ABSTRACT
Tropomyosin receptor kinase (Trk) inhibitors are investigated as a novel targeted therapy in various cancers. We investigated the in vitro effects of the pan-Trk inhibitor GNF-5837 in human neuroendocrine tumor (NET) cells. The human neuroendocrine pancreatic BON1, bronchopulmonary NCI-H727 and ileal GOT1 cell lines were treated with GNF-5837 alone and in combination with everolimus. Cell viability decreased in a time- and dose-dependent manner in GOT1 cells in response to GNF-5837 treatment, while treatment in BON1 and NCI-H727 cells showed no effect on cellular viability. Trk receptor expression determined GNF-5837 sensitivity. GNF-5837 caused downregulation of PI3K-Akt-mTOR signaling, Ras-Raf-MEK-ERK signaling, the cell cycle and increased apoptotic cell death. The combinational treatment of GNF-5837 with everolimus showed a significant enhancement in inhibition of cell viability vs single substance treatments, due to a cooperative PI3K-Akt-mTOR and Ras-Raf-MEK-ERK pathway downregulation, as well as an enhanced cell cycle component downregulation. Immunohistochemical staining for Trk receptors were performed using a tissue microarray containing 107 tumor samples of gastroenteropancreatic NETs. Immunohistochemical staining with TrkA receptor and pan-Trk receptor antibodies revealed a positive staining in pancreatic NETs in 24.2% (8/33) and 33.3% (11/33), respectively. We demonstrated that the pan-Trk inhibitor GNF-5837 has promising anti-tumoral properties in human NET cell lines expressing the TrkA receptor. Immunohistochemical or molecular screening for Trk expression particularly in pancreatic NETs might serve as predictive marker for molecular targeted therapy with Trk inhibitors.
Subject(s)
Neuroendocrine Tumors/drug therapy , Protein Kinase Inhibitors/therapeutic use , Receptor, trkA/antagonists & inhibitors , Humans , Neuroendocrine Tumors/pathology , Protein Kinase Inhibitors/pharmacologyABSTRACT
BACKGROUND/AIMS: The tumor suppressor p53 is depleted in many tumor cells by the E3 ubiquitin ligase mouse double minute 2 homolog (MDM2) through MDM2/p53 interaction. A novel target for inhibiting p53 degradation and for causing reexpression of p53wild type is inhibition of MDM2. The small molecule NVP-CGM097 is a novel MDM2 inhibitor. We investigated MDM2 inhibition as a target in neuroendocrine tumor cells in vitro. METHODS: Human neuroendocrine tumor cell lines from the pancreas (BON1), lung (NCI-H727), and midgut (GOT1) were incubated with the MDM2 inhibitor NVP-CGM097 (Novartis) at concentrations from 4 to 2,500 nM. RESULTS: While p53wild type GOT1 cells were sensitive to NVP-CGM097, p53mutated BON1 and p53mutated NCI-H727 cells were resistant to NVP-CGM097. Incubation of GOT1 cells with NVP-CGM097 at 100, 500, and 2,500 nM for 96 h caused a significant decline in cell viability to 84.9 ± 9.2% (p < 0.05), 77.4 ± 6.6% (p < 0.01), and 47.7 ± 9.2% (p < 0.01). In a Western blot analysis of GOT1 cells, NVP-CGM097 caused a dose-dependent increase in the expression of p53 and p21 tumor suppressor proteins and a decrease in phospho-Rb and E2F1. Experiments of co-incubation of NVP-CGM097 with 5-fluorouracil, temozolomide, or everolimus each showed additive antiproliferative effects in GOT1 cells. NVP-CGM097 and 5-fluorouracil increased p53 and p21 expression in an additive manner. CONCLUSIONS: MDM2 inhibition seems a promising novel therapeutic target in neuroendocrine tumors harboring p53wild type. Further investigations should examine the potential role of MDM2 inhibitors in neuroendocrine tumor treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Fluorouracil/pharmacology , Isoquinolines/pharmacology , Neuroendocrine Tumors/drug therapy , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Drug Therapy, Combination , E2F1 Transcription Factor/metabolism , Humans , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Retinoblastoma Protein/metabolism , Signal Transduction/drug effects , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND/AIMS: The hepatocyte growth factor/transmembrane tyrosine kinase receptor c-Met has been defined as a potential target in antitumoral treatment of various carcinomas. We aimed to investigate the direct effect of c-Met inhibition on neuroendocrine tumor cells in vitro. METHODS: The effects of the multi-tyrosine kinase inhibitors cabozantinib and tivantinib and of the highly specific c-Met inhibitor INC280 were investigated in human pancreatic neuroendocrine BON1, bronchopulmonary NCI-H727 and midgut GOT1 cells in vitro. RESULTS: INC280, cabozantinib and tivantinib inhibited c-Met phosphorylation, respectively. However, while equimolar concentrations (10 µM) of cabozantinib and tivantinib inhibited cell viability and cell migration, INC280 had no inhibitory effect. Knockdown experiments with c-Met siRNA also did not demonstrate effects on cell viability. Cabozantinib and tivantinib caused a G2 arrest in neuroendocrine tumor cells. CONCLUSIONS: Our in vitro data suggest that c-Met inhibition alone is not sufficient to exert direct antitumoral or antimigratory effects in neuroendocrine tumor cells. The multi-tyrosine kinase inhibitors cabozantinib and tivantinib show promising antitumoral and antimigratory effects in neuroendocrine tumor cells, which are most probably 'off-target' effects, not mediated by c-Met.
