VEGFR3 inhibition chemosensitizes ovarian cancer stemlike cells through down-regulation of BRCA1 and BRCA2

Abstract

In ovarian cancer, loss of BRCA gene expression in tumors is associated with improved response to chemotherapy and increased survival. A means to pharmacologically downregulate BRCA gene expression could improve the outcomes of patients with BRCA wild-type tumors. We report that vascular endothelial growth factor receptor 3 (VEGFR3) inhibition in ovarian cancer cells is associated with decreased levels of both BRCA1 and BRCA2. Inhibition of VEGFR3 in ovarian tumor cells was associated with growth arrest. CD133(+) ovarian cancer stemlike cells were preferentially susceptible to VEGFR3-mediated growth inhibition. VEGFR3 inhibition-mediated down-regulation of BRCA gene expression reversed chemotherapy resistance and restored chemosensitivity in resistant cell lines in which a BRCA2 mutation had reverted to wild type. Finally, we demonstrate that tumor-associated macrophages are a primary source of VEGF-C in the tumor microenvironment. Our studies suggest that VEGFR3 inhibition may be a pharmacologic means to downregulate BRCA genes and improve the outcomes of patients with BRCA wild-type tumors.

Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

Figures

Figure W1

Maz51 does not induce apopotosis. PI/Annexin FACS analysis of control and Max51 treated OVCAR8 cells demonstrating no increase in Annexin stain with Maz51 treatment.

Figure 1

Expression of VEGF-C, VEGF-D, and VEGFR3 in ovarian cancer. (A and B) qRT-PCR evaluation of VEGF-C mRNA and VEGFR3 mRNA in VLCs, TECs, and primary ovarian tumor cells (TCs) is presented. (C) Western blot confirms high VEGF-C protein expression in VLCs. (D) FACS analysis of VEGFR3 and CD133 in A2780 and OVCAR8 cells is presented. (E) Immunohistochemistry demonstrating VEGFR3 expression in human ovary is primarily in vessels and rare stromal cells, whereas VEGFR3 expression in primary ovarian tumors is in vascular structures and tumor cells (original magnification, × 100). (F) p-VEGFR3 Western blot of A2780 tumor cells treated with VEGF-C is shown.

Figure 2

VEGFR3 inhibition results in ovarian cancer cell growth arrest and preferentially targets CD133+ cells. (A) OVCAR8 and A2780 tumor cell number (relative to untreated controls) after treatment with the indicated doses of Maz51 is presented. (B) Propidium iodide FACS cell cycle analysis of control and Maz51-treated OVCAR8 and A2780 cells is presented. (C) Quantification of BrdU incorporation in control and Maz5-treated OVCAR8 and A2780 cells is presented. (D) Total cell numbers (normalized to untreated control) of FACS-isolated CD133+versus CD133− OVCAR8 and A2780 cells after treatment with the indicated doses of Maz51 are presented. (E) Quantification and representative images of tumor spheres formed from three independent primary ovarian cancer cell specimens in the absence and presence of increasing doses of Maz51 are shown.

Figure 3

VEGFR3 inhibition decreases BRCA1 and BRCA2 gene expression through p-ERK and E2F1. (A) (i) Phosphoprotein Western blot analysis of control and VEGF-C–treated A2780 and OVCAR8 cells in the presence or absence of Maz51 showing VEGF-C treatment is associated with increased p-ERK. (ii) Western blot demonstrating Maz51 treatment is associated with decreased BRCA1 and E2F1 in both A2780 and OVCAR8 cells. (B) qRT-PCR demonstrating treatment with (i) Maz51 or (ii) MEK inhibition is associated with down-regulation of BRCA1 and BRCA2 mRNA. (C). Comparison of cell growth inhibition of Maz51 in (i) two sets of isogenic control and BRCA1 knockout murine ovarian cancer cell lines and in (i) BRCA2 mutant PEO1 and BRCA2 revertant PEO4 cells.

Figure 4

VEGFR3 inhibition with Maz51 chemosensitizes in vitro in a BRCA-dependent manner. (A) Absolute tumor cell number of OVCAR8 and A2780 cells treated with Maz51 only, cisplatin chemotherapy only, cisplatin followed by Maz51, Maz51 and cisplatin concurrently, or Maz51 followed by cisplatin is presented. (B) Normalized cell counts of PEO1 (BRCA2 mutant) and PEO4 cells (BRCA2 revertant) treated with the indicated agents are presented.

Figure 5

VEGFR3 inhibitor restricts tumor growth and increases chemosensitivity. (A and B) Tumor growth curves for OVCAR8 and A2780 cell–derived tumors treated with single agent Maz51 are presented. (B) (i) Tumor growth curves for A2780 tumors treated with cisplatin or Maz51 before and concurrent with cisplatin. Arrows indicate doses of cisplatin. Red bars indicate timing of daily Maz51 treatment. (ii) IHC analysis and quantification of Ki67 in cisplatin- and cisplatin + Maz51–treated A2780 tumors are presented. (C) (i) FACS evaluation of CD133 and ALDH expression in cisplatin- and Maz51 + cisplatin–treated tumors and (ii) absolute numbers of CD133+ and ALDH + cells in cisplatin- and Maz51 + cisplatin–treated tumors are presented.