Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1.

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: Yujiro Hayashi, Michael R Bardsley, Yoshitaka Toyomasu, Srdjan Milosavljevic, Gabriella B Gajdos, Kyoung Moo Choi, K Marie Reid-Lombardo, Michael L Kendrick, Juliane Bingener-Casey, Chih-Min Tang, Jason K Sicklick, Simon J Gibbons, Gianrico Farrugia, Takahiro Taguchi, Anu Gupta, Brian P Rubin, Jonathan A Fletcher, Abhijit Ramachandran, Tamas Ordog
• Journal: Gastroenterology (Gastroenterology) Vol. 149 Issue 2 Pg. 420-32.e16 (Aug 2015) ISSN: 1528-0012 [Electronic] United States
• PMID: 25865047 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

Chemical References

• Benzamides
• Benzimidazoles
• Biomarkers, Tumor
• DNA-Binding Proteins
• Etv1 protein, mouse
• Nucleic Acid Precursors
• Piperazines
• Piperidines
• Pyrimidines
• Transcription Factors
• Imatinib Mesylate
• Proto-Oncogene Proteins c-kit
• Receptor, Platelet-Derived Growth Factor alpha
• Receptor, Platelet-Derived Growth Factor beta
• crenolanib

Topics

• Animals
• Benzamides (metabolism)
• Benzimidazoles (metabolism)
• Biomarkers, Tumor (metabolism)
• Cell Line, Tumor
• Cell Proliferation (genetics)
• DNA-Binding Proteins (genetics)
• Flow Cytometry
• Gastrointestinal Stromal Tumors (genetics, metabolism)
• Gene Knockdown Techniques (methods)
• Humans
• Imatinib Mesylate
• Immunohistochemistry
• Mice
• Mice, Inbred BALB C
• Mutation
• Nucleic Acid Precursors (genetics)
• Phosphorylation (genetics)
• Piperazines (metabolism)
• Piperidines (metabolism)
• Proto-Oncogene Proteins c-kit (genetics, metabolism)
• Pyrimidines (metabolism)
• Receptor, Platelet-Derived Growth Factor alpha (genetics, metabolism)
• Receptor, Platelet-Derived Growth Factor beta (metabolism)
• Signal Transduction (genetics)
• Transcription Factors (genetics)