Inhibition of vascular endothelial growth factor A and hypoxia-inducible factor 1α maximizes the effects of radiation in sarcoma mouse models through destruction of tumor vasculature

Abstract

Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma.

Methods and materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines.

Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm(3) within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm(3) for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays.

Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

Conflict of interest statement

Conflict of interest: none

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Figure 1

(A) HT1080 xenografts. Groups were treated with control IgG, DC101, RT (8 Gy × 1), and/or metronomic doxorubicin (Dox). Graph and photos of total apoptosis (B), EC-specific apoptosis (C), and microvessel density (D) in HT1080 tumor groups. Arrows point to TUNEL and CD31 positive cells. Scale bar = 10-50 μm. Bars represent standard deviation. *p

Figure 2

(A) Extremity STS in LSL-KrasG12D/+/Trp53fl/fl mice (KP mice). Groups were treated with control IgG, DC101, RT 10 Gy times 2, and/or metronomic doxorubicin (Dox). Graph of microvessel density and photos of CD31 (B), nuclear HIF-1α (C), and CA9 (D) in KP mice tumor groups. Scale bar = 20-50 μm. *p<0.05 compared to control IgG group, **p<0.05 compared to all other groups.

Figure 3

(A) HT1080 cells transduced with HIF-1α shRNA (sh.HIF-1α) or scrambled shRNA (sh.Scr) followed by subcutaneous flank injection in athymic nude mice. Groups were treated with DC101 or control IgG. Some groups received RT 8 Gy × 1. Graphs of EC-specific apoptosis (B), microvessel density (C), and CA9 expression (D) in HT1080 tumor groups. Bars represent standard deviation. *p

Figure 4

(A) Proliferation of tumor EC 3 days after withdrawal of VEGF (No VEGF), RT (6 Gy), and/or low dose doxorubicin (Dox, 0.005 μM). (B) Colony formation of tumor EC 14 days after withdrawal of VEGF (No VEGF), RT (0, 2, 4 and 6 Gy), and/or low dose doxorubicin (Dox, 0.005 μM). (C) Immunofluorescence photos of Comet assay, γH2AX expression, and cleaved caspase 3. Experiments were performed in normoxia (21% O2) and hypoxia (1% O2). *p<0.05 compared to all other groups.

Figure 5

(A) Western blot analysis of HIF-1α in HT1080 cells transduced with HIF-1α shRNA (sh.HIF-1α) or scrambled (Scr) shRNA. Actin blot serves as loading control. (B) Proliferation of HT1080 cells after RT (6 Gy) and/or transduction with HIF-1α shRNA (sh.HIF-1a) or scrambled shRNA (sh.Scr). (C) Proliferation of four STS cell lines 3 days after RT and/or low dose doxorubicin (0.005 μM). (D) Colony formation of four STS cell lines in normoxia and hypoxia after RT (0, 2, 4 and 6 Gy), and/or low dose doxorubicin (0.005 μM). All experiments were performed in normoxia and hypoxia. *p