Abstract P2-09-04: Development of novel combination therapy of IGF-1R/InsR and MEK inhibitors in triple-negative breast cancer (TNBC)

Abstract

Abstract Recent studies found that TNBC had a highly activated profile in the insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (InsR) pathway. Patients in whom the IGF-1R/InsR pathway was activated had a worse prognosis than did those in whom the pathway was not activated. We also previously found that the tumor-initiating cells in mouse TNBCs had a highly activated IGF-1R pathway. On the basis of these findings, we tested an IGF-1R/InsR dual kinase inhibitor, KW-2450, in TNBC. Results: To investigate the antitumor effects of KW-2450 in TNBC, we first confirmed the high IGF-1R and low InsR expression in TNBC cell lines (e.g., SUM149, MDA-MB-231, MDA-MB-468). An in vitro growth inhibition assay revealed that KW-2450 inhibited cell growth (all IC50 <0.5 μM), and an agar assay for colony formation confirmed this antitumor effect in TNBC cells. We also tested KW-2450's inhibitory effect against cancer stem cell (CSC) activity. With KW-2450 treatment of SUM149 and MDA-MB-231 cells, proportions of CSCs, profiled as CD44+CD24−, were significantly reduced in a dose-dependent manner. Indeed, KW-2450 dose-dependently inhibited mammosphere-forming, a hallmark of CSC activity. Cell cycle analysis revealed that KW-2450 induced mitotic accumulation and apoptosis in TNBC cells. Interestingly, MDA-MB-468 cells were the most susceptible to death, and their sensitivity to KW-2450 was associated with the high activation level of the mitotic checkpoint, the levels of which were determined by accumulation of cyclin B1 (on Western blot) and of phospho-histone H3–positive cells, a mitosis marker (on FACS). In contrast, SUM149 and MDA-MB-231 cells, which are relatively unsusceptible to death from KW-2450, exited from mitosis (as indicated by an accumulation of 8N; octaploidy) without significant cell death. This variable sensitivity to KW-2450 was also observed in in vivo studies. We confirmed that MDA-MB-468 tumors that were treated with 80 mg/kg of KW-2450 in vivo were stable and had many more mitotic cells than did those treated with vehicle control, which suggests that mitotic accumulation is a key process for this antitumor effect. Since it is known that any of the MAPKs (e.g., JNKs, p38 kinase, ERKs) become activated at the mitotic phase in mammalian cells, we next investigated whether the activation levels of MAPKs play critical roles in either mitotic progression or mitotic death in TNBC. Western blot analysis revealed that KW-2450 activated ERKs, but not JNKs or p38 kinase, in KW-2450–insensitive MDA-MB-231 and SUM149 cells, suggesting that ERK activation may promote mitotic progression but not mitotic death. Indeed, the combination of a MEK inhibitor (AZD6244), which inhibits ERK activation, and KW-2450 significantly reduced the 8N fraction and increased cell death in SUM149 and MDA-MB-231 cells. Conclusion: KW-2450 had significant antitumor effects in vitro and in vivo in TNBC cells. KW-2450–induced cell death, accompanied by mitotic accumulation, depended on mitotic checkpoint activity. TNBC cells refractory to KW-2450 were sensitized to KW-2450 by the addition of a MEK inhibitor. This novel combination therapy targeting IGF-1R/InsR and MEK in TNBCs, whose mitotic checkpoints are commonly abrogated, needs to be developed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-04.