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Posthumadeboer J.,VU University Amsterdam | Piersma S.R.,VU University Amsterdam | Pham T.V.,VU University Amsterdam | Van Egmond P.W.,VU University Amsterdam | And 10 more authors.
British Journal of Cancer

Background:Osteosarcoma (OS) is the most common bone tumour in children and adolescents. Despite aggressive therapy regimens, treatment outcomes are unsatisfactory. Targeted delivery of drugs can provide higher effective doses at the site of the tumour, ultimately improving the efficacy of existing therapy. Identification of suitable receptors for drug targeting is an essential step in the design of targeted therapy for OS.Methods:We conducted a comparative analysis of the surface proteome of human OS cells and osteoblasts using cell surface biotinylation combined with nano-liquid chromatography - tandem mass spectrometry-based proteomics to identify surface proteins specifically upregulated on OS cells. This approach generated an extensive data set from which we selected a candidate to study for its suitability as receptor for targeted treatment delivery to OS. First, surface expression of the ephrin type-A receptor 2 (EPHA2) receptor was confirmed using FACS analysis. Ephrin type-A receptor 2 expression in human tumour tissue was tested using immunohistochemistry. Receptor targeting and internalisation studies were conducted to assess intracellular uptake of targeted modalities via EPHA2. Finally, tissue micro arrays containing cores of human OS tissue were stained using immunohistochemistry and EPHA2 staining was correlated to clinical outcome measures.Results:Using mass spectrometry, a total of 2841 proteins were identified of which 156 were surface proteins significantly upregulated on OS cells compared with human primary osteoblasts. Ephrin type-A receptor 2 was highly upregulated and the most abundant surface protein on OS cells. In addition, EPHA2 was expressed in a vast majority of human OS samples. Ephrin type-A receptor 2 effectively mediates internalisation of targeted adenoviral vectors into OS cells. Patients with EPHA2-positive tumours showed a trend toward inferior overall survival.Conclusion:The results presented here suggest that the EPHA2 receptor can be considered an attractive candidate receptor for targeted delivery of therapeutics to OS. © 2013 Cancer Research UK. All rights reserved. Source

De Boer J.P.,VU University Amsterdam | van Egmond P.W.,Medisch Centrum Alkmaar MCA | Helder M.N.,VU University Amsterdam | Helder M.N.,Research Institute MOVE Skeletal Tissue Engineering Group Amsterdam STEGA | And 8 more authors.

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Despite aggressive therapy, survival outcomes remain unsatisfactory, especially for patients with metastatic disease or patients with a poor chemotherapy response. Chemoresistance contributes to treatment failure. To increase the efficacy of conventional chemotherapy, essential survival pathways shouldbe targeted concomitantly. Here, we performed a loss-of-function siRNA screen of the human kinome in SaOS-2 cells to identify critical survival kinases after doxorubicin treatment. Gene silencing of JNK-interacting-protein-1 (JIP1) elicited the most potent sensitisation to doxorubicin. This candidate was further explored as potential target for chemosensitisation in OS. A panel of OS cell lines and human primary osteoblasts was examined for sensitisation to doxorubicin using small molecule JIP1-inhibitor BI-78D3. JIP1 expression and JIP1-inhibitor effects on JNKsignalling were investigated by Western blot analysis. JIP1 expression in human OS tumours was assessed by immunohistochemistry on tissue micro arrays. BI-78D3 blocked JNK-signalling and sensitised three out of four tested OS cell lines, but not healthy osteoblasts, to treatment with doxorubicin. Combination treatment increased the induction of apoptosis. JIP1 was found to be expressed in two-thirds of human primary OS tissue samples. Patients with JIP1 positive tumours showed a trend to inferior overall survival. Collectively, JIP1 appears a clinically relevant novel target in OS to enhance the efficacy of doxorubicin treatment by means of RNA interference or pharmacological inhibition. © Posthuma De Boer et al. Source

PosthumaDeBoer J.,VU University Amsterdam | Wurdinger T.,VU University Amsterdam | Wurdinger T.,Harvard University | Graat H.C.A.,VU University Amsterdam | And 6 more authors.
BMC Cancer

