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Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: implications for xenograft models of human prostate cancer.
Exp Cell Res 2015; 333(1):136-46EC

Abstract

BACKGROUND

Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis.

METHODS

An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing.

RESULTS

At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site.

CONCLUSION

These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.

Authors+Show Affiliations

Institute of Health and Biomedical Innovation and the Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology, Translational Research Institute, Woolloongabba 4102, QLD, Australia.Institute of Health and Biomedical Innovation and the Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology, Translational Research Institute, Woolloongabba 4102, QLD, Australia.Institute of Health and Biomedical Innovation and the Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology, Translational Research Institute, Woolloongabba 4102, QLD, Australia.Institute of Health and Biomedical Innovation and the Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology, Translational Research Institute, Woolloongabba 4102, QLD, Australia.Institute of Health and Biomedical Innovation and the Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology, Translational Research Institute, Woolloongabba 4102, QLD, Australia.Institute of Health and Biomedical Innovation and the Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology, Translational Research Institute, Woolloongabba 4102, QLD, Australia.Institute of Health and Biomedical Innovation and the Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology, Translational Research Institute, Woolloongabba 4102, QLD, Australia. Electronic address: s.stephenson@qut.edu.au.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25724897

Citation

Lisle, J E., et al. "Murine, but Not Human, ephrin-B2 Can Be Efficiently Cleaved By the Serine Protease Kallikrein-4: Implications for Xenograft Models of Human Prostate Cancer." Experimental Cell Research, vol. 333, no. 1, 2015, pp. 136-46.
Lisle JE, Mertens-Walker I, Stephens CR, et al. Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: implications for xenograft models of human prostate cancer. Exp Cell Res. 2015;333(1):136-46.
Lisle, J. E., Mertens-Walker, I., Stephens, C. R., Stansfield, S. H., Clements, J. A., Herington, A. C., & Stephenson, S. A. (2015). Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: implications for xenograft models of human prostate cancer. Experimental Cell Research, 333(1), pp. 136-46. doi:10.1016/j.yexcr.2015.02.014.
Lisle JE, et al. Murine, but Not Human, ephrin-B2 Can Be Efficiently Cleaved By the Serine Protease Kallikrein-4: Implications for Xenograft Models of Human Prostate Cancer. Exp Cell Res. 2015 Apr 10;333(1):136-46. PubMed PMID: 25724897.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: implications for xenograft models of human prostate cancer. AU - Lisle,J E, AU - Mertens-Walker,I, AU - Stephens,C R, AU - Stansfield,S H, AU - Clements,J A, AU - Herington,A C, AU - Stephenson,S-A, Y1 - 2015/02/24/ PY - 2014/10/30/received PY - 2014/12/24/revised PY - 2015/02/14/accepted PY - 2015/3/1/entrez PY - 2015/3/1/pubmed PY - 2015/6/16/medline KW - EphB4 ligand KW - Ephrin-B2 KW - In silico modelling KW - Kallikrein 4 KW - Protease KW - Receptor tyrosine kinase SP - 136 EP - 46 JF - Experimental cell research JO - Exp. Cell Res. VL - 333 IS - 1 N2 - BACKGROUND: Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. METHODS: An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. RESULTS: At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. CONCLUSION: These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations. SN - 1090-2422 UR - https://www.unboundmedicine.com/medline/citation/25724897/Murine_but_not_human_ephrin_B2_can_be_efficiently_cleaved_by_the_serine_protease_kallikrein_4:_implications_for_xenograft_models_of_human_prostate_cancer_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0014-4827(15)00061-0 DB - PRIME DP - Unbound Medicine ER -