title: Esophageal Adenocarcinoma Cells and Xenograft Tumors Exposed to Erb-b2 Receptor Tyrosine Kinase 2 and 3 Inhibitors Activate Transforming Growth Factor Beta Signaling, Which Induces Epithelial to Mesenchymal Transition.
abstract: Drugs that inhibit the erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) are the standard treatment of patients with different types of cancer, including HER2-overexpressing gastroesophageal tumors. Unfortunately, cancer cells become resistant to these drugs, so overall these drugs provide little benefit to patients with these tumors. We investigated mechanisms that mediate resistance of esophageal adenocarcinoma (EAC) cells and patient-derived xenograft tumors to ERBB inhibitors.
  
  
  
  We cultured primary tumor cells, isolated from EAC patient samples, and OE19 and OE33 EAC cell lines with trastuzumab (an inhibitor of HER2), with or without pertuzumab (which inhibits dimerization of HER2 with HER3) or a specific antibody against HER3 (anti-HER3). HER2 was knocked down by expression of small hairpin RNAs. In addition, cells were incubated with NRG1-β, a mediator of HER2-HER3 signaling, or A83-01, an inhibitor of transforming growth factor beta (TGFβ) signaling. Cells were analyzed for markers of the epithelial to mesenchymal transition (EMT) using flow cytometry, immunofluorescence, and quantitative reverse transcription polymerase chain reaction. We performed limiting dilution, transwell, and cell viability assays to study the functional effects of HER2 and HER3 inhibition and reactivation. We analyzed publicly available EAC gene expression datasets to correlate expression of ERBB genes with genes encoding epithelial and mesenchymal proteins. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were given subcutaneous injections of AMC-EAC-007B cells and also given injections of single or combined inhibitors; growth of these patient-derived xenograft tumors was quantified.
  
  
  
  EAC cells incubated with trastuzumab decreased expression of epithelial markers (CD24, CD29, and CDH1) and increased expression of mesenchymal markers (CXCR4, VIM, ZEB1, SNAI2, and CDH2), compared with cells not exposed to trastuzumab, indicating induction of EMT. Addition of NRG1-β to these cells restored their epithelial phenotype. Incubation of EAC cells with trastuzumab and pertuzumab accelerated the expression of EMT markers, compared with cells incubated with trastuzumab alone. EAC cells cultured for 2 months with a combination of trastuzumab and pertuzumab became resistant to chemotherapeutic agents (5-fluoruracil, carboplatin, cisplatin, eribulin, and paclitaxel), based on their continued viability, which was accompanied with an enhanced migratory capacity in transwell assays and clonogenicity in limiting dilution analyses. In comparisons of EAC gene expression patterns, we associated high expression of ERBB3 with an epithelial gene expression signature; expression of TGFβ correlated with expression of EMT-related genes, and we found an inverse correlation between expression of TGFB1 and ERBB3. EAC cells incubated with ERBB inhibitors began to secrete ligands for the TGFβ receptor and underwent EMT. Incubation of EAC cells with trastuzumab, followed by 10 days of incubation with the TGFβ receptor inhibitor in the presence of trastuzumab, caused cells to regain an epithelial phenotype. EAC patient-derived xenograft tumors grew more slowly in mice given the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor than in mice given single agents or a combination of trastuzumab and pertuzumab. Tumors exposed to trastuzumab and pertuzumab expressed EMT markers and were poorly differentiated, whereas tumors exposed to the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor expressed epithelial markers and were more differentiated.
  
  
  
  EAC cells become resistant to trastuzumab and pertuzumab by activating TGFβ signaling, which induces EMT. Agents that block TGFβ signaling can increase the anti-tumor efficacies of trastuzumab and pertuzumab.
authors.affiliation: Cancer Center Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands; Cancer Center Amsterdam, Department of Medical Oncology, Academic Medical Center, Amsterdam, The Netherlands. Electronic address: e.a.ebbing@amc.uva.nl.
authors.forename: Eva A
authors.initials: EA
authors.lastname: Ebbing
authors.affiliation: Cancer Center Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands; Cancer Center Amsterdam, Department of Medical Oncology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Anne
authors.initials: A
authors.lastname: Steins
authors.affiliation: Cancer Center Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Fessler
authors.affiliation: Cancer Center Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands; Cancer Center Amsterdam, Department of Medical Oncology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Phylicia
authors.initials: P
authors.lastname: Stathi
authors.affiliation: The University of Western Australia, Crawley, WA, Australia.
authors.forename: Willem Joost
authors.initials: WJ
authors.lastname: Lesterhuis
authors.affiliation: Cancer Center Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands; Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Kausilia K
authors.initials: KK
authors.lastname: Krishnadath
authors.affiliation: Cancer Center Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Louis
authors.initials: L
authors.lastname: Vermeulen
authors.affiliation: Cancer Center Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands; Cancer Genomics Center, Center for Molecular Medicine, Utrecht, The Netherlands.
authors.forename: Jan Paul
authors.initials: JP
authors.lastname: Medema
authors.affiliation: Cancer Center Amsterdam, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Maarten F
authors.initials: MF
authors.lastname: Bijlsma
authors.affiliation: Cancer Center Amsterdam, Department of Medical Oncology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Hanneke W M
authors.initials: HWM
authors.lastname: van Laarhoven
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy* / Q000473:pathology; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology* / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002470:Cell Survival; D004347:Drug Interactions; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D004938:Esophageal Neoplasms / Q000188:drug therapy* / Q000473:pathology; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D009368:Neoplasm Transplantation; D020890:Neuregulin-1 / Q000494:pharmacology; D061251:Primary Cell Culture; D011720:Pyrazoles / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D020893:Receptor, ErbB-3 / Q000037:antagonists & inhibitors / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D013882:Thiosemicarbazones / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000235:genetics; D000068878:Trastuzumab / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D020890:Neuregulin-1; D011720:Pyrazoles; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D013882:Thiosemicarbazones; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D000068878:Trastuzumab; C507011:A-83-01; C117467:neuregulin beta; C485206:pertuzumab
keywords: Anti-Tumor Agent; Biologic; Cancer Stem Cell (CSC); Targeted Therapy