Notch-1 Confers Resistance to ErbB-2 Targeted Inhibition by Regulating PTEN, p27, and IGF-1R.

Abstract

Abstract Background: Amplification, over-expression, and/or hyperactivity of ErbB-2 occur in 30% of breast tumors. Metastatic breast tumors that overexpress ErbB-2 are difficult to treat and generally resistant to trastuzumab or lapatinib. We have recently published that overexpression of ErbB-2 suppresses Notch-1 activity and this is reversed by inhibitors of ErbB-2, trastuzumab or a tyrosine kinase inhibitor (Osipo et al., 2008. Oncogene). Furthermore, we demonstrated that trastuzumab resistance is reversed when Notch-1 is downregulated by siRNA or when cells are treated with a gamma-secretase inhibitor. Overexpression of IGF-1R and/or downregulation of PTEN, and p27Kip1 have been shown to be very sensitive markers of trastuzumab resistance. Based on our results and what is known from the literature, we asked by what mechanism Notch-1 contributes to trastuzumab resistance and can a combination therapy prevent or reverse the resistant phenotype by using a pre-clinical in vivo model.Methods: Using a pcDNA3.1-NIC construct expressing activated Notch-1 and a Notch-1 siRNA, we either increased NIC levels in trastuzumab sensitive or downregulated Notch-1 in trastuzumab resistant BT474 cells, respectively and measured protein and mRNA expressions of PTEN, p27Kip1, and the IGF-1R. In addition, BT474 breast tumor xenografts were generated in athymic mice and randomized to vehicle, MRK-003 or Ly411,575 gamma-secretase inhibitor, trastuzumab, or trastuzumab plus a gamma-secretase inhibitor. Tumor size was measured up to 14 weeks with treatments and up to 12 weeks after stopping treatments.Results: The results showed that intracellular, active Notch-1 (ICN) increased mRNA and protein expression of the IGF-1R, while simultaneously decreased protein expressions of PTEN and p27Kip1 in ErbB-2 positive breast cancer cells sensitive to trastuzumab. Conversely, knockdown of Notch-1 by siRNA in resistant cells decreased IGF-1R, while increasing PTEN and p27Kip1. The xenograft studies demonstrated that combining a GSI with trastuzumab either completely prevented tumor recurrence (MRK-003 GSI) or significantly delayed tumor recurrence (Ly 411,575 GSI).Conclusions: Taken together, these results suggest that Notch-1 potentially contributes to trastuzumab resistance by upregulating the IGF-1R and downregulating PTEN and p27Kip1 in vitro. The benefit of inhibiting Notch activity using a GSI in combination with trastuzumab is prevention of resistance and tumor recurrence. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3123.