Background:We reported that Notch-1, a potent breast oncogene, is activated in response to trastuzumab and contributes to trastuzumab resistance in vitro. We sought to determine the preclinical benefit of combining a Notch inhibitor (γ-secretase inhibitor (GSI)) and trastuzumab in both trastuzumab-sensitive and trastuzumab-resistant, ErbB-2-positive, BT474 breast tumours in vivo. We also studied if the combination therapy of lapatinib plus GSI can induce tumour regression of ErbB-2-positive breast cancer.Methods:We generated orthotopic breast tumour xenografts from trastuzumab- or lapatinib-sensitive and trastuzumab-resistant BT474 cells. We investigated the antitumour activities of two distinct GSIs, LY 411 575 and MRK-003, in vivo.Results:Our findings showed that combining trastuzumab plus a GSI completely prevented (MRK-003 GSI) or significantly reduced (LY 411 575 GSI) breast tumour recurrence post-trastuzumab treatment in sensitive tumours. Moreover, combining lapatinib plus MRK-003 GSI showed significant reduction of tumour growth. Furthermore, a GSI partially reversed trastuzumab resistance in resistant tumours.Conclusion:Our data suggest that a combined inhibition of Notch and ErbB-2 signalling pathways could decrease recurrence rates for ErbB-2-positive breast tumours and may be beneficial in the treatment of recurrent trastuzumab-resistant disease.

IntroductionWomen with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells.MethodsReal-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both.ResultsHerein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G1 phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo.ConclusionsTaken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes.

Amplification, over-expression, and/or hyperactivity of ErbB-2 occur in 15-30% of breast tumors which include both luminal B and the HER2+ subtypes. Metastatic breast tumors that overexpress ErbB-2 are difficult to treat and generally resistant to trastuzumab. Lapatinib, a dual EGFR/ErbB-2 tyrosine kinase inhibitor, is approved for the treatment of ErbB-2-positive breast cancer that has advanced during or after trastuzumab treatment. However, resistance to lapatinib occurs in cell culture models in vitro. 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. 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 (MRK-003, GSI). Additionally, in an independent study we showed that GSI also prevented resistance to lapatinib. Since lapatinib is a very potent inhibitor of ErbB-2 tyrosine kinase activity in vitro, we asked whether a combination therapy based on lapatinib and GSI can prevent the resistant phenotype by using a pre-clinical in vivo model. BT474 breast tumor xenografts were generated in athymic mice and randomized to vehicle, GSI, lapatinib, or lapatinib plus a GSI. Tumor size was measured up to 13 weeks with treatments. The xenograft studies using BT474 lapatinib sensitive cells demonstrated that lapatinib treatment alone inhibited tumor growth by 40%. GSI alone had no effect on growth of tumors compared to vehicle control. However, GSI plus lapatinib significantly reduced tumor growth where regression was observed. Tumor histology of lapatinib plus GSI-treated tumors displayed a significant decrease in Ki67, a proliferation marker, and an increase in apoptotic cells as measured by TUNEL. Furthermore, both ERK1/2 and AKT1 phosphorylated proteins were undetectable by western blot in tumors treated with lapatinib plus GSI. These results suggest that both ErbB-2 and Notch activities must be inhibited to induce tumor regression of ErbB-2 positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2553. doi:10.1158/1538-7445.AM2011-2553

Notch-1 is a breast oncogene that is overexpressed with its ligand Jagged-1 in human breast cancers-associated with the poorest overall survival. Notch signaling can be inhibited by a gamma-secretase inhibitor (GSI). We have recently determined that the estrogen receptor-alpha or ErbB-2 inhibits Notch-1 activation in breast cancer cells. In the current study, we sought to uncover the complex crosstalk between ER, ErbB-2, and Notch-1 and ask whether triple combination treatment can disrupt this complex cross-talk model to inhibit growth of ER positive breast cancer cells (MCF-7) and possibly prevent anti-hormone resistance. The results demonstrated that following 1 week of estrogen deprivation, MCF-7 cells develop resistance and regrow. Canonical Notch gene targets: Hes-1, Hey-1, Hes-5, and Deltex-1 are significantly increased at this 1 week time point. Similarly, ErbB-2 mRNA increased significantly after 72 hours of estrogen deprivation. Upon treatment with fulvestrant, a pure ant-estrogen, ER transcriptional activity as measured by PS2 mRNA is inhibited after one week. Interestingly, the combination of fulvestrant and lapatinib, an anti-ErbB-2 tyrosine kinase inhibitor, synergistically increased both Deltex-1 and ErbB-2 mRNAs which were reduced upon GSI treatment. Western blot analysis showed an increase in ER and tyrosine-phosphorylated ErbB-2 and total ErbB-2 proteins. This increase in protein expression was further increased upon dual treatment with fulvestrant and lapatinib. Conversely, treatment with a Notch inhibitor, GSI caused a decrease in both total and tyrosine-phosphorylated ErbB-2. Cell cycle analysis showed that fulvestrant treatment significantly growth arrested cells in G1 while decreasing cells in S-phase. However, lapatnib or GSI or the combination treatments did not further enhance the growth inhibitory effects of fulvestrant. Similar results were observed using Annexin-V analysis by flow cytometry as a measure of early apoptosis. These results suggest that while inhibiting ER and ErbB-2 simultaneously increases Notch activation in a synergistic manner, this dramatic increase in Notch activity in the short term does not sensitize ER positive breast cancer cells to a Notch inhibitor to undergo cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2284. doi:10.1158/1538-7445.AM2011-2284