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Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1.
Gastroenterology. 2015 Aug; 149(2):420-32.e16.G

Abstract

BACKGROUND & AIMS

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+Show Affiliations

Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota.Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota.Department of Surgery, Mayo Clinic, Rochester, Minnesota.Department of Surgery, Mayo Clinic, Rochester, Minnesota.Department of Surgery, Mayo Clinic, Rochester, Minnesota.Division of Surgical Oncology, Moores Cancer Center, University of California, San Diego, California.Division of Surgical Oncology, Moores Cancer Center, University of California, San Diego, California.Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota.Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota; Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota.Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Kochi, Japan.Departments of Pathology and Molecular Genetics, Lerner Research Institute and Taussig Cancer Center, Cleveland Clinic, Cleveland, Ohio.Departments of Pathology and Molecular Genetics, Lerner Research Institute and Taussig Cancer Center, Cleveland Clinic, Cleveland, Ohio.Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.AROG Pharmaceuticals, LLC, Dallas, Texas.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.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25865047

Citation

Hayashi, Yujiro, et al. "Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT By Stabilizing ETV1." Gastroenterology, vol. 149, no. 2, 2015, pp. 420-32.e16.
Hayashi Y, Bardsley MR, Toyomasu Y, et al. Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1. Gastroenterology. 2015;149(2):420-32.e16.
Hayashi, Y., Bardsley, M. R., Toyomasu, Y., Milosavljevic, S., Gajdos, G. B., Choi, K. M., Reid-Lombardo, K. M., Kendrick, M. L., Bingener-Casey, J., Tang, C. M., Sicklick, J. K., Gibbons, S. J., Farrugia, G., Taguchi, T., Gupta, A., Rubin, B. P., Fletcher, J. A., Ramachandran, A., & Ordog, T. (2015). Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1. Gastroenterology, 149(2), 420-e16. https://doi.org/10.1053/j.gastro.2015.04.006
Hayashi Y, et al. Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT By Stabilizing ETV1. Gastroenterology. 2015;149(2):420-32.e16. PubMed PMID: 25865047.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1. AU - Hayashi,Yujiro, AU - Bardsley,Michael R, AU - Toyomasu,Yoshitaka, AU - Milosavljevic,Srdjan, AU - Gajdos,Gabriella B, AU - Choi,Kyoung Moo, AU - Reid-Lombardo,K Marie, AU - Kendrick,Michael L, AU - Bingener-Casey,Juliane, AU - Tang,Chih-Min, AU - Sicklick,Jason K, AU - Gibbons,Simon J, AU - Farrugia,Gianrico, AU - Taguchi,Takahiro, AU - Gupta,Anu, AU - Rubin,Brian P, AU - Fletcher,Jonathan A, AU - Ramachandran,Abhijit, AU - Ordog,Tamas, Y1 - 2015/04/09/ PY - 2014/08/13/received PY - 2015/04/02/revised PY - 2015/04/06/accepted PY - 2015/4/14/entrez PY - 2015/4/14/pubmed PY - 2015/10/20/medline KW - Cancer KW - Receptor Tyrosine Kinase KW - Signal Transduction KW - Stem Cells SP - 420 EP - 32.e16 JF - Gastroenterology JO - Gastroenterology VL - 149 IS - 2 N2 - BACKGROUND & AIMS: 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. SN - 1528-0012 UR - https://www.unboundmedicine.com/medline/citation/25865047/Platelet_Derived_Growth_Factor_Receptor_α_Regulates_Proliferation_of_Gastrointestinal_Stromal_Tumor_Cells_With_Mutations_in_KIT_by_Stabilizing_ETV1_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0016-5085(15)00497-7 DB - PRIME DP - Unbound Medicine ER -