Matrix-Gla protein promotes osteosarcoma lung metastasis and associates with poor prognosis
Osteosarcoma (OS) is the most prevalent osseous tumour in children and adolescents and, within this, lung metastases remain one of the factors associated with a dismal prognosis. At present, the genetic determinants driving pulmonary metastasis are poorly understood. We adopted a novel strategy usin...
Saved in:
| Published in: | The Journal of pathology Vol. 239; no. 4; pp. 438 - 449 |
|---|---|
| Main Authors: | , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Chichester, UK
John Wiley & Sons, Ltd
01-08-2016
Wiley Subscription Services, Inc |
| Subjects: | |
| Online Access: | Get full text |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | Osteosarcoma (OS) is the most prevalent osseous tumour in children and adolescents and, within this, lung metastases remain one of the factors associated with a dismal prognosis. At present, the genetic determinants driving pulmonary metastasis are poorly understood. We adopted a novel strategy using robust filtering analysis of transcriptomic profiling in tumour osteoblastic cell populations derived from human chemo‐naive primary tumours displaying extreme phenotypes (indolent versus metastatic) to uncover predictors associated with metastasis and poor survival. We identified MGP, encoding matrix‐Gla protein (MGP), a non‐collagenous matrix protein previously associated with the inhibition of arterial calcification. Using different orthotopic models, we found that ectopic expression of Mgp in murine and human OS cells led to a marked increase in lung metastasis. This effect was independent of the carboxylation of glutamic acid residues required for its physiological role. Abrogation of Mgp prevented lung metastatic activity, an effect that was rescued by forced expression. Mgp levels dramatically altered endothelial adhesion, trans‐endothelial migration in vitro and tumour cell extravasation ability in vivo. Furthermore, Mgp modulated metalloproteinase activities and TGFβ‐induced Smad2/3 phosphorylation. In the clinical setting, OS patients who developed lung metastases had high serum levels of MGP at diagnosis. Thus, MGP represents a novel adverse prognostic factor and a potential therapeutic target in OS. Microarray datasets may be found at: http://bioinfow.dep.usal.es/osteosarcoma/ Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. |
|---|---|
| Bibliography: | American Society for Bone and Mineral Research (ASBMR) Formación de Profesorado Universitario (FPU) Unión Temporal de Empresas (UTE) istex:D5C93A40B8F546460DA5E8C828CC4E6F672ED83D Mari Paz Jimenez Casado Foundation RTICC - No. RD12/0036/0040; No. RD12/0036/0068; No. PI042282 Redes Temáticas de Investigación Cooperativa en Cáncer (RETICC) Asociación Española Contra el Cáncer (AECC) Ministerio de Economía y Competitividad (MINECO) - No. PTQ-10-04248 Spanish Research Sarcoma Group Instituto de Salud Carlos III GEIS - No. FIS PI13/01476 Ministerio de Educación, Cultura y Deporte - No. AP2010-2197 ArticleID:PATH4740 ark:/67375/WNG-CMC9S20P-B Appendix S1. Supplementary materials and methodsAppendix S2. Supplementary figure legendsFigure S1. In vitro cell proliferation and in vivo tumour growth of Mgp-expressing cells. (a) In vitro cell growth kinetics of murine (P1.15) and human (143B) cells overexpressing murine mgp-myc and human MGP, respectively, evaluated by MTS assay. (b) In vivo tumour growth after subcutaneous injection of cells overexpressing murine mgp-myc in p1.15 cells and human MGP in 143B cells by retroviral transduction in the flanks of athymic nude miceFigure S2. Mgp confers lung prometastatic activity in murine osteosarcoma RF379 cells. (a) Western blot analysis of Mgp levels after retroviral transduction of a murine Mgp-myc sequence using an anti-myc antibody in the RF379 murine osteosarcoma cell line. (b) (Left panel) Bioluminescence measurements of the thoracic cavity in athymic nude animals (eight mice/group) after intratibial (i.t.) injection of control and Mgp-myc RF379 cells at day 15 post-injection; (right panel) representative images of bioluminescence; arrow, lung metastasis. (c) Quantification by image analysis of nodule area and number of nodules in H&E lung sections derived from RF379 cells. (d) Representative H&E lung sections of lungs derived from animals injected with RF379 cells. Bar = 4 mmFigure S3. In vitro cell proliferation and in vivo tumour growth of Mgp-silenced cells. (a) In vitro proliferation assay of P1.15 cells with silenced Mgp assessed by MTS assay. (b) In vivo tumour growth after subcutaneous injection of cells with silenced levels of Mgp following lentiviral transduction of different shRNAs and control cells in the flanks of athymic nude miceFigure S4. γ-Carboxylation of glutamic residues does not affect tumour growth or metastasis. (a) Outline of the experiment of treatment starting 7 days post-injection. (b) (Upper panel) Quantification of the number and area of the lung nodules in H&E sections of athymic nude animals after intratibial (i.t.) injection of mgp-myc P1.15 cells; the mice were subsequently allocated to three groups (eight mice/group), control (vehicle), mice treated with vitamin K (15 mg/kg) twice weekly and warfarin-treated (in drinking water at 7.5 g/l for 2 days, 5 g/l for 2 days, 7.5 g/l for 2 days and water for 2 days) at day 18 post-injection; (lower panel) representative images of the excised lungs. (c) (Upper panel) Quantification of the osteolytic area in the hind limbs of treated animals 18 days post-injection; (lower panel) representative images of X-ray images of the hind limbs. (d) Outline of the experiment of treatment starting 2 days post-injection. (e) Quantification of bioluminescence imaging of the thoracic cavity (left panels) and the hind limbs (right panels) at days 7 and 12 post-injection. (f) Quantification of the number and area of metastatic nodules in the lungs of treated animals at day 14 post-injection. (g) (Upper panel) Quantification of the osteolytic area in the hind limbs of treated animals 14 days post-injection; (lower panel) representative images of X-ray images of the hind limbsFigure S5. Activation of BMP-4 signalling in cells with different Mgp levels. Western blot analysis of phosphorylated Smad1/5/8 and total Smad in P1.15 cells overexpressing Mgp (mgp-myc) (left panel), with silenced levels of Mgp (sh-mgp) and rescued levels (sh-Δ-Mgp) (right panel) treated with BMP-4 (80 ng/ml) for the indicated times or 30 minTable S1. Clinical features of OS patients from whom biopsy material was obtained for the transcriptomic analysisTable S2. The top-scoring genes of the screening strategyTable S3. Clinical features of OS patients from whom sera were extracted for MGP analysis ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0022-3417 1096-9896 |
| DOI: | 10.1002/path.4740 |