Journal: Breast Cancer Research : BCR

Article Title: Parity predisposes breasts to the oncogenic action of PAPP-A and activation of the collagen receptor DDR2

doi: 10.1186/s13058-019-1142-z

Figure Lengend Snippet: PAPP-A promotes DDR2 activation through collagen mRNA stabilization and IGF signaling in vitro. a Schematic of the proposed mechanism of activation of DDR2/Snail signaling axis of invasion . b Representative images of Snail IHC (Snail: red; nuclei: blue) of virgin, involuting, or post-partum mammary glands from non-transgenic or PAPP-A transgenic mice. n = 5 mice per group. Quantification of Snail IHC is shown, n = 5 mice per group. Mean ± SEM, unpaired t test with Welch’s correction (comparisons between the score 3 group only). * p < 0.05, ** p < 0.005. c Immunoblot of indicated markers in MCF-7 or MCF-7 PAPP-A cells co-cultured with and without collagen. Scale bar 100 μm. d Representative images of 48 h Transwell in vitro invasion assays of MCF-7 and MCF-7 PAPP-A cells. Experiments repeated in technical triplicate. Scale bar 100 μm. Quantification is shown, unpaired t test with Welch’s correction. * p < 0.05. e Relative fold change of col1a1 mRNA transcript in MCF-7 PAPP-A over MCF-7 cells measured by real-time qPCR and normalized to actin. Mean + SEM, triplicate experiment. Unpaired t test with Welch’s correction. * p < 0.05. f Relative fold change of col1a1 mRNA transcript in MCF-7 cells treated in vitro with or without PAPP-A media for 24 h. PAPP-A media concentration is at 10 ng/mL of PAPP-A protein (quantified by ELISA). Measured by real-time qPCR and normalized to actin. Mean + SEM, triplicate experiment. Unpaired t test with Welch’s correction. ** p < 0.005. g Immunoblot of LARP6 in MCF-7 cells treated in vitro with or without PAPP-A media for 24 h. PAPP-A media concentration is at 10 ng/mL of PAPP-A protein (quantified by ELISA). h Representative immunoblot of LARP6 in the mammary glands from non-transgenic or PAPP-A transgenic mice during virgin, involution, or late post-partum. n = 5 mice per group. i Immunoblot of rIGFBP-5 following a 3-h incubation in culture media from MCF-7 (CTL), MCF-7 PAPP-A (PA), or MCF-7 overexpressing proteolytic-dead mutant PAPP-A E483Q (E483Q). Immunoblot of PAPP-A secreted in culture media from MCF-7, MCF-7 PAPP-A , and MCF-7 PAPP-A/E483Q cells. j Immunoblot of MCF-7 cells treated in vitro with culture media from MCF-7 (CTL), MCF-7 PAPP-A (PA), or MCF-7 PAPP-A/E483Q (E483Q) cells for 24 h. k Immunoblot of DDR2 and phospho-DDR2 in MCF-7 and MCF-7 PAPP-A cells treated in vitro with recombinant IGF-1 at 10 nM for indicated time points

Article Snippet: Thirty micrograms total protein in 1× Laemmli buffer per sample was separated on a 10% SDS-glycine polyacrylamide gels ran at 80 V for 30 min and 200 V for 45 min. Proteins were transferred to nitrocellulose membranes (GE Healthcare) for 1.5 h at 85 V. Membranes were blocked in 5% milk in TBS-T and incubated on a rotator overnight at 4 °C in the following primary antibodies: DDR2 1:250 (Cell Signaling Technology #12133S), p-DDR2 Y740 1:600 (R&D Systems, #MAB25382), Snail 1:1000 (Cell Signaling Technology L70G2 #3895), Larp6 1:600 (Abnova, #H00055323-B01P), IGFBP-5 1:1000 (EMD Millipore, #06-110), PAPP-A 1:200 (Santa Cruz Biotechnology, # sc-50518), phospho-Akt (Ser473) 1:1000 (Cell Signaling Technology 587F11 #4051), Akt 1:10,000 (Cell Signaling Technology #9272), and actin 1:10,000 (EMB Millipore #MAB1501R).

