A role for IGFBP-2 in DNA repair in breast cancer cells

Abstract

Insulin-like growth factor binding protein-2 (IGFBP-2) mediates chemoresistance in prostate and breast cancer cells, as we have previously shown. Also, we and others have demonstrated that IGFBP-2 upregulates DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in double-stranded break repair (DSBR) in prostate and oesophageal cancer cells, respectively and that IGFBP-2 might interact with the DNA damage response in breast cancer. Hence, this study aimed to determine the role of IGFBP-2 in the DNA-damage response induced by etoposide in breast cancer cells. Cells (MCF-7; high levels of IGFBP-2 and MDA-MB-231; low levels of IGFBP-2) were treated with etoposide in the absence (IGFBP-2 siRNA for MCF-7) or presence (exogenous IGFBP-2; in MDA-MB-231) of IGFBP-2. To determine if the effects of IGFBP-2 were IGF-dependent or independent, the effects of IGF-I in MCF-7 cells and Arginyl-glycyl-aspartic acid (RGD) in MDA-MB-231 cells on the DNA damage response were investigated. Western blotting of whole cell lysates was performed to monitor changes in protein abundance of DNA damage/repair markers, γH2AX and P-DNA-PKcs, respectively. With MCF-7 cells, silencing IGFBP-2 alone had no effect on the levels of P-DNA-PKcs compared to the non-silencing siRNA control, but γH2AX levels increased significantly. Etoposide alone caused an increase in γH2AX that was enhanced by silencing IGFBP-2, whereas etoposide-induced P-DNA-PKcs levels were reduced when IGFBP-2 was silenced. With or without IGFBP-2, IGF-I significantly reduced the DNA damage response and repair in MCF-7 cells. Accordingly, IGFBP-2 was acting in an IGF-independent manner. Immunoprecipitation was used to determine how IGFBP-2 interacts with DNA repair molecules. It was observed that IGFBP-2 is associated with Ku80, one of the key molecules in non-homologous end joining (NHEJ). In MDA-MB-231 cells, the exogenous addition of IGFBP-2 alone had no effect on γH2AX or P-DNA-PKcs levels. The addition of RGD and IGFBP-2 alone and in combination enhanced DNA repair and reduced DNA damage in the presence of etoposide. This suggested that IGFBP-2 and RGD were acting in a similar integrin-mediated manner. In summary, these studies revealed that IGFBP-2 reduced DNA damage by increasing the DNA repair mechanism to protect breast cancer cells. Understanding how IGFBP-2 is involved in the DNA damage response may facilitate more effective targeting and treatment regimens in breast cancer.