Supplementary Material for: Mechanisms of Targeting the MDM2-p53-FOXM1 Axis in Well-Differentiated Intestinal Neuroendocrine Tumors

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.