OR04-5 Prohibitin-2 is a binding protein for IGFBP-6 on rhabdomyosarcoma cell membranes and modulates IGFBP-6-dependent migration

Abstract

to examine whether phosphorylation of IGFBP-3 by DNA-PK is necessary for its apoptotic effect. ATM, which phosphorylates similar sites to DNA-PK, was also investigated. Methods: Two triple-negative breast cancer cell lines (Hs578T and MDAMB468) were treated for 1–48 hours with doxorubicin (0–10 mM), etoposide (0–50 mM) or ionising radiation (0–10 Gy), alone or after pretreatment (30–60 min) with inhibitors of ATM (KU55933; 0–30 mM) or DNA-PK (NU7441; 0–10 mM). DSB were monitored by gH2AX formation, DNA repair processes by kinase autophosphorylation and p53 activation, and apoptosis by caspase-3 activity. Endogenous IGFBP-3, ATM and DNA-PK were downregulated using siRNA. Knockdown (≥80%) was confirmed by qRT-PCR and immunoblot. Results: Although IGFBP3 is reported to be p53-inducible, IGFBP-3 protein was downregulated by doxorubicin treatment in MDAMB468 cells and upregulated in Hs578T cells, even though p53 was activated in both cell lines. This may be due to the different p53 mutations in each cell line. DSB were induced within 1 hour after addition of DNA-damaging agents. DNA damage-stimulated gH2AX and p53 phosphorylation was inhibited by KU55933 but increased by NU7441. DNA damage-induced apoptosis was significantly enhanced in both cell lines by kinase inhibition. In IGFBP-3-silenced cells, doxorubicin-induced apoptosis was significantly attenuated (20%), but neither KU55933 nor NU7441 altered the decreased apoptotic response, suggesting that IGFBP-3 does not need to be phosphorylated by ATM or DNA-PK for its apoptotic effect. In DNA-PK-silenced cells, IGFBP-3 knockdown no longer attenuated doxorubicin-induced apoptosis, suggesting that IGFBP-3 cannot carry out its apoptotic functions in the absence of DNA-PK. Conclusion: We conclude that DNA damaging therapy in breast cancer cells is enhanced by DNA repair inhibitors and is most effective in the presence of both DNA-PK and IGFBP-3. This suggests that cancers positive for IGFBP-3 may be most amenable to this therapy. Further work will employ mass spectrometric methods to determine the specific sites on IGFBP-3 that may become phosphorylated during these treatments. This work was supported by the Australian Research Council (DP0984232) (RCB); and a University of Sydney Postgraduate Award (KAM).