Abstract 2851: Metronomic doxorubicin blocks hypoxia inducible factor-mediated responses to hypoxia in sarcomas and augments the effects of anti-VEGF therapy

Abstract

Abstract Introduction: Levels of hypoxia and hypoxia inducible factors (HIFs) in human sarcomas correlate with tumor progression and metastasis. Prolonged anti-angiogenic therapy of sarcomas can inhibit tumor growth but may also increase hypoxia and HIF activation. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (dox), was recently found to block HIF binding to DNA at low metronomic doses. Methods: 20 patients with soft tissue sarcomas were treated with anti-VEGF antibody (bevacizumab). The tumors, before and after treatment, were examined for changes in microvessel density using CD31 immunohistochemical staining and global gene expression using microarrays. Patients’ tumors were then treated with a combination of bevacizumab and radiation followed by surgical resection. Expression of specific HIF-target genes in the response to hypoxia and/or dox was examined in vitro in 4 sarcoma cell lines by q-RT-PCR. HT1080 human sarcoma xenografts and sarcomas in genetically engineered LSL-KrasG12D/+Trp53fl/fl mice were treated with anti-VEGF receptor antibody (DC101) and/or metronomic dox and analyzed for levels of hypoxia, HIFs, and HIF-target genes. Results: Bevacizumab alone reduced microvessel density in sarcomas by a median of 53% (p<0.05). Gene set enrichment analysis of microarrays found the Gene Ontology category “Response to hypoxia” (which includes HIF-1a, HIF-2a, and ARNT) was upregulated in sarcomas with a poor response to combination therapy (bevacizumab plus radiation). Dox at 1-10 uM blocked HIF induction of VEGF and CA-9 in all 4 sarcoma cell lines by 83.7-97.0% and 88.6-97.6%, respectively, while inhibition of other HIF-target genes including c-MET and FOXM1 was variable. HT1080 sarcoma xenografts and LSL-KrasG12D/+Trp53fl/fl sarcomas demonstrated increasing hypoxia with larger tumor size and with treatment with an anti-VEGFR-2 antibody (DC101). Increasing hypoxia corresponded to greater nuclear expression of HIF-1α and HIF-2α, and metronomic Dox lowered expression of HIF-depended genes by up to 92.3%. Combining VEGFR-2 antibody and metronomic Dox therapies had a synergistic effect in suppressing tumor growth. Conclusion: Human sarcomas respond to increased hypoxia by expressing HIF-target genes which promote compensatory responses. This hypoxic response may be exacerbated by anti-VEGF therapies and promote resistance to such therapies. Low metronomic doses of dox can block activation of HIF-target genes and works synergistically with anti-VEGF therapies to inhibit tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2851. doi:10.1158/1538-7445.AM2011-2851