Patritumab

To induce the phosphorylation of HER3, the cells were stimulated with HRG-β1 (R&D Systems, Milan, Italy), at the concentration of 10 ng/ml for the indicated times. The anti-HER3 antibody patritumab was kindly provided from Daiichi Sankyo (Tokyo, Japan) was used at the concentration of 10 μg/ml. The chemotherapeutic drugs 5-FU and Ox were purchased from Teva Italia (Assago, Mi) and used at the concentration of 50 and 20 μM, respectively. The anti-MEK inhibitor trametinib (GSK-1120212) (Selleckchem, Milan, Italy) was used at the concentration of 5 nM. The treatment were performed at the indicated times.

The DiFi human colorectal cancer cell line was kindly provided by P. A. Janne (Dana Farber Cancer Institute). DiFi cells were maintained under a humidified atmosphere of 5% CO₂ in air at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing high glucose and supplemented with Ham's F-12 and 10% fetal bovine serum (FBS). Cetuximab was obtained from Merck Serono, and patritumab was kindly provided by Daiichi-Sankyo (Tokyo, Japan). Recombinant human heregulin (NRG1-β1/HRG1-β1 extracellular domain) was obtained from R&D Systems.

Human SKBR3, BT474, MDA‐MB‐453, and HCC1419 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA). The human JIMT‐1 cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Braunschweig, Germany). SKBR3 cells transfected with heregulin (SKBR3‐HRG) and SKBR3 cells transfected with the corresponding empty vector (SKBR3‐Mock) were previously established.14 We also established BT474‐HRG and BT474‐Mock cells transfected with heregulin or empty vector, respectively, as previously described.14 Cells were maintained in a humidified atmosphere of 5% CO₂/95% air at 37°C in Roswell Park Memorial Institute 1640 medium (for JIMT‐1 cells) or Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (FBS). Cells were routinely tested for mycoplasma using MycoAlert (LT07; Lonza, Basel, Switzerland) and were negative. Trastuzumab and pertuzumab were purchased from Chugai Pharmaceuticals (Tokyo, Japan). Patritumab was provided by Daiichi‐Sankyo Co., Ltd. (Tokyo, Japan).