Techniques: Activation Assay, In Vitro, Transgenic Assay, Western Blot, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Mutagenesis, Recombinant

Journal: Breast Cancer Research : BCR

Article Title: Parity predisposes breasts to the oncogenic action of PAPP-A and activation of the collagen receptor DDR2

doi: 10.1186/s13058-019-1142-z

Figure Lengend Snippet: Loss of DDR2 abolishes cell invasion, Snail expression, and tumor growth by PAPP-A. a Immunoblot of indicated markers in MCF-7 PAPP-A CTL and MCF-7 PAPP-A DDR2 −/− cells. b Representative images of 48 h Transwell in vitro invasion assays of MCF-7 CTL, MCF-7 DDR2 −/− , MCF-7 PAPP-A CTL, and MCF-7 PAPP-A DDR2 −/− cells. Experiments repeated in technical triplicate. Scale bar 100 μm. Quantification is shown, unpaired t test with Welch’s correction. * p < 0.05. c Immunoblot of indicated markers in MCF-7 CTL or MCF-7 DDR2 −/− cells treated with or without PAPP-A media for 24 h. d Relative tumor growth of MCF-7, MCF-7 PAPP-A , and MCF-7 PAPP-A DDR2 −/− xenografts in a Matrigel-collagen mixture. n = 5 mice per group, ten tumors each. Each point represents the average of five mice, mean ± SEM. Unpaired t test at end point (MCF-7 PAPP-A vs. MCF-7 PAPP-A DDR2 −/− ). * p < 0.05 e Representative images of Snail IHC on MCF-7, MCF-7 PAPP-A , and MCF-7 PAPP-A DDR2 −/− xenograft tumors in Matrigel-collagen mixture. n = 3 mice, six tumors total per group. Scale bar 100 μm. f Representative images of Masson’s trichrome collagen stain (blue) and second-harmonic generation (SHG) imaging of collagen (lower panels). Yellow dotted lines indicate the mammary tumor borders. Corresponding regions of interest (ROI) on magnified histological sections of MCF-7, MCF-7 PAPP-A , or MCF-7 PAPP-A DDR2 −/− xenograft tumors in a Matrigel-collagen mixture. n = 3 mice per group. Scale bar 100 μm. g Quantification of TACS3 per total curvelets analyzed in MCF-7, MCF-7 PAPP-A , or MCF-7 PAPP-A DDR2−/− xenograft tumors in a Matrigel-collagen mixture using the CurveAlign software. TACS3 characterized as curvelet angles 60–90 relative to the ductal border. n = 3 mice, six tumors total per group. Mean + SEM, unpaired t test with Welch’s correction (comparisons between TACS3 group only, MCF-7 PAPP-A vs. MCF-7 PAPP-A DDR2 −/− ). * p < 0.05. h Immunoblot of indicated markers in MCF-7 PAPP-A cells treated for 48 h at the indicated concentrations of PQ401 and imatinib. i Proliferation assay of MCF-7 and MCF-7 PAPP-A cells treated with imatinib at the indicated concentrations for 48 h. j Proliferation assay of MCF-7 and MCF-7 PAPP-A cells treated with PQ401 at the indicated concentrations for 48 h. k Proliferation assay of MCF-7 and MCF-7 PAPP-A cells treated with PQ401 at 7.5 μM in addition to the indicated concentrations of imatinib for 48 h

Article Snippet: Thirty micrograms total protein in 1× Laemmli buffer per sample was separated on a 10% SDS-glycine polyacrylamide gels ran at 80 V for 30 min and 200 V for 45 min. Proteins were transferred to nitrocellulose membranes (GE Healthcare) for 1.5 h at 85 V. Membranes were blocked in 5% milk in TBS-T and incubated on a rotator overnight at 4 °C in the following primary antibodies: DDR2 1:250 (Cell Signaling Technology #12133S), p-DDR2 Y740 1:600 (R&D Systems, #MAB25382), Snail 1:1000 (Cell Signaling Technology L70G2 #3895), Larp6 1:600 (Abnova, #H00055323-B01P), IGFBP-5 1:1000 (EMD Millipore, #06-110), PAPP-A 1:200 (Santa Cruz Biotechnology, # sc-50518), phospho-Akt (Ser473) 1:1000 (Cell Signaling Technology 587F11 #4051), Akt 1:10,000 (Cell Signaling Technology #9272), and actin 1:10,000 (EMB Millipore #MAB1501R).

