title: Sustained Inhibition of HER3 and EGFR Is Necessary to Induce Regression of HER2-Amplified Gastrointestinal Carcinomas.
abstract: OBJECTIVE
  Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities.
  
  
  METHODS
  The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays.
  
  
  RESULTS
  Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib-or, importantly, monotherapy with afatinib-resulted in overt tumor shrinkage.
  
  
  CONCLUSIONS
  Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519-31. ©2015 AACR.
authors: Department of Oncology, University of Turin Medical School, Turin, Italy. Laboratory of Translational Cancer Medicine, Candiolo Cancer Institute-FPO IRCCS, Turin, Italy.
authors: Simonetta M
authors: SM
authors: Leto
authors: Laboratory of Translational Cancer Medicine, Candiolo Cancer Institute-FPO IRCCS, Turin, Italy.
authors: Francesco
authors: F
authors: Sassi
authors: Department of Oncology, University of Turin Medical School, Turin, Italy. Laboratory of Translational Cancer Medicine, Candiolo Cancer Institute-FPO IRCCS, Turin, Italy.
authors: Irene
authors: I
authors: Catalano
authors: Laboratory of Methodology for Biomedical Research, Oncology Department, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy.
authors: Valter
authors: V
authors: Torri
authors: Department of Oncology, University of Turin Medical School, Turin, Italy. Laboratory of Translational Cancer Medicine, Candiolo Cancer Institute-FPO IRCCS, Turin, Italy.
authors: Giorgia
authors: G
authors: Migliardi
authors: Department of Oncology, University of Turin Medical School, Turin, Italy. Laboratory of Translational Cancer Medicine, Candiolo Cancer Institute-FPO IRCCS, Turin, Italy.
authors: Eugenia R
authors: ER
authors: Zanella
authors: Merus B.V., CH Utrecht, the Netherlands.
authors: Mark
authors: M
authors: Throsby
authors: Department of Oncology, University of Turin Medical School, Turin, Italy. Laboratory of Translational Cancer Medicine, Candiolo Cancer Institute-FPO IRCCS, Turin, Italy. Istituto Nazionale di Biostrutture e Biosistemi, INBB, Rome, Italy. andrea.bertotti@unito.it livio.trusolino@unito.it.
authors: Andrea
authors: A
authors: Bertotti
authors: Department of Oncology, University of Turin Medical School, Turin, Italy. Laboratory of Translational Cancer Medicine, Candiolo Cancer Institute-FPO IRCCS, Turin, Italy. andrea.bertotti@unito.it livio.trusolino@unito.it.
authors: Livio
authors: L
authors: Trusolino
mesh_terms: D000077716:Afatinib; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002277:Carcinoma / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005770:Gastrointestinal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D005784:Gene Amplification; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D000077341:Lapatinib; D051379:Mice; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011799:Quinazolines / Q000008:administration & dosage / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000068878:Trastuzumab / Q000008:administration & dosage / Q000494:pharmacology; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D000077716:Afatinib; D000970:Antineoplastic Agents; D066246:ErbB Receptors; D000077341:Lapatinib; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D000068878:Trastuzumab