Bonferroni post-test. * or †, p<0.05; ***, p<0.001.</p

E-peptides affect IGF-IR downstream signaling.

<p><b>A–C</b>. C2C12 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045588#pone-0045588-g002" target="_blank">Figure 2</a>, with 0 nM or 2 nM IGF-I and optimal doses of the E-peptides (EA 1 µM, EB 10 nM) for 20 minutes. <b>B–C</b>. Quantification of Akt and ERK1/2 phosphorylation after EA (B) or EB (C) treatment. EA and EB alone are compared to NoTx, while IGF-I with E-peptides are compared to 2 nM IGF-I alone. Data are presented as the effect on phosphorylation after E-peptide treatment compared to NoTx or NoPeptide plus IGF-I. Bars represent means ± s.e.m. of N = 3–4 replicates. †, p<0.05 for comparisons of 0 nM IGF samples to their 2 nM IGF-I counterparts via student t-tests.</p

Synthetic E-peptide sequences.

<p><b>A</b>. Rodent <i>Igf1</i> 3′ splicing leads to two mRNA isoforms. While mature IGF-I is encoded by exons 3 and 4, the E-peptides are encoded by exons 4, 5, and/or 6. EA isoforms exclude exon 5, while EB isoforms retain exon 5, leading to an altered reading frame and earlier stop codon in exon 6. Exons not drawn to scale <b>B</b>. Synthetic E-peptide amino acid sequences. EA and EB are less than 50% identical. Scr = Scrambled peptide. * = potential glycosylation sites in EA. The portion of EB that corresponds to MGF is <u>underlined</u>.</p

EB increases in myoblast proliferation and migration are MAPK and IGF-IR signaling dependent.

<p><b>A</b>. C2C12 cells were plated in 96 well plates, starved for 6 hours, and treated with synthetic E-peptides. A BrdU plate assay was used to quantify proliferating cells, where increased absorbance is correlated with increased proliferation. Bars represent means ± s.e.m. of N = 10 wells. <b>B</b>. A similar BrdU assay was used to visualize the proliferating cells on slides. Cells were treated as above (EA and Scr 100 nM, EB and IGF 10 nM), fixed, and stained with BrdU and DAPI. Total cells and proliferating (BrdU positive) cells were counted from 3 10× fields for each slide, and bars represent means ± s.e.m. of N = 5 slides. For A and B, *, p<0.05; ***, p<0.001 for comparisons to NoTx via 1-way ANOVA followed by a Tukey post-hoc test. <b>C</b>. C2C12 cells were tested as in A, except an inhibitor of MEK, PD 098059 (PD, 50 µM) or IGF-IR (NVP, 100 nM) was included in the cell media. Bars represent means ± s.e.m. of N = 18 wells for No Inhibitor (No Inh) and N = 8 for with inhibitors. <b>D</b>. C2C12 cells were plated in the upper chamber of 24-well plate trans-well migration inserts in 0% serum media. Cells were allowed to migrate for 5 hours and stained with DAPI, imaged and counted. Synthetic E-peptides (100 nM) were added to upper and lower chambers with or without inhibitors (PD 50 µM, NVP 100 nM). Images were taken as in B, and bars represent means ± s.e.m. of N = 4 slides. For C and D, *, p<0.05; ***, p<0.001 for comparisons to NoTx via 2-way ANOVA followed by a Bonferroni post-test. †, p<0.05 for comparisons to No Inh via 2-way ANOVA followed by a Bonferroni post-test.</p

EA and EB increase MAPK signaling in C2C12 cells.

