Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1α–mediated tumor apoptotic switch
Claire Magnon, Paule Opolon, Marcel Ricard, Elisabeth Connault, Patrice Ardouin, Ariane Galaup, Didier Métivier, Jean-Michel Bidart, Stéphane Germain, Michel Perricaudet, Martin Schlumberger
Claire Magnon, Paule Opolon, Marcel Ricard, Elisabeth Connault, Patrice Ardouin, Ariane Galaup, Didier Métivier, Jean-Michel Bidart, Stéphane Germain, Michel Perricaudet, Martin Schlumberger
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Research Article Oncology

Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1α–mediated tumor apoptotic switch

Abstract

Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1–regulated (HIF-1–regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1–dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1α increases the activity of the canstatin-induced αvβ5 signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1α activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy.

Authors

Claire Magnon, Paule Opolon, Marcel Ricard, Elisabeth Connault, Patrice Ardouin, Ariane Galaup, Didier Métivier, Jean-Michel Bidart, Stéphane Germain, Michel Perricaudet, Martin Schlumberger

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Figure 4

HIF-1 is required for potentiation of integrin-mediated tumor apoptotic signaling pathway induced by CanHSA.

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HIF-1 is required for potentiation of integrin-mediated tumor apoptotic ...
The expression levels of both HIF-1α and caspase-3 were assessed by Western blotting of whole-cell extracts prepared from AdCO1- or AdCanHSA-infected MDA-MB-231 cells, in the presence or absence of DFO (A) at different time points following infection (B) with several MOIs (2,000, 10,000, or 16,000 particle viral [PV]) or (C) at different concentrations of DFO (1, 10, 50, and 100 μM [D1, D10, D50, and D100]). Immunoblots of whole-cell extracts, from noninfected (NI) or AdCO1- or AdCanHSA-infected RCC cells expressing VHL mutated (D) or VHL WT (E) in the presence or absence of DFO, confirmed that HIF-1 expression potentiates caspase-3 cleavage following infection with AdCanHSA. (F) Western blotting of protein extracts from HUVECs showed that HIF-1α is not required to induce the CanHSA-mediated apoptotic process in endothelial cells. Note that HIF-1α expression slightly accelerated caspase-3 cleavage. Staurosporine (St), a known inducer of caspase-dependent apoptosis, was used as a positive control. To determine whether αvβ5 integrins promote the CanHSA-induced HIF-1α signaling tumor apoptotic pathway, expression of αvβ5 was silenced through siRNA β5 on MDA-MB-231 cells expressing or not expressing HIF-1α (FACS analysis as previously described; ref. 27) (G) and Western blotting was done on whole-cell extracts from AdCO1- or AdCanHSA-infected MDA-MB-231 cells, which were transfected with random siRNA (siRNA scrambled) or siRNA β5 (H). (I) In parallel, to investigate whether αvβ3 integrins take part in the HIF-1–dependent tumor apoptotic process, MDA-MB-231 cells were also transfected with siRNA β3 and then infected with AdCO1 and AdCanHSA. Each experiment was done twice. To confirm whether CanHSA triggers an HIF-1–dependent apoptotic process, we transfected an siRNA HIF-1 within the MDA-MB-231 cells.