Abstract 503: Instability of ABCG2-associated side population expression in human A549 lung cancer cells and its contribution to drug resistance

Abstract

Abstract Dynamic changes in tumour subpopulations may contribute to post-therapeutic repopulation and treatment failure. Human tumours and established cell lines can maintain ‘side’ populations (SPs) – identified by a reduced accumulation of Hoechst dye 33342 attributable to ABCG2-mediated efflux. Progenitors in SP fractions present a challenge for therapies that attempt to target active S phase if they also comprise agents subject to active cellular efflux. The current study addresses the impact of the SP phenotype on drug resistance characteristics with a focus on a predicted resistance to the S-phase targeting anticancer drug DNA topoisomerase I poison topotecan (TPT). The human lung cancer A549 cell line system was used to isolate subclones that initially presented a range of SP expression (< 0.5 – >30 %) and a positive correlation was found between the size of the SP fraction and reduced TPT accumulation associated with ABCG2 expression. Representative clones were established with an initially low SP fraction (<1% SP; A549LowSP) or an initially high SP fraction (>30% SP; A549HighSP) compared with the A549parent (∼7% SP). Using RT-PCR, cells were compared for the expression of drug resistance-associated ATP binding cassette transporters (ABCG2, ABCB1). Increased ABCG2, but not ABCB1, expression, correlated with the degree of SP expression. A database survey of 59 human tumor NCI60 panel cell lines suggested that expression of the cancer stem cell marker aldehyde dehydrogenase (ALDH1A1) may augment TPT resistance in cells with low-level expression of ABCG2. However, parental, A549LowSP and A549HighSP cells showed similar co-expression patterns for ALDH genes (ALDH1A1, ALDH2, and ALDH3A1). A549LowSP increased its SP fraction during continued culture while A549HighSP maintained the SP fraction. The extent of the SP fraction reflected the degree of TPT efflux that was sensitive to the iABCG2-inhibitor Fumitremorgin C. To address whether innate SP expression provided an initial advantage for recovery from TPT exposure we studied the dynamic changes in the SP under in vitro selection of A549parent cells. We found that human SPs have a selective advantage, with enrichment of >20-fold, in cell populations recovering from multiple rounds of TPT treatment. Clonal analysis of TPT recovering populations found no evidence of long-term enrichment of SP fraction suggesting innate instability in SP expression. The data suggest that A549 non-SP cell populations can re-create SP fractions indicating that they are likely to arise by variations that provide a proliferative advantage rather than a unique stem-like progenitor. Innate SP fractions provide the tumor population with a short term selective advantage following TPT exposure related to their enhanced ABCG2 expression, and become enriched under dose fractionation. (LC is funded by UK-EPSRC and KP, LHP by Yorkshire Cancer Research) 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 503. doi:10.1158/1538-7445.AM2011-503