Mechanisms of Targeting the MDM2-p53-FOXM1 Axis in Well-Differentiated Intestinal Neuroendocrine Tumors

Abstract

Background/Aims: The tumor suppressor p53 is rarely mutated in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) but they frequently show a strong expression of negative regulators of p53, rendering these tumors excellent targets for a p53 recovery therapy. Therefore, we analyzed the mechanisms of a p53 recovery therapy on intestinal neuroendocrine tumors in vitro and in vivo.Methods: By Western blot and immunohistochemistry, we found that in GEP-NEN biopsy material overexpression of MDM2 was present in intestinal NEN. Therefore, we analyzed the effect of a small-molecule inhibitor, nutlin-3a, in p53 wild-type and mutant GEP-NEN cell lines by proliferation assay, flow cytometry, immunofluorescence, Western blot, and by multiplex gene expression analysis. Finally, we analyzed the antitumor effect of nutlin-3a in a xenograft mouse model in vivo. During the study, the tumor volume was determined. Results: The midgut wild-type cell line KRJ-I responded to the treatment with cell cycle arrest and apoptosis. By gene expression analysis, we could demonstrate that nutlins reactivated an antiproliferative p53 response. KRJ-I-derived xenograft tumors showed a significantly decreased tumor growth upon treatment with nutlin-3a in vivo. Furthermore, our data suggest that MDM2 also influences the expression of the oncogene FOXM1 in a p53-independent manner. Subsequently, a combined treatment of nutlin-3a and cisplatin (as chemoresistance model) resulted in synergistically enhanced antiproliferative effects. Conclusion: In summary, MDM2 overexpression is a frequent event in p53 wild-type intestinal neuroendocrine neoplasms and therefore recovery of a p53 response might be a novel personalized treatment approach in these tumors.

Inhibition of mTOR's Catalytic Site by PKI-587 Is a Promising Therapeutic Option for Gastroenteropancreatic Neuroendocrine Tumor Disease

“…The cells were incubated with PKI-587 or RAD001 at low (IC 50 ) and high (C max ) concentrations or with DMSO as a control (in triplicate) for 12 and 36 h. A fluorescence reagent mix for detection of viability and cytotoxicity (different wave lengths) and, later, a chemiluminescence reagent for detection of caspase 3/7 activity were added following the manufacturer's instructions (ApoTox-Glo Triplex Assay; Promega). Measurements were conducted using Tecan Infinite M200 (fluorescence) and Berthold Centro LB 960 plate readers.…”

“…Cells were seeded into 3.5-cm dishes, at 125,000-500,000 cells per dish, and were left to adhere overnight and incubated with PKI-587 or RAD001 at low (IC 50 ) and high (C max ) concentrations or with DMSO as a control for 48 h (all cell lines) and 96 h (just QGP-1 and LCC-18), respectively, in four biological replicates. Fixing and staining of each sample was accomplished as described previously [35] .…”

“…3 c). 50 , C mid , and C max were determined by nonlinear fitting of the dose-response curves (also refer to the online suppl. material).…”

“…Although the clinical incidence of p53 mutations is low in GEP-NENs, recent studies have revealed several loss-of-function mutations in cell lines, leading to conformational changes in the DNA-binding domain of p53 (LCC-18) or to truncated (BON) or completely lost expression of the p53 protein (QGP-1) [49][50][51][52] . QGP-1 cells in particular seem to be incapable of inducing apoptotic processes.…”

Immunohistochemical Study of Mitosis-regulatory Proteins in Gastroenteropancreatic Neuroendocrine Neoplasms

“…Our results indicate that aurora B inhibition might be even more beneficial in high-grade tumors and carcinomas than in the analyzed cell lines, which were generated from welldifferentiated primary material. Nevertheless, the untypical mutation patterns of both cell lines (23,24) brings into question whether these cell lines represent well-differentiated tumors. Furthermore, a variety of aurora inhibitors have already been made available (25).…”