Abstract 2065: Heterogeneity of tumor response to hypoxia: Carbonic anhydrase 9 induction defines subpopulation of hypoxic cells with high Wnt signalling, stem cell properties and drug resistance
Authors: Ledaki Ioanna , Wigfield Simon , Mcintyre Alan , McGowan Simon , Waugh Craig , Harris Adrian 
Published: July 2012

Abstract
Carbonic anhydrase IX [CA9], a membrane-associated protein with an extracellular CA domain is strongly induced by hypoxia and regulates tumour pH. CA9 expression is associated with poor outcome. The aim of this study was to use CA9 to investigate heterogeneity of the hypoxic response, the molecular pathways specifically activated in the CA9+ population and their phenotype in terms of stem cell behaviour and drug resitance.
Methods: MCF7, MDA231 breast cancer cell lines and HCT116 and SW1222 colon cancer cell lines under normoxic or hypoxic (0.1% O2, 72hrs) were sorted into CA9 + and – populations using a CA9-FITCH conjugated antibody. mRNA expression profiles were measured using a Deep Sequence arrays. Quantitative RT-PCR and western blot analysis was used to confirm changes. The ability of 2 populations to form mammospheres, 3D spheroids and soft agar colonies was investigated, as was drug sensitivity testing using the SRB assays.
Results: Our data showed that under hypoxic conditions there were two populations which differentially express the hypoxic marker CAIX in four cell lines tested. The percentage of CA9 + cells ranged from 30% to 50% in the four cell lines examined. Among the genes significantly upregulated in the CA9+ populations were those implicated in stem cell maintainance such as ALDHA1 (fold change (fc) 2.28,), IGF1 (fc 2.12), LIN28 (fc 2.28). Genes involved in the epithelial mesenchymal transition and multi drug resistant such as WNT2 (fc 1.78), TWIST1 (fc 2.5), and ABCC2 (fc 1.65), were also significantly upregulated. The expression of HIF1 alpha and many downstream genes such as LDHA, PDK1 and AK4 were equally expressed in the 2 populations. The CA9+ population formed larger mammospheres (p&lt;0.01), 3D spheroids (p&lt;0.01) and more soft agar colonies (p&lt;0.01). Only the CAIX positive population had the ability to recapitulate the original phenotype and reform both populations in long term culture. The CA9 positive cells were more resistant to 5-FU (CA9 + IC50 236.2 μM, CA9- IC50 77.45μM) but surprisingly, using the histone deacetylase inhibitor SAHA resulted greater sensitivity of the CA9+ population survival (CA9 + IC50 0.2μM, CA9- IC50 0.4 μM).
Conclusions: Our results indicate differential expression of the hypoxia transcriptome in MCF7, MDA231, HCT116 and SW1222 cancer cell lines, with major phenotypic differences defined in the CA9+ and – populations. There is a strong relation between CA9 expression and stem cell markers, but it is expressed in a much larger population. The basis for the regulation is not clear but the differential sensitivity to HDAC inhibitors suggests an epigenetic mechanism and the role may be to provide a wider hypoxia tolerant microenvironment to protect the stem cell population.
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 2065. doi:10.1158/1538-7445.AM2011-2065