Techniques: Expressing, Western Blot, In Vitro, Staining, Imaging, Software, Proliferation Assay

Journal: Breast Cancer Research : BCR

Article Title: Parity predisposes breasts to the oncogenic action of PAPP-A and activation of the collagen receptor DDR2

doi: 10.1186/s13058-019-1142-z

Figure Lengend Snippet: PAPP-A/SNAI1/COL1A1 gene signature correlates with distant metastasis in breast cancer patients. a Heatmap of patients clustered based on their score for the PAPP-A/SNAI1/COL1A1 signature (categorized according to the mean score value). Each row represents one patient, n = 327. b Kaplan-Meier curve for time to distant metastasis according to the PAPP-A/SNAI1/COL1A1 score as defined in a . Number of patients at risk at each time point over a 150-month period is recorded below in table. c Heatmap representing the relative expression of a selected panel of 78 genes (FDR < .05) in the PAPP-A/SNAI1/COL1A1 -low and PAPP-A/SNAI1/COL1A1 -high patient populations. Genes are organized by pathways, as labeled adjacent to the gene names. The red text highlights relevant genes investigated in this study ( PAPP-A , DDR2 , SNAI1 , COL1A1 , and LARP6 ). Each column represents a patient, and each row represents a gene. d Chart of GSEA pathways significantly enriched in the PAPP-A/SNAI1/COL1A1 -high patient population. Each bar represents the normalized enrichment score of the indicated pathway

Article Snippet: Thirty micrograms total protein in 1× Laemmli buffer per sample was separated on a 10% SDS-glycine polyacrylamide gels ran at 80 V for 30 min and 200 V for 45 min. Proteins were transferred to nitrocellulose membranes (GE Healthcare) for 1.5 h at 85 V. Membranes were blocked in 5% milk in TBS-T and incubated on a rotator overnight at 4 °C in the following primary antibodies: DDR2 1:250 (Cell Signaling Technology #12133S), p-DDR2 Y740 1:600 (R&D Systems, #MAB25382), Snail 1:1000 (Cell Signaling Technology L70G2 #3895), Larp6 1:600 (Abnova, #H00055323-B01P), IGFBP-5 1:1000 (EMD Millipore, #06-110), PAPP-A 1:200 (Santa Cruz Biotechnology, # sc-50518), phospho-Akt (Ser473) 1:1000 (Cell Signaling Technology 587F11 #4051), Akt 1:10,000 (Cell Signaling Technology #9272), and actin 1:10,000 (EMB Millipore #MAB1501R).

Techniques: Expressing, Labeling

Journal: Breast Cancer Research : BCR

Article Title: Parity predisposes breasts to the oncogenic action of PAPP-A and activation of the collagen receptor DDR2

doi: 10.1186/s13058-019-1142-z

Figure Lengend Snippet: Model of PAPP-A-driven PABC. Schematic of our current understanding of PAPP-A-driven PABC, where red boxes indicate factors that have been established to activate the pathway, blue boxes indicate factors inhibiting the pathway, and green boxes factors that are hypothesized to affect the pathway. In this model, elevated content in collagen is necessary for PAPP-A to cleave IGFBP-5 in the mammary gland. The elevated level of collagen can be provided through distinct avenues including involution and, as demonstrated in the current study, post-partum environment. We postulate that high breast density may contribute the activity of PAPP-A. Conversely, extended breastfeeding appears to inhibit PAPP-A. Upon activation of PAPP-A and cleavage of IGFBP-5, free IGFs are released and able to bind and activate the IGF receptor, which results in increased IGF signaling, increased collagen deposition, and extended involution, in the case of an involuting mammary gland. We show that LARP6 is activated following PAPP-A overexpression and as a chaperone of the mRNA of collagen, LARP6 contributes to the elevation in collagen deposition in PAPP-A-driven mammary tumors. In addition, the collagen receptor DDR2 is activated, which leads to the DDR2/Snail axis of metastasis reported by others. In a mechanism that remains unclear, DDR2 promotes the formation of TACS3 collagen, which facilitates metastasis

Article Snippet: Thirty micrograms total protein in 1× Laemmli buffer per sample was separated on a 10% SDS-glycine polyacrylamide gels ran at 80 V for 30 min and 200 V for 45 min. Proteins were transferred to nitrocellulose membranes (GE Healthcare) for 1.5 h at 85 V. Membranes were blocked in 5% milk in TBS-T and incubated on a rotator overnight at 4 °C in the following primary antibodies: DDR2 1:250 (Cell Signaling Technology #12133S), p-DDR2 Y740 1:600 (R&D Systems, #MAB25382), Snail 1:1000 (Cell Signaling Technology L70G2 #3895), Larp6 1:600 (Abnova, #H00055323-B01P), IGFBP-5 1:1000 (EMD Millipore, #06-110), PAPP-A 1:200 (Santa Cruz Biotechnology, # sc-50518), phospho-Akt (Ser473) 1:1000 (Cell Signaling Technology 587F11 #4051), Akt 1:10,000 (Cell Signaling Technology #9272), and actin 1:10,000 (EMB Millipore #MAB1501R).