<p><b>A</b>. Cells were starved in media without serum, and treated with synthetic E-peptides at concentrations indicated for 20 minutes. Protein lysates were separated via SDS-PAGE and immunoblotted for Phosphorylated ERK 1 and 2 (P-ERK1/2), stripped, and blotted for Total ERK 1 and 2 (T-ERK1/2). <b>B–C</b>. Quantification of A. <b>D</b>. Cells were treated as above at optimal doses (EA and Scr 1 µM, EB 10 nM) for times indicated. <b>E–F</b> Quantification of C. NoTx at 30 minutes was included in each experiment for normalization between blots. For B–C and E–F, bars represent means ± s.e.m. of N = 3 replicates. *, p<0.05; ***, p<0.001, for comparisons to NoTx via 2-way ANOVA followed by a Bonferroni post-test.</p

Supplemental Material, DS1_VET_10.1177_0300985818776054 - Characteristics of the Epithelial-Mesenchymal Transition in Primary and Paired Metastatic Canine Mammary Carcinomas

<p>Supplemental Material, DS1_VET_10.1177_0300985818776054 for Characteristics of the Epithelial-Mesenchymal Transition in Primary and Paired Metastatic Canine Mammary Carcinomas by Talita M. M. Raposo-Ferreira, Becky K. Brisson, Amy C. Durham, Renee Laufer-Amorim, Veronica Kristiansen, Ellen Puré, Susan W. Volk, and Karin Sorenmo in Veterinary Pathology</p

Identification of prognostic collagen signatures and potential therapeutic stromal targets in canine mammary gland carcinoma

<div><p>Increasing evidence indicates that the tumor microenvironment plays a critical role in regulating the biologic behavior of breast cancer. In veterinary oncology, there is a need for improved prognostic markers to accurately identify dogs at risk for local and distant (metastatic) recurrence of mammary gland carcinoma and therefore would benefit from adjuvant therapy. Collagen density and fiber organization have been shown to regulate tumor progression in both mouse and human mammary tumors, with certain collagen signatures predicting poor outcomes in women with breast cancer. We hypothesized that collagen signatures in canine mammary tumor biopsies can serve as prognostic biomarkers and potential targets for treatment. We used second harmonic generation imaging to evaluate fibrillar collagen density, the presence of a tumor-stromal boundary, tumor associated collagen signatures (TACS) and individual collagen fiber characteristics (width, length and straightness) in grade I/II and grade III canine mammary tumors. Collagen density, as well as fiber width, length and straightness, were inversely correlated with patient overall survival time. Notably, grade III cases were less likely to have a tumor-stromal boundary and the lack of a boundary predicted poor outcome. Importantly, a lack of a defined tumor-stromal boundary and an increased collagen fiber width were associated with decreased survival even when tumor grade, patient stage, ovariohysterectomy status at the time of mammary tumor excision, and histologic evidence of lymphovascular invasion were considered in a multivariable model, indicating that these parameters could augment current methods to identify patients at high risk for local or metastatic progression/recurrence. Furthermore, these data, which identify for the first time, prognostic collagen biomarkers in naturally occurring mammary gland neoplasia in the dog, support the use of the dog as a translational model for tumor-stromal interactions in breast cancer.</p></div

Canine mammary tumor clinical parameters predict outcome.

<p>Kaplan-Meier survival curves and Cox regression univariate analysis was used to evaluate whether clinical parameters significantly impacted survival. A. Grade: grade I/II vs. grade III mammary tumors (p<0.001, hazard ratio (HR) 10.106, 95%CI 3.071–33.254). B. Stage: Stage 1/2 vs. 3/4 mammary tumors (p = 0.042, HR 2.778, 95%CI 1.039–7.420). C. Lymphovascular Invasion: Evidence vs. No Evidence (p<0.001, HR 7.462, 95%CI 2.359–23.603). D. Excision Completeness: Complete vs. Incomplete (p = 0.014, HR 4.255, 95%CI 1.335–13.558). E. OHE: At time of excision vs. prior to excision or not performed (p = 0.009, HR 0.133, 95%CI 0.029–0.604).</p

Collagen density predicts poor outcome in canine mammary tumors.

<p>Integrated density of collagen signal from SHG images was quantified using Fiji (Image J) software. Graph represents averages from 5 images per tumor from 11 grade I/II and 9 grade III mammary tumors. *p<0.05 via an unpaired Mann-Whitney test (A). Kaplan-Meier survival curve for 18 dogs with collagen integrated density higher or lower than the mean integrated density value (B). The log-rank test was used to evaluate whether the collagen density significantly impacted survival (p = 0.013, HR 4.099, 95%CI 0.860–19.520).</p