Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling PI3K/Akt and MAPK/ERK pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation.

IR-induced YB-1 phosphorylation in K-RAS wild type (K-RASwt) and K-RAS mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using gamma-H2AX foci and standard colony formation assays the function of YB-1 in repair of IR-induced DNA-double strand breaks (DNA-DSB) and post-irradiation survival was investigated.

The average level of phosphorylation of YB-1 in breast cancer cell lines SKBr3, MCF7, HBL100 and MDA-MB-231 was significantly higher than in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, a lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similar to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 due to irradiation or due to K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS-siRNA as well as YB-1-siRNA blocked repair of DNA-DSB. Likewise, YB-1-siRNA increased radiation sensitivity.

IR induces YB-1 phosphorylation. YB-1 phosphorylation induced by oncogenic K-RAS or IR enhances repair of DNA-DSB and post-irradiation survival via erbB1 downstream PI3K/Akt and MAPK/ERK signaling pathways.

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