Abstract P5-07-05: Insulin-like growth factor binding protein-3 is a key component of the breast cancer cell response to DNA-damaging therapy

Abstract

Abstract Introduction. Breast cancer cells frequently develop resistance to DNA-damaging therapies, such as radiation and the cytotoxic drugs doxorubicin and etoposide, and activation of the epidermal growth factor receptor (EGFR) may play an important role in this process. However many estrogen receptor (ER)-negative breast tumors respond poorly to DNA-damaging drugs even when combined with an EGFR inhibitor. 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). In previous studies we have shown that the growth-regulatory protein, insulin-like growth factor binding protein-3 (IGFBP-3), can translocate to the nucleus, is a substrate for DNA-PKcs, and can transactivate EGFR. The aim of this study was to delineate the role of IGFBP-3 in the response of breast cancer cells to DSB-inducing chemotherapeutic agents. Results. In the triple-negative breast cancer cell lines MDA-MB-468 and Hs578T, which express IGFBP-3 highly, nuclear localization of EGFR and IGFBP-3, and of their complex as measured by coimmunoprecipitation (coIP), was enhanced by exposure to etoposide or doxorubicin, peaking 2 h after etoposide treatment. This effect was blocked by 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. Similar to the nuclear DNA-PKcs-EGFR complex, the nuclear DNA-PKcs-IGFBP-3 complex (seen by both coIP and PLA) was greatest 4 h after treatment. DNA-PKcs activation at serine-2056, required for the repair of DNA DSB and stimulated by etoposide treatment, was decreased by IGFBP-3 downregulation using two separate siRNAs, and this treatment also attenuated the enhanced DNA-PKcs-EGFR complexes seen in response to etoposide. Collectively these data suggest that IGFBP-3 has an obligatory role in the DNA repair response to DNA-damaging therapy through its interactions with EGFR and DNA-PKcs. Conclusion. IGFBP-3 co-translocation to the nucleus of breast cancer cells and its complex formation with DNA-PKcs and EGFR in response to DNA damage demonstrate its previously unrecognized involvement in the regulation of 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 the Australian Research Council. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-05.