Abstract 3943: Invasion and degradation of extracellular matrix decreases in HIF-1α and HIF-2α-silenced MDA-MB-231 human breast cancer cells
Authors: Shah Tariq , Krishnamachary Balaji , Wildes Flonne , Mironchik Yelene , Bhujwalla Zaver M. 
Published: August 2012

The hypoxia inducible factor (HIF) is a master regulator of hypoxia inducible genes several of which facilitate invasion and metastasis. Here, using our Magnetic Resonance (MR) compatible cell perfusion assay, we have investigated the effect of silencing either HIF-1α or HIF-1 and 2α (doubly silenced), on the ability of MDA-MB-231 breast cancer cells to degrade ECM. Experiments were performed under normoxic or hypoxic conditions at oxygen tensions of 20% and 1% respectively. MR data were acquired on a 9.4 T MR spectrometer every 12h for a period of 2 days. Degradation of ECM by cancer cells was determined at the 24h time point relative to the initial time point from the proton (1H) images. Degradation of the ECM gel was estimated by drawing a region of interest (ROI) around the ECM region NIH ImageJ software. The degradation index was defined as (ROI0-ROI24)/ROI0. One dimensional 1H MR profiles of intracellular water were acquired along the length (z-axis) of the sample by diffusion-weighted MRI. These profiles were used to derive an invasion index by quantifying the number of cells invading into the ECM. Intracellular metabolite levels were also derived from unlocalized 1H MR spectra. T1-weighted 1H MR images demonstrated degradation of ECM gel by 48 h under normoxic conditions by parental MDA-MB-231 cells. Under hypoxic conditions, MDA-MB-231 cells completely degraded ECM within 24 instead of 48 h. This enhanced degradation of ECM under hypoxia may be driven by the upregulation of a number of proteolytic enzymes involved in ECM degradation. HIF-1α silenced cells showed similar degradation as parental cells under normoxia and hypoxia. On the other hand doubly silenced cells showed significantly reduced (p &lt; 0.01) degradation of ECM compared to parental cells under normoxia and hypoxia. These cells also showed significant decreased invasion even under normoxic conditions compared to parental cells. The absence of any significant difference in ECM degradation between HIF-1α silenced and parental cells, coupled with a significant difference observed in the doubly silenced cells indicates the importance of HIF-2α in these cells, and is currently under investigation. Quantitative analyses of metabolites demonstrated significantly decreased total choline (p &lt; 0.07) and phosphocholine (p &lt; 0.05) in HIF-1α silenced and doubly silenced cells compared to parental cells, consistent with our earlier observations of transcriptional regulation of choline kinase by hypoxia. These results suggest that in vivo, cells in or near hypoxic regions are likely to be more invasive and degrade the ECM, which can influence metastasis as well as drug delivery. These data also indicate that targeting HIF-1α alone is not sufficient to attenuate the invasiveness of these triple negative breast cancer cells under hypoxia and both HIF-1α and HIF-2α downregulation is required to inhibit invasion and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3943. doi:1538-7445.AM2012-3943