Introduction and Aims. Radiotherapy and certain chemotherapy drugs act by inducing double-strand breaks (DSB) in DNA, resulting in cell senescence or death unless the DNA damage is repaired. Since the ability of cancer cells to repair damaged DNA contributes to their chemo- or radio-resistance, it is important to understand the mechanisms involved and how to overcome them. We recently reported that IGFBP-3, a growth-regulatory protein associated with poor prognosis in women with basal-like breast cancer, is an important component of the cellular DSB repair mechanism (1).  This study aimed to discover new pathways of DNA damage repair in basal-like breast cancer, with the ultimate goal of improving responsiveness to DNA-damaging therapies.

Methods and Results. The basal-like breast cancer cell line MDA-MB-468 (triple-negative for ER, PR and HER2) was treated with 20 µM etoposide for 2 h to induce DNA DSB. To discover IGFBP-3-associated repair pathways, proteins that interact with IGFBP-3 were immunoprecipitated from cell lysates using IGFBP-3 antibodies and subjected to proteomic analysis by nanoflow-liquid-chromatography, MALDI-TOF/TOF mass spectrometry, and MASCOT database searching. Of  >200 proteins found to be associated with IGFBP-3, ~40 were unique to the etoposide-treated cells (i.e. not immunoprecipitated from untreated cells), almost all identified as phosphoproteins by MS/MS. Gene ontogeny analysis of IGFBP-3-associated phosphoproteins indicated association with chromatin organisation, histone modification, cell cycle regulation and other key pathways in addition to DNA damage response.

Discussion. Chemotherapy is a front-line treatment for breast cancers of the basal-like molecular subtype, for which few targeted therapies are available. This study has identified novel phosphoproteins that associate with IGFBP-3 in response to chemotherapy, implicating IGFBP-3 in previously unrecognised DNA damage response pathways. Confirmation of these interactions will lead to new approaches to counteract chemoresistance in breast cancer.

Supported by NHMRC, SU Cancer Research Fund, NTCRU and Cancer Institute NSW.