We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulinlike growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 ϳ2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P 1 ) or S1P 3 , but not S1P 2 , abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P 1 and S1P 3 . These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P 1 /S1P 3 , suggesting that S1P, the product of SphK activity and ligand for S1P 1 and S1P 3 , is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3. Insulin-like growth factor-binding protein-3 (IGFBP-3)2 is one of the family of six IGFBPs that bind the peptide growth factors IGF-I and IGF-II with high affinity and regulate their bioactivity (1). As the predominant carrier of IGFs in the endocrine system, IGFBP-3 regulates the movement of these growth factors from the circulation to target tissues and inhibits their proliferative and antiapoptotic cellular effects by blocking their activation of the type 1 IGF receptor (IGFR1) at the cell surface. In vitro studies in a variety of cell types have revealed that IGFBP-3 may also impact on cell growth and survival independently of modulating IGF bioactivity, inducing cell cycle arrest and apoptosis by regulation of apoptotic effector proteins (2-4) and interaction with nuclear receptors (5-7).There is, however, also evidence of an association between IGFBP-3 and enhanced cell proliferation. Some clinical studies in breast, prostate, pancreatic, renal cell, and non-small cell lung cancers have shown that a high level of tissue expression of IGFBP-3 correlates with increased tumor growth or malignancy (8 -13). Although the mechanism linking IGFBP-3 with growth stimulation in vivo remains unclear, we and others have shown that, in vitro, IGFBP-3 can enhance the effects of stimulatory growth factors. Human and bovine skin fibroblasts exposed to low concentrations of exogenous IGFBP-3 exhibit enhanced IGF-stimulated DNA synthesis (14, 15), and similarly, exogenous and endogenous IGFBP-3 enhanced the growth response to IGF-I in the MCF-7 breast cancer cell line (16). We have also shown previously that IGFBP-3 is inh...

The type I EGF receptor (EGFR or ErbB1) and insulin-like growth factor-binding protein-3 (IGFBP-3) are highly expressed in triple-negative breast cancer (TNBC), a particularly aggressive disease that cannot be treated with conventional therapies targeting the estrogen or progesterone receptors (ER and PR), or HER2. We have shown previously in normal breast epithelial cells that IGFBP-3 potentiates growth-stimulatory signaling transduced by EGFR, and this is mediated by the sphingosine kinase-1 (SphK1)/sphingosine 1-phosphate (S1P) system. In this study, we investigated whether cotargeting the EGFR and SphK1/S1P pathways in TNBC cells results in greater growth inhibition compared with blocking either alone, and might therefore have novel therapeutic potential in TNBC. In four TNBC cell lines, exogenous IGFBP-3 enhanced ligand-stimulated EGFR activation, associated with increased SphK1 localization to the plasma membrane. The effect of exogenous IGFBP-3 on EGFR activation was blocked by pharmacologic inhibition or siRNA-mediated silencing of SphK1, and silencing of endogenous IGFBP-3 also suppressed EGF-stimulated EGFR activation. Real-time analysis of cell proliferation revealed a combined effect of EGFR inhibition by gefitinib and SphK1 inhibition using SKi-II. Growth of MDA-MB-468 xenograft tumors in mice was significantly inhibited by SKi-II and gefitinib when used in combination, but not as single agents. We conclude that IGFBP-3 promotes growth of TNBC cells by increasing EGFR signaling, that this is mediated by SphK1, and that combined inhibition of EGFR and SphK1 has potential as an anticancer therapy in TNBC in which EGFR and IGFBP-3 expression is high. Mol Cancer Ther; 13(2); 316-28. Ó2013 AACR.

Following exposure to radiation and chemotherapeutic agents, the epidermal growth factor receptor (EGFR) can modulate the repair of DNA double-strand breaks (DSB) by forming protein complexes that include the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). This is one of the key mechanism by which tumors become resistant to DNA-damaging therapies. Our previous studies have shown that insulin-like growth factor binding protein-3 (IGFBP-3) is a substrate for DNA-PKcs, and can transactivate EGFR. We therefore questioned whether IGFBP-3 might interact with the EGFR-DNA-PK complex that regulates the DNA damage response. 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. In the estrogen receptor-negative breast cancer cell lines MDA-MB-468 and Hs578T, which express IGFBP-3 highly, nuclear localization of EGFR and IGFBP-3 was enhanced by treatment with cytotoxic drugs etoposide or doxorubicin and reduced by the EGFR kinase inhibitor gefitinib. Enhanced association among IGFBP-3, EGFR and DNA-PKcs, following the exposure to DNA-damaging drugs was supported by both co-immunoprecipitation analysis and direct visualization by proximity ligation assay. The activation of DNA-PKcs at Ser2056, DNA repair as measured by a nonhomologous end-joining assay, and the increase in EGFR and DNA-PKcs interaction induced by DNA-damaging agents, were all decreased by IGFBP-3 silencing, suggesting that IGFBP-3 has an obligatory role in the DNA repair response to DNA-damaging therapy. In conclusion, IGFBP-3 co-translocation to the nucleus of breast cancer cells and its formation of a complex with DNA-PKcs and EGFR in response to DNA damage shows its potential involvement in the regulation of DNA repair. This suggests the possibility of a therapeutic approach for sensitizing breast cancer to chemo- or radiotherapy by targeting the DNA repair function of IGFBP-3.