Techniques: Activity Assay, Activation Assay, Over Expression

Journal: Breast Cancer Research : BCR

Article Title: Parity predisposes breasts to the oncogenic action of PAPP-A and activation of the collagen receptor DDR2

doi: 10.1186/s13058-019-1142-z

Figure Lengend Snippet:

Article Snippet: Thirty micrograms total protein in 1× Laemmli buffer per sample was separated on a 10% SDS-glycine polyacrylamide gels ran at 80 V for 30 min and 200 V for 45 min. Proteins were transferred to nitrocellulose membranes (GE Healthcare) for 1.5 h at 85 V. Membranes were blocked in 5% milk in TBS-T and incubated on a rotator overnight at 4 °C in the following primary antibodies: DDR2 1:250 (Cell Signaling Technology #12133S), p-DDR2 Y740 1:600 (R&D Systems, #MAB25382), Snail 1:1000 (Cell Signaling Technology L70G2 #3895), Larp6 1:600 (Abnova, #H00055323-B01P), IGFBP-5 1:1000 (EMD Millipore, #06-110), PAPP-A 1:200 (Santa Cruz Biotechnology, # sc-50518), phospho-Akt (Ser473) 1:1000 (Cell Signaling Technology 587F11 #4051), Akt 1:10,000 (Cell Signaling Technology #9272), and actin 1:10,000 (EMB Millipore #MAB1501R).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Parity predisposes breasts to the oncogenic action of PAPP-A and activation of the collagen receptor DDR2

doi: 10.1186/s13058-019-1142-z

Figure Lengend Snippet:

Article Snippet: Thirty micrograms total protein in 1× Laemmli buffer per sample was separated on a 10% SDS-glycine polyacrylamide gels ran at 80 V for 30 min and 200 V for 45 min. Proteins were transferred to nitrocellulose membranes (GE Healthcare) for 1.5 h at 85 V. Membranes were blocked in 5% milk in TBS-T and incubated on a rotator overnight at 4 °C in the following primary antibodies: DDR2 1:250 (Cell Signaling Technology #12133S), p-DDR2 Y740 1:600 (R&D Systems, #MAB25382), Snail 1:1000 (Cell Signaling Technology L70G2 #3895), Larp6 1:600 (Abnova, #H00055323-B01P), IGFBP-5 1:1000 (EMD Millipore, #06-110), PAPP-A 1:200 (Santa Cruz Biotechnology, # sc-50518), phospho-Akt (Ser473) 1:1000 (Cell Signaling Technology 587F11 #4051), Akt 1:10,000 (Cell Signaling Technology #9272), and actin 1:10,000 (EMB Millipore #MAB1501R).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Expression of the Discoidin Domain Receptor Family Depended on Glucose and Their High Expression in Arterial Tissues in the Rat Model of Type 2 Diabetes.

doi: 10.1248/bpb.b24-00245

Figure Lengend Snippet: Fig. 4. Expression Levels of DDR2, DDR1, EGFR, and α-SMA in Arterial Tissues of Control and Diabetic Rats

Article Snippet: Reagents, Antibodies, and Western Blotting A polyclonal antibody against human DDR2 (AF2538) was purchased from R&D systems.

Techniques: Expressing, Control

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. Western blot analysis of DDR2 and Twist1 protein expression in established ovarian cancer cell lines B. A2780 or ES2 ovarian cancer cell lines were infected with lentivirus expressing the indicated shRNAi. Western blotting preformed using the indicated antibodies. C. Western blot of indicated proteins from whole cell lysates of OVCAR3 cells treated with 2ng/mL TGF-B to biochemically induce EMT for indicated times. D. ES2 ovarian cancer spheroids expressing the indicated shRNAi labeled with CMTCX dye (red) were added to a confluent monolayer of primary mesothelial cells that were labeled with CMFDA dye (green) and monitored over 7 hours. Images show representative mesothelial clearance at 0 and 7 hours. E. Quantification of experiment in D. >5 spheroids averaged per condition. Error bars denote SEM. **p<0.01, Student’s t test.

