The role of IGFBP-3 in breast cancer cell response to DNA-damaging therapy

Abstract

Background: Triple-negative breast cancer is an aggressive form of breast cancer, not treatable by current targeted therapies and with a tendency to acquire resistance to conventional therapies. It is therefore crucial to negate the drivers of these malignancies in order to improve therapeutic sensitivity. A major cause of resistance to radiotherapy and some chemotherapy is the ability of breast cancer cells to aberrantly repair DNA double strand breaks (DSBs), fueling tumour progression. Irreparable cancer cells may either die or develop pro-survival pathways. Insulin-like growth factor binding protein-3 (IGFBP-3) was previously shown to translocate into the nucleus following DNA damage, where it forms complexes with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and epidermal growth factor receptor (EGFR). This finding, demonstrated in triple-negative breast cancer cells, was the first connection drawn between IGFBP-3 and DNA repair. Aims: The first aim of this study was to ascertain the point at which IGFBP-3 becomes involved in the DNA damage response. The second aim concerned IGFBP-3 as a determinant of cell fate following the initial DNA repair. Methods: The Hs578T human breast cancer cell line was used in this study as these cells express high endogenous levels of IGFBP-3 and phenotypically represent triple-negative breast cancer. The role of endogenous IGFBP-3 was determined using a stable loss-of-function approach, in which IGFBP-3 expression was silenced using short-hairpin RNA (shRNA). Neocarzinostatin (a radiomimetic agent) and etoposide (a topoisomerase II inhibitor) were used to induce DNA DSBs. Results: Etoposide and neocarzinostatin activated the ATM-p53-H2AX axis early following drug exposure, as measured by immunoblotting. This was followed by the phosphorylation of EGFR at Tyr1068. Autophosphorylation of DNA-PKcs at Ser2056 occurred at a later stage of DNA damage, whereas its phosphorylation at Thr2609 varied depending on the DNA-damaging agent used. Using stable knockdown of IGFBP-3, no evidence was found for the regulation of DNA damage and repair signalling by endogenous IGFBP-3. However, transient IGFBP-3 knockdown significantly inhibited DNA-PKcs phosphorylation four hours after etoposide treatment, consistent with previous findings. IGFBP-3 appeared to have no role in the activation of ATM or its substrates p53 and H2AX, suggesting that its involvement in DNA damage repair does not occur upstream of EGFR-DNA-PK activation. IGFBP-3 stable knockdown potentiated the etoposide- and neocarzinostatin-induced loss of cell viability, measured by the MTS assay. However, cell colony formation did not appear to be regulated by endogenous IGFBP-3. With prolonged exposure to etoposide, IGFBP-3 knockdown potentiated PARP-1 cleavage while, surprisingly, it inhibited caspase-3 cleavage. Autophagic flux, measured as LC3-II, increased with etoposide treatment, the increase being attenuated in IGFBP-3 knockdown cells concomitantly with increased PARP-1 cleavage. Conclusions: The data presented in this thesis suggest that chronic IGFBP-3 downregulation is not inhibitory to DNA damage and repair signalling, implying that a compensatory mechanism occurs to negate the effect seen with short-term IGFBP-3 knockdown. They also indicate that the involvement of IGFBP-3 in DNA damage repair does not occur earlier than DNA-PK activation. It is concluded that under some conditions IGFBP-3 may inhibit the pro-apoptotic effects of chemo- and radiotherapeutic agents, culminating in cell survival, consistent with the poor prognosis of women with ER-negative breast cancers with high IGFBP-3 expression.