title: Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1.
abstract: OBJECTIVE
  In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1.
  
  
  METHODS
  We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation.
  
  
  RESULTS
  KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA.
  
  
  CONCLUSIONS
  In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.
authors: Yujiro
authors: Y
authors: Hayashi
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.
authors: Michael R
authors: MR
authors: Bardsley
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.
authors: Yoshitaka
authors: Y
authors: Toyomasu
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.
authors: Srdjan
authors: S
authors: Milosavljevic
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.
authors: Gabriella B
authors: GB
authors: Gajdos
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota.
authors: Kyoung Moo
authors: KM
authors: Choi
authors: Department of Surgery, Mayo Clinic, Rochester, Minnesota.
authors: K Marie
authors: KM
authors: Reid-Lombardo
authors: Department of Surgery, Mayo Clinic, Rochester, Minnesota.
authors: Michael L
authors: ML
authors: Kendrick
authors: Department of Surgery, Mayo Clinic, Rochester, Minnesota.
authors: Juliane
authors: J
authors: Bingener-Casey
authors: Division of Surgical Oncology, Moores Cancer Center, University of California, San Diego, California.
authors: Chih-Min
authors: CM
authors: Tang
authors: Division of Surgical Oncology, Moores Cancer Center, University of California, San Diego, California.
authors: Jason K
authors: JK
authors: Sicklick
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota.
authors: Simon J
authors: SJ
authors: Gibbons
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota.
authors: Gianrico
authors: G
authors: Farrugia
authors: Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Kochi, Japan.
authors: Takahiro
authors: T
authors: Taguchi
authors: Departments of Pathology and Molecular Genetics, Lerner Research Institute and Taussig Cancer Center, Cleveland Clinic, Cleveland, Ohio.
authors: Anu
authors: A
authors: Gupta
authors: Departments of Pathology and Molecular Genetics, Lerner Research Institute and Taussig Cancer Center, Cleveland Clinic, Cleveland, Ohio.
authors: Brian P
authors: BP
authors: Rubin
authors: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.
authors: Jonathan A
authors: JA
authors: Fletcher
authors: AROG Pharmaceuticals, LLC, Dallas, Texas.
authors: Abhijit
authors: A
authors: Ramachandran
authors: Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota; Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota. Electronic address: ordog.tamas@mayo.edu.
authors: Tamas
authors: T
authors: Ordog
mesh_terms: D000818:Animals; D001549:Benzamides / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005434:Flow Cytometry; D046152:Gastrointestinal Stromal Tumors / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques / Q000379:methods; D006801:Humans; D000068877:Imatinib Mesylate; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D009694:Nucleic Acid Precursors / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D010879:Piperazines / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000235:genetics / Q000378:metabolism*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D001549:Benzamides; D001562:Benzimidazoles; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; C490641:Etv1 protein, mouse; D009694:Nucleic Acid Precursors; D010879:Piperazines; D010880:Piperidines; D011743:Pyrimidines; D014157:Transcription Factors; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit; D020796:Receptor, Platelet-Derived Growth Factor alpha; D020797:Receptor, Platelet-Derived Growth Factor beta; C577197:crenolanib
keywords: Cancer; Receptor Tyrosine Kinase; Signal Transduction; Stem Cells