1. ¹Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada
2. ²Department of Cell Biology, School of Medicine, Fukuoka University, Fukuoka, Japan
3. ³Research Institute, International Medical Center of Japan, Tokyo, Japan
4. ⁴Department of Pediatrics, Montreal Children's Hospital, McGill University, Montreal, Quebec, Canada

Correspondence: Dr BL Coomber, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1. E-mail: bcoomber@uoguelph.ca

Received 19 September 2007; Revised 27 November 2007; Accepted 6 December 2007; Published online 28 January 2008.

Abstract

Antiangiogenic therapies are promising approaches to cancer control, but the details of their effects on subsequent tumor progression are not fully understood. Such therapies have the potential to eventually generate extensive amounts of tumor ischemia, and we previously demonstrated that ischemic conditions induce K-ras mutations in cells with deficient mismatch repair (MMR) mechanisms. This suggested that similar effects on oncogene mutagenesis may accompany antiangiogenic therapy. To test this, MMR-deficient colorectal cancer cells (Dks-8) were xenografted into immune-deficient mice and treated with the antiangiogenic regimen of low-dose/metronomic cyclophosphamide for 2 weeks followed by a 2-week recovery period without therapy. This treatment resulted in transient tumor growth inhibition, increased hypoxia, and decreased microvessel density, and cancer cells from treated tumors acquired activating mutations of the K-ras oncogene (K-ras^G13D). In vitro exposure of Dks-8 cells to the active metabolite of cyclophosphamide (4-hydroxycyclophosphamide) had no effect on the K-ras status, indicating that there was no direct action of this alkylating agent on K-ras mutagenesis. In addition, cells sorted from hypoxic regions of Dks-8 tumors were enriched in K-ras^G13D mutants. Collectively, our studies suggest that increases in tumor hypoxia induced by antiangiogenic treatment may lead to K-ras mutation and consequently tumor progression, especially in susceptible individuals.