Efficacy and antivascular effects of EphA2 reduction with an agonistic antibody in ovarian cancer.
BACKGROUND
EphA2 is an oncoprotein and tyrosine kinase receptor that is overexpressed in ovarian and many other cancers. We investigated the effects of reduced EphA2 levels on tumor growth and the tumor microenvironment in an orthotopic ovarian cancer model.


METHODS
The effect of the EphA2-agonistic monoclonal antibody EA5, alone or in combination with paclitaxel, on the growth of ovarian cancer cells (SKOV3ip1, HeyA8, and HeyA8MDR [taxane-platinum resistant]) was determined in vitro and in vivo by immunoblotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and immunohistochemical analysis. Expression of EphA2 and markers of angiogenesis (CD31, vascular endothelial growth factor [VEGF], and basic fibroblast growth factor), proliferation (proliferating cell nuclear antigen), and endothelial cell apoptosis (CD31-terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick-end labeling colocalization) and phosphorylation of Src were analyzed by immunoblotting, immunohistochemistry, immunofluorescence, and in situ hybridization in tumors from treated mice. Statistical tests were two-sided.


RESULTS
EA5 antibody treatment led to a more than 90% reduction in EphA2 expression in HeyA8 tumors in vivo. In mice bearing orthotopic SKOV3ip1 or HeyA8 tumors, 4 weeks of EA5 treatment resulted in tumors that weighed 31% and 45% less, respectively, than those in control (IgG-treated) mice (95% confidence interval [CI] = -0.09% to 71% and 20% to 70%, P = .27 and .01, respectively). Combination therapy with EA5 and paclitaxel reduced tumor weight by 77% and 80% (95% CI = 63% to 91% and 68% to 91%), respectively, compared with paclitaxel alone and by 92% and 88% (95% CI = 87% to 97% and 80% to 94%), respectively, compared with IgG alone. Combination therapy also reduced the weight of HeyA8MDR tumors by 47% (95% CI = 24% to 72%) compared with paclitaxel. Mice bearing SKOV3ip1 or HeyA8 tumors that were treated with combination therapy survived longer than those treated with paclitaxel alone (median survival = 144 versus 69 days and 46 versus 37 days, respectively). EA5-treated tumors had reduced microvascular density, proliferation, and VEGF protein and mRNA levels, with increased endothelial cell apoptosis. EphA2 was associated with Src, which was rapidly dephosphorylated after EA5 treatment.


CONCLUSIONS
EA5 in combination with paclitaxel decreased tumor growth in an orthotopic ovarian cancer mouse model through antiangiogenic mechanisms associated with reduced levels of VEGF and phosphorylated Src. Humanized antibody constructs against EphA2 are worthy of future study.
Landen
Charles N
CN
Department of Gynecologic Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
Lu
Chunhua
C
Han
Liz Y
LY
Coffman
Karen T
KT
Bruckheimer
Elizabeth
E
Halder
Jyotsnabaran
J
Mangala
Lingegowda S
LS
Merritt
William M
WM
Lin
Yvonne G
YG
Gao
Changhou
C
Schmandt
Rosemarie
R
Kamat
Aparna A
AA
Li
Yang
Y
Thaker
Premal
P
Gershenson
David M
DM
Parikh
Nila U
NU
Gallick
Gary E
GE
Kinch
Michael S
MS
Sood
Anil K
AK
D000704:Analysis of Variance; D020533:Angiogenesis Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology* / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D017403:In Situ Hybridization; D020287:In Situ Nick-End Labeling; D051379:Mice; D009389:Neovascularization, Pathologic / Q000188:drug therapy* / Q000517:prevention & control; D010051:Ovarian Neoplasms / Q000098:blood supply* / Q000188:drug therapy*; D036104:Receptor, EphA2 / Q000037:antagonists & inhibitors* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000187:drug effects
D020533:Angiogenesis Inhibitors; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D042461:Vascular Endothelial Growth Factor A; D036104:Receptor, EphA2