Role of Y-Box Binding Protein-1 mediated cell signaling in proliferation and radiotherapy resistance of breast cancer cells

Abstract

The Y-box binding protein-1 (YB-1) is a multifunctional protein that binds to nucleic acids and proteins to regulate essential cellular functions. YB-1 is overexpressed in cancers from different entities and is constitutively phosphorylated in KRAS-mutated breast cancer cells. In the current study, the signaling pathways involved in the phosphorylation of YB-1 and the role of phospho-YB-1 in cell proliferation, double strand break (DSB) repair and radioresistance of KRAS(G13D)-mutated breast cancer were investigated. Pharmacological inhibitors, siRNA/shRNA knockdown, CRISPR/Cas9 YB-1 knockout and plasmid based overexpression approaches were used. Expression pattern of the components of YB-1 signaling pathway in normal and tumor tissues from breast cancer patients was analyzed by immunofluorescence staining. Proteomic analysis was performed to evaluate the YB-1 regulated proteins involved in DSB repair pathways. In KRAS(G13D)-mutated cells, the constitutive high level of phospho-YB-1 occurred predominantly through the MEK/ERK pathway or PI3K pathway depending on the additional PTEN mutation. Compared to single targeting of PI3K or MEK, dual targeting of both kinases synergistically inhibited YB-1 phosphorylation and attenuated expression of EGFR. Upregulated phospho-YB-1 and related signaling pathways that were identified in vitro were also observed in tumor tissues but not in the normal breast tissue from breast cancer patients ex vivo. Furthermore, YB-1 expression was seen to be crucial regulator of cell proliferation in KRAS(G13D) mutated breast cancer cells. It was further shown that YB-1 is essential for DSB repair and post-irradiation cell survival. YB-1 knockout downregulated expression of number of proteins involved in DSB repair pathways, among them KAP-1 was the most important one. This study uncovered the novel mechanism by which YB-1 regulates DSB repair through HR and A-NHEJ pathway and mediates radioresistance. Thus, YB-1 may be an efficient target to overcome radiotherapy resistance of solid tumors. Along with the clinical significance, finding from this study also sheds light on the cytoplasmic signalling cascades that mediate DNA repair.