title: Overcoming acquired resistance to cetuximab by dual targeting HER family receptors with antibody-based therapy.
abstract: BACKGROUND
  Cetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers. However, many patients who initially respond to cetuximab acquire resistance. To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (Ctx(R)) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226. Previous studies characterizing this model revealed that: 1) EGFR was robustly overexpressed in Ctx(R) clones due to decreased EGFR ubiquitination and degradation and 2) Ctx(R) clones expressed increased HER2 and HER3 activation resulting in constitutive activation of the PI3K/AKT signaling axis. These findings suggest that dual targeting HER family receptors would be highly beneficial in the Ctx(R) setting.
  
  
  RESULTS
  Since HER3 has been implicated in resistance to EGFR inhibitors, the efficacy of dually targeting both EGFR and HER3 in Ctx(R) models was evaluated. First, EGFR and HER3 expression were knocked down with siRNAs. Compared to the Ctx(S) parental cell line (HP), all Ctx(R) clones exhibited robust decreases in cell proliferation upon dual knockdown. Analysis of Ctx(R) clones indicated that neuregulin-1 was highly overexpressed compared to HP cells. Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab. Next, dual treatment of Ctx(R) clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects. Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT. Treatment with both mAbs resulted in knockdown of both signaling pathways simultaneously. HER2 was also strongly inactivated upon dual mAb therapy, suggesting that this treatment regimen can diminish signaling from three HER family receptors. De novo CtxR H226 mouse xenografts were established to determine if dual therapy could overcome acquired resistance to cetuximab in vivo. Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only. Combinatorial-treated xenograft tumors expressed decreased Ki67 and increased cleaved caspase-3 levels compared to tumors treated with either monotherapy.
  
  
  CONCLUSIONS
  These studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.
authors.forename: Mari
authors.initials: M
authors.lastname: Iida
authors.forename: Toni M
authors.initials: TM
authors.lastname: Brand
authors.forename: Megan M
authors.initials: MM
authors.lastname: Starr
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Huppert
authors.forename: Neha
authors.initials: N
authors.lastname: Luthar
authors.forename: Harsh
authors.initials: H
authors.lastname: Bahrar
authors.forename: John P
authors.initials: JP
authors.lastname: Coan
authors.forename: Hannah E
authors.initials: HE
authors.lastname: Pearson
authors.forename: Ravi
authors.initials: R
authors.lastname: Salgia
authors.affiliation: Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Wisconsin Institute for Medical Research, 1111 Highland Ave,, Madison, WI 53705, USA. dlwheeler@wisc.edu.
authors.forename: Deric L
authors.initials: DL
authors.lastname: Wheeler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized / Q000627:therapeutic use*; D057134:Antibodies, Neutralizing; D000970:Antineoplastic Agents / Q000627:therapeutic use; D000080908:Broadly Neutralizing Antibodies; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000068818:Cetuximab; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing; D000970:Antineoplastic Agents; D000080908:Broadly Neutralizing Antibodies; D000068818:Cetuximab; D066246:ErbB Receptors; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; C585471:patritumab; C581292:ERBB3 protein, human