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Western Blot, Expressing, Infection, Labeling

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A, B . Matrigel invasion assays. A. ES2 and B. A2780 cells were depleted of DDR2 or control (SCRM) and added to Matrigel coated Boyden chambers (8um pore size).Representative images and quantification (Means and S.D. for 3 individual chambers per group plotted as number of cells invading through to lower surface 4 per high powered field (hpf)). Repeated with statistical significance in 3 independent experiments. Unpaired t-test, **p<0.01, ***p<0.001. C. Collagen I invasion assays of ES2 shDDR2 or control (shSCRM) cells. Representative photographs taken at day 3 and day 6. D. Quantification of collagen invasion. 20 cells per well, 3 wells per condition quantified. Mean and s.d. are shown, unpaired t-test **p<0.01

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Control, Pore Size

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. ES2 or B. A2780 cells depleted of DDR2 or control (SCRM) were added to collagen I coated (+) or uncoated (−) plates for 6 hours. Western blotting with the indicated antibodies was performed on cell extract. Data representative of 3 independent experiments. C, D. Q-PCR analysis of mRNA isolated from C. ES2 or D. A2780 shRNA-depleted of DDR2 or transduced with scrambled control (SCRM). Three replicates of each gene were done for each experiment. Data are representative of three independent experiments. *p<0.05 student’s t test E. Gelatin zymography was conducted on the supernatants from ES2 cells depleted of DDR2 or control (SCRM) that had been plated on collagen I coated (+) or uncoated (−) plates for 24 hours. Representative zymogram and quantitation of 3 independent experiments shown.

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Control, Western Blot, Isolation, shRNA, Transduction, Zymography, Quantitation Assay

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. ES2 or A2780 shRNAi-depleted of DDR2 or transduced with scrambled control (SCRM) were cultured for 6 hours under serum free conditions on 2mg/mL of collagen I, and cell free media was incubated with recombinant human fibronectin (FN). Extent of cleavage was measured at time intervals of 0.25, 1, and 16 hours by SDS Page followed by coomassie staining. B, C. Quantification of cleavage of intact FN by B. A2780 or C. ES2 plotted as a fraction of remaining intact FN as compared to control. Amount of remaining intact FN (~220 kDa) quantified by densitometry. Data from 3 independent experiments are plotted. D. Quantification of cell area when plated on fibronectin coated hydrogels for 3 hrs. Dots represent individual cell areas, with box plot overlay depicting the median and interquartile ranges of area spread. n>200 cells quantified for each group. E. Representative images of ES2 shSCRM or shDDR2 cells plated on fibronectin coated hydrogels.

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Transduction, Control, Cell Culture, Incubation, Recombinant, SDS Page, Staining

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. A2780 cells stably expressing shRNA targeting sequence for scramble control (shSCRM) or DDR2 (shDDR2). Beta-Actin was used as a protein loading control. B–D. Representative images and quantification of Balb/c Nu mice injected IP with 7.5×106 A2780 shSCRM or shDDR2 cells. Tumor burden was assessed at 14 days post injection in the B entire peritoneal cavity C. Mesentary only D. Omentum only. N=10 mice per group. Means and s.d. Unpaired t-test, *P<0.05, **P≤0.01

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Stable Transfection, Expressing, shRNA, Sequencing, Control, Injection

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. Overall survival of advanced-stage III, IV serous ovarian cancers by Kaplan-Meier methods and log rank test. MST: Median survival time. B. Representative images of DDR2 immunohistochemical staining in i) early stage IA primary tumor ii) advanced stage IV primary tumor iii)mesenteric metastatic implants C. Classification of immunohistochemical analysis of DDR2 staining in normal (benign) ovary, human primary ovarian tumors, and metastatic implants.

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Immunohistochemical staining, Staining

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. Western blot of patient derived primary ovarian tumor (POV) cells for expression of indicated proteins. B, C . A subset of POV cells were added to Matrigel coated Boyden chambers to assay invasion. B. Representative images of inserts and C. quantification plotted as number of cells invading through to lower surface per high powered field (hpf). Means and s.d. are shown for 3 individual chambers per group, 4 hpf per chamber. D. Proposed role of DDR2 in ovarian cancer metastasis

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Western Blot, Derivative Assay, Expressing