Abstract 5002: Intermittent hypoxia selects for increased carcinogenicity in breast epithelial cells.

The hypoxic regions of solid tumors are hotbeds of drug resistance and selection. Tumors with highly hypoxic regions correlate with poor prognosis and increased aggressiveness. The exact molecular mechanisms of selection have not been elucidated. Recent studies have demonstrated that reoxygenation causes widespread DNA damage. Additionally, hypoxia suppresses the DNA damage machinery. Because cells within a solid tumor do not receive a consistent amount of oxygen, cycles of acute hypoxia and reoxygenation occur, a phenomenon termed intermittent hypoxia (IH). We predict that this may lead to widespread unrepaired DNA damage that consistently selects for populations adapted to survive within harsh tumor microenvironmental conditions. We tested three breast cell lines - one aggressive carcinoma (MB 231), one DCIS line, and one non-transformed epithelial cell line (MCF10A) - for DNA damage and transformation after multiple cycles of IH. Cells were incubated under 0.2% hypoxia for 16 hours and reoxygenated for 8, for multiple cycles. Multiple clones of each cell line selected under IH conditions were then isolated. MCF10A IH-selected clones exhibit increased resistance to etoposide relative to unselected clones. Two of these clones were selected for additional phenotyping, and exhibited constituitive carbonic anhydrase 9 expression, as measured by qRT-PCR. Resistance to methotrexate, docetaxel, and hypoxia induced cell death was also observed, but growth-factor independent proliferation was not detected. Additionally, genomic analyses have revealed loss of the tp53 and e-cadherin loci after intermittent hypoxia. Consistent with this, we detect loss of p53 and e-cadherin mRNA levels and anchorage independent growth. These data increase our understanding of why hypoxia creates more aggressive cancers and poorer prognoses, and how de novo drug resistance arises. Citation Format: Daniel Verduzco, Robert Gatenby, Robert Gillies. Intermittent hypoxia selects for increased carcinogenicity in breast epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5002. doi:10.1158/1538-7445.AM2013-5002