Background: The use of radiotherapy in osteosarcoma (OS) is controversial due to its radioresistance. OS patients currently treated with radiotherapy generally are inoperable, have painful skeletal metastases, refuse surgery or have undergone an intralesional resection of the primary tumor. After irradiation-induced DNA damage, OS cells sustain a prolonged G 2cell cycle checkpoint arrest allowing DNA repair and evasion of cell death. Inhibition of WEE1 kinase leads to abrogation of the G 2arrest and could sensitize OS cells to irradiation induced cell death.Methods: WEE1 expression in OS was investigated by gene-expression data analysis and immunohistochemistry of tumor samples. WEE1 expression in OS cell lines and human osteoblasts was investigated by Western blot. The effect of WEE1 inhibition on the radiosensitivity of OS cells was assessed by cell viability and caspase activation analyses after combination treatment. The presence of DNA damage was visualized using immunofluorescence microscopy. Cell cycle effects were investigated by flow cytometry and WEE1 kinase regulation was analyzed by Western blot.Results: WEE1 expression is found in the majority of tested OS tissue samples. Small molecule drug PD0166285 inhibits WEE1 kinase activity. In the presence of WEE1-inhibitor, irradiated cells fail to repair their damaged DNA, and show higher levels of caspase activation. The inhibition of WEE1 effectively abrogates the irradiation-induced G 2arrest in OS cells, forcing the cells into premature, catastrophic mitosis, thus enhancing cell death after irradiation treatment.Conclusion: We show that PD0166285, a small molecule WEE1 kinase inhibitor, can abrogate the G 2checkpoint in OS cells, pushing them into mitotic catastrophe and thus sensitizing OS cells to irradiation-induced cell death. This suggests that WEE1 inhibition may be a promising strategy to enhance the radiotherapy effect in patients with OS. © 2011 PosthumaDeBoer et al; licensee BioMed Central Ltd. Source

Jurgens W.J.F.M.,VU University Amsterdam | Jurgens W.J.F.M.,Research Institute MOVE Skeletal Tissue Engineering Group Amsterdam STEGA | Lu Z.,VU University Amsterdam | Lu Z.,Research Institute MOVE Skeletal Tissue Engineering Group Amsterdam STEGA | And 8 more authors.
Journal of Tissue Engineering and Regenerative Medicine

Apart from soluble growth factors, various other biophysicochemical cues are known to promote chondrogenesis. Under physiological conditions, cartilage in the joint comprises a hyperosmotic and hypoxic environment. Therefore, in this study, we examined the inductive effects of hyperosmotic and/or hypoxic conditions on adipose stem cells (ASCs) and compared them with conventional TGFβ1-induction. After encapsulation in collagen type II hydrogels and specific induction, ASCs were assessed for viability, proliferation, morphology and chondrogenic differentiation potential. Viability was similar under all conditions, with low proliferative activity. After 4days, hypoxia and/or hyperosmolarity did not affect round cell morphology, while cells were mainly stretched in the TGFβ1-induced group. At 21days, the TGFß1-treated group had aggregated into a cell nodule. Hyperosmolarity mimicked this aggregation to a lesser extent, whereas cells under hypoxia stretched out after 21days, with a combined effect in the hypoxic/hyperosmotic group. Both individual and combined hyperosmotic and/or hypoxic conditions significantly upregulated SOX5, SOX9, COMP and Link-p gene expression compared with the non-induced group, and to similar levels as the TGFβ1-induced group. GAG synthesis in both hydrogel and medium was increased under hypoxic conditions, whereas hyperosmolarity decreased GAG formation in the hydrogels, but increased GAG formation in the medium. We conclude that in a joint mimicking the three-dimensional (3D) micro-environment, a combination of hyperosmolarity and hypoxia is able to induce chondrogenesis to the same extent as TGFβ1. This might lead to an interesting alternative when considering short-term triggering in a one-step surgical procedure for the treatment of cartilaginous defects. © 2011 John Wiley & Sons, Ltd. Source

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