title: EGFR inhibition fails to suppress vascular proliferation and tumor growth in a Ewing's sarcoma model.
abstract: BACKGROUND
  Expression of epidermal growth factor receptor (EGFR), a potent regulator of cellular homeostasis, is associated with aggressive tumor behavior. The mechanism by which EGFR inhibition functions is unclear, with controversial results demonstrating an effect on the tumor cells, endothelial cells, or pericytes. EGFR activation has been linked to the expression of vascular endothelial growth factor (VEGF), a known mitogen of angiogenesis, but the relationship between these factors and their effect on tumor vessel development is vague. We hypothesized that using an EGFR inhibitor on a human Ewing's sarcoma model would inhibit tumor growth by suppressing vessel proliferation.
  
  
  METHODS
  A cell proliferation assay was performed on the Ewing's sarcoma (SK-NEP-1) cell line. Tumor cells were implanted intrarenally in athymic mice. Animals received daily gavage with vehicle or gefitinib 1 wk following implantation. Mice (n = 12/cohort) were euthanized 6 wk following implantation. Remaining mice were maintained without treatment for 2 wk. Vascular changes were assessed by angiography and immunohistochemically. EGFR and vascular endothelial growth factor (VEGF) expression were quantified using quantitative polymerase chain reaction (qPCR).
  
  
  RESULTS
  Gefitinib suppressed in vitro cell growth with an IC(50) = 1.36 μM. Minimal tumor growth suppression was noted at 6 wk (6.01 ± 1.2 g in control versus 4.61 ± 0.9 g treated, P = 0.36). After cessation of gefitinib, tumor growth was increased in both groups (7.37 ± 1.62 g versus 6.77 ± 1.53 g, P = 0.79). Microvessel density was unchanged despite EGFR inhibition (161,000 ± 16,000 pixels versus 135,000 ± 18,000 pixels, P = 0.31). At 6 wk, the vascular maturity index was similar in both groups (3.63 ± 1.12 versus 4.09 ± 1.71, P = 0.83). A downward trend in EGFR expression (49% of control) and an upward trend in VEGF levels (50% of control) occurred in the treated group.
  
  
  CONCLUSIONS
  EGFR expression was suppressed in cultured cells and xenograft tumors. Despite a cytotoxic effect on cell lines, gefitinib had little effect on tumor growth. No effects on the tumor vasculature were noted in the setting of EGFR suppression, suggesting that angiogenesis induced by SK-NEP-1 cells is refractory to EGFR inhibition. Interestingly, the resulting increase in VEGF expression following EGFR blockade, provides an alternative pro-angiogenic pathway promoting tumor survival.
authors.affiliation: Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039, USA.
authors.forename: Artur
authors.initials: A
authors.lastname: Chernoguz
authors.forename: Kelly
authors.initials: K
authors.lastname: Crawford
authors.forename: Eileen
authors.initials: E
authors.lastname: Donovan
authors.forename: Abbey
authors.initials: A
authors.lastname: Vandersall
authors.forename: Caroline
authors.initials: C
authors.lastname: Berglund
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Cripe
authors.forename: Jason S
authors.initials: JS
authors.lastname: Frischer
mesh_terms: D000792:Angiography; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D001859:Bone Neoplasms / Q000098:blood supply / Q000188:drug therapy / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D005260:Female; D000077156:Gefitinib; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000503:physiopathology*; D011799:Quinazolines / Q000494:pharmacology* / Q000627:therapeutic use; D012512:Sarcoma, Ewing / Q000098:blood supply* / Q000188:drug therapy / Q000473:pathology*; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D011799:Quinazolines; D042461:Vascular Endothelial Growth Factor A; D066246:ErbB Receptors; D000077156:Gefitinib