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: