Metronomic doxorubicin blocks HIF-1α-mediated responses in sarcomas and augments the effects of anti-VEGF-A therapy

Increased levels of hypoxia and hypoxia inducible factor 1α (HIF-1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged anti-angiogenic therapy of tumors can delay tumor growth but may also increase hypoxia and HIF-1α activity. In our recent clinical trial, treatment with the anti-vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene set enrichment analysis of microarrays from pre-treatment biopsies found the Gene Ontology category “Response to hypoxia” was upregulated in poor responders, and hierarchical clustering based on 140 hypoxia-responsive genes separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF-1α binding to DNA at low metronomic doses. We thus examined Dox treatment in 4 sarcoma cell lines, and found Dox at low concentrations (1-10 uM) blocked HIF-1α induction of VEGF-A by 84-97%, while inhibition of other HIF-1α-target genes including CA9, c-Met and FOXM1 was variable. HT1080 sarcoma xenografts had increased hypoxia and/or HIF-1α activity with increasing tumor size and with anti-VEGF receptor antibody (DC101) treatment. Combining DC101 and metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, primarily via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF-1α-target genes which may promote resistance to anti-angiogenic and other therapies. Metronomic Dox can block HIF-1α activation of target genes and works synergistically with anti-VEGF therapy to inhibit sarcomas. Pre-treatment biopsies were collected from 16 human sarcoma. The gene expression analysis was performed using Illumina platform.