Introduction. Breast cancer cells frequently develop resistance to DNA-damaging therapy, and epidermal growth factor receptor (EGFR) may play an important role in this process. EGFR can modulate the repair of DNA double strand breaks (DSB) by non-homologous end-joining (NHEJ), by forming protein complexes that include the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). However many ER-negative breast tumors still respond poorly to DNA-damaging drugs even when combined with an EGFR inhibitor. IGFBP-3, an intracellular and secreted protein, is highly expressed in these tumors. Since we previously showed that IGFBP-3 can transactivate EGFR and translocate to the nucleus, the aim of this study was to determine whether IGFBP-3 forms part of the EGFR-DNA-PK complex that regulates the DNA damage response to DSB-inducing chemotherapeutic agents.

Results. In the triple-negative breast cancer cell lines MDA-MB-468 and Hs578T, which express IGFBP-3 highly, protein complex formation and nuclear localization of EGFR, DNA-PKcs and IGFBP-3 were enhanced by exposure to cytotoxic drugs etoposide or doxorubicin and reduced by IGFBP-3 knockdown or the EGFR kinase inhibitor gefitinib. Concomitantly, the co-location of EGFR-IGFBP-3 complexes with lipid rafts, visualized by proximity ligation assay (PLA) and confocal microscopy, decreased in response to drug treatment, consistent with the loss of this complex from the plasma membrane. Furthermore, the phosphorylation of DNA-PKcs at Ser2056 and its substrate Artemis nuclease at S516, and DNA repair capacity as measured by a non-homologous end-joining assay, were all decreased by IGFBP-3 silencing, while H2AX phosphorylation was increased, suggesting that IGFBP-3 has an obligatory role in the DNA repair response to DNA-damaging therapy.

Conclusion. The increased intranuclear interaction between EGFR and DNA-PKcs in response to DNA damage, and the subsequent stimulation of NHEJ activity, require the presence of IGFBP-3, which also forms part of the nuclear complex. This demonstrates a previously unrecognized “caretaker” role for IGFBP-3 in regulating DNA DSB repair by NHEJ. These novel findings suggests the possibility of a therapeutic approach for sensitizing ER-negative breast cancers to chemo- or radiotherapy by targeting the DNA repair function of IGFBP-3. (Supported by Australian Research Council grant DP0984232 to RCB).

Citation Format: Mike Zhen Lin, Hasanthi C. de Silva, Janet Lee Martin, Robert Charles Baxter. Insulin-like growth factor binding protein-3 (IGFBP-3) facilitates the response to DNA-damaging chemotherapy in triple-negative breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 616. doi:10.1158/1538-7445.AM2013-616

©2013 American Association for Cancer Research
2013