P3.01-058 Demethylation of CXCR4 and Stemness Acquisition in Lung Cancer: Topic: Stem Cells in Lung Cancer

Abstract

As a cancer stem cell marker, CXCR4 has been known to be closely associated with cell survival and stemness acquisition. Previous studies reported that the level of CXCR4 is increased after hypoxic condition in several types of cancer. However, the mechanism of the increased CXCR4 expression has not been well understood. We investigated whether aberrant promoter demethylation could induce CXCR4 activation by using hypoxic stress in lung cancer. Human normal lung cell (BEAS-2B) and lung cancer cell lines (A549, H292, H226 and H460) were incubated under hypoxic condition. Transcriptome and methylation analysis using next-generation sequencing were performed by HiSeq 2500 system. For further validation, CXCR4 expression was analyzed by RT-PCR and western blotting. To determine whether CXCR4 is reactivated, cell lines were treated with a DNA methyltransferase inhibitor (AZA). Hypoxia-induced DNA demethylation was identified by methylation-specific PCR and bisulfite sequencing. Stem cell characteristics were assessed by sphere formation assay and in vivo mice tumor model. Next-generation sequencing results revealed that CXCR4 expression was increased after hypoxic condition, whereas CXCR4 methylation was reduced. CXCR4 was activated by either hypoxic condition and treatment with AZA. MSP showed decreased CXCR4 promoter methylation in hypoxic condition compared with normoxic condition. Functional stem cell assay indicated that hypoxic stress increased sphere formation and in vivo mice tumor formation. These results suggest that hypoxia induces stem cell characteristics which are related with CXCR4 reactivation by promoter demethylation.