Arole 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.

Date of Award	20 Jun 2023
Original language	English
Awarding Institution	University of Bristol
Sponsors	Government of Saudi Arabia
Supervisor	Claire M Perks (Supervisor), Jeffrey Holly (Supervisor) & Kalina M Biernacka (Supervisor)