The functional role of AP 1 is to recruit and direct appropriate factors to regulate gene expres sion and promote proliferation, differentiation, inhibitor CHIR99021 inflam mation and or apoptosis. Previous investigations have determined that overex pression of Notch 1 and Notch 4 plays a critical role in breast tumorigenesis and Inhibitors,Modulators,Libraries that PEA3 overexpression is associated with aggressive breast cancers, particularly the triple negative subtype. Herein we provide novel evidence of a link between two pathways that are overexpressed in breast cancer. PEA3 is a transcriptional activator of Notch 1 and Notch 4 and a repressor of Notch 2 in MDA MB 231 cells, an example of triple negative breast cancer cells. PEA3 mediated Notch 1 transcription is AP 1 independent, while Notch 4 tran scription requires both PEA3 and c JUN.

PEA3 and or Notch signaling are essential for proliferation, Inhibitors,Modulators,Libraries survival and tumor growth of MDA MB 231 cells. Furthermore, PEA3 Inhibitors,Modulators,Libraries is a transcriptional activator of both Notch 1 and Notch 4 in other breast cancer cells. Thus we hypothe sized that targeting of the PEA3 and or Notch pathways might provide a new therapeutic strategy for triple nega tive breast cancer as well as possibly other breast cancer subtypes where PEA3 regulates Notch 1 and or Notch 4. Materials and methods Cell culture and reagents MDA MB 231, SKBr3, BT474 and MCF 7 breast cancer cells were purchased from the American Type Culture Collection. All cell lines were supplemented with 100 umol nonessential amino acids and 1% L glutamine. SKBr3 cells and MDA MB 231 cells were maintained in Iscoves Minimal Essential Medium.

MCF 7 cells were maintained in DMEM Hams Nutrient Mixture F 12. BT474 cells were maintained in DMEM. All cells were cultured in a 37 C incubator with 5% CO2. MRK 003 GSI was kindly provided by Merck Oncology International, Inc. MRK 003 GSI was dissolved in dimethylsulfoxide and stored at 80 C until use. Lactacystin Inhibitors,Modulators,Libraries was purchased from Sigma Aldrich, dissolved in deionized water and stored at 20 C until use. The pcDNA3. 1 expression vector and the PEA3 pcDNA3. 1 expression vector were kindly provided by Dr Mein Chie Hung. The pHMB empty and pHMB TAM 67 expression vectors were kindly provided Inhibitors,Modulators,Libraries by Dr Richard Schultz, Department of Immunology and Microbiology. RNA interference and reagents Control scrambled siRNA a, Notch 1 siRNA, and a smart pool of three distinct PEA3 siRNA were purchased from Santa Cruz Biotechnology.

An unre lated control siRNA and PEA3 siRNA were purchased from Ambion. A smart pool of four distinct c Jun siRNA were purchased from Santa Cruz Biotechnol ogy. Transfection reagents though used were Lipofectamine 2000 and Lipofectamine RNAiMAX, which were purchased from Invitrogen, and FuGENE 6 was purchased from Roche Diag nostics Corporation. Protocols were performed as described by the manufacturers. Antibodies Notch 1, Notch 4, PEA3, c JUN were purchased from Santa Cruz Biotechnology.