EGFL7 inhibits tumor angiogenesis via its Emilin-like domain

• Author / Creator

  Kadam, Alisha

• Epidermal Growth Factor-like domain 7 (EGFL7) is a secreted angiogenic factor that is almost exclusively expressed by endothelial cells. High EGFL7 expression occurs during physiological angiogenesis, under pathological conditions, and in solid tumor angiogenesis. Evidence suggests that EGFL7 expression is elevated in several human tumors, such as glioma, hepatocellular carcinoma, and colon cancer. EGFL7 is composed of three domains: an Emilin-like (EMI) domain, two Epidermal Growth Factor-like (EGF) repeats and a Matrilin-like (MAT) domain. While EGFL7 is a known regulator of physiological angiogenesis, little is known about its function in tumors. In this study, we attempted to clarify EGFL7’s role in the regulation of tumor angiogenesis and its mechanism of action. EGFL7 was stably expressed in HT1080 fibrosarcoma cells as either full length protein or as a mutant with one domain deleted per cell line (HT1080 EGFL7ΔEMI, HT1080 EGFL7ΔEGF and HT1080 EGFL7ΔMat). Cell proliferation was assessed using an MTT-based proliferation assay. A tumor growth assay and two angiogenesis assays were carried out to elucidate the effect of EGFL7 and its domains on angiogenic sprouting in tumors. Contrary to expectation, the cancer-specific expression of EGFL7 in fibrosarcoma cells led to the suppression of tumor growth in vivo. Importantly, there was no significant difference in their ability to proliferate in vitro. Both in vitro and ex ovo angiogenesis assay showed significant reduction in the vascular density for the EGFL7 overexpressing cells. Interestingly, both the reduction in tumor size and inhibition of angiogenesis were dependent upon the presence of the Emilin-like domain in EGFL7. To study EGFL7’s mechanism of action, EGFL7 from tumor cell lysates was immunoprecipitated using antibody coated protein-A beads and co-precipitated proteins were identified using western blotting. Integrin β1 and Thrombospondin-1 were discovered to be interacting partners for EGFL7 using this method. Finally, extracellular vesicles (EVs) were isolated from enriched medium of HT1080 cells (Control, EGFL7 and deletion mutants) and analysed using flow cytometry, western blotting, TEM, and confocal imaging. EGFL7 was observed to be secreted via EVs from EGFL7-expressing cancer cells, as confirmed by the TEM imaging. Interestingly, deleting the EGF-like domain significantly reduced the number of EGFL7-expressing EVs that were released from cancer cells. To summarize, EGFL7 is a potent inhibitor of tumor angiogenesis and its anti-angiogenic phenotype is medium ted via the Emilin-like domain. The EGF-like domain is also functional as it regulates the EV-mediated secretion of EGFL7 from cancer cells. Additionally, two important angiogenic mediators, Integrin β1 and Thrombospondin-1, were identified as protein interactors of the EGFL7 protein and may be involved in EGFL7’s inhibition of tumor angiogenesis.

• Subjects / Keywords

  • EGFL7
  • Tumor biology
  • Angiogenesis

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3RX93W1S

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.