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.

Background: Cytokine production is critical in ischemia/reperfusion (IR) injury. Acetylcholine binds to macrophages and inhibits cytokine synthesis, through the cholinergic anti-inflammatory pathway. This study examined the role of the cholinergic pathway in cytokine production and hepatic IR-injury.

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Purpose Breast cancer is the second leading cause of cancer mortality among women worldwide. The major problem with current treatments is tumor resistance, recurrence, and disease progression. ErbB-2 positive breast tumors are aggressive and frequently become resistant to trastuzumab or lapatinib. We showed previously that Notch-1 is required for trastuzumab resistance in ErbB-2 positive breast cancer. Experimental Design Here, we sought to elucidate mechanisms by which ErbB-2 attenuates Notch signaling and how this is reversed by trastuzumab or lapatinib. Results The current study elucidates a novel Notch inhibitory mechanism by which PKCα downstream of ErbB-2: 1. Restricts the availability of Jagged-1 at the cell surface to trans activate Notch; 2. Restricts the critical interaction between Jagged-1 and Mindbomb-1, an E3 ligase that is required for Jagged-1 ubiquitinylation and subsequent Notch activation; 3. Reverses trastuzumab resistance in vivo; and 4. Predicts better outcome in women with ErbB-2 positive breast cancer. Conclusions The clinical impact of these studies is PKCα is potentially a good prognostic marker for low Notch activity and increased trastuzumab sensitivity in ErbB-2 positive breast cancer. Moreover, women with ErbB-2 positive breast tumors expressing high Notch activation and low PKCα expression could be the best candidates for anti-Notch therapy.