Abstract 1041: Restoration of p53 function in soft tissue sarcomas correlates with increased sensitivity to TNFα

Abstract

Abstract Background: Sarcomas are rare, poorly understood cancers, highly aggressive and refractory to standard therapies. In these tumors mutations of p53 or Mdm2 amplification are frequently found. Isolated limb perfusion (ILP) with TNFα and melphalan is an innovative approach to treat unresectable soft tissue sarcomas (STS) of extremities. TNF is known to increase endothelium permeability for melphalan, and to disrupt tumoral vessels. However, the direct apoptotic effect of TNF on sarcoma cells remains controversial. Several reports indicate an association of sensitivity to TNF with p53 activity in in vitro models. In the present study we attempted to test the ability of TNF to induce apoptosis of sarcoma cells and investigated the role of p53 in the regulation of this effect. Methods: Ex vivo study was performed on sarcoma tumor pieces from patients. Tumor slices were treated with TNF and cytotoxic effect was assessed with TUNEL method. In vitro studies were carried on human soft tissue sarcoma cell lines with different p53 status and Mdm2 expression, representing 5 histological types. Cytotoxic effect of TNF was assessed with DiOC6/PI test. Activation of p53 was verified by the detection of expression of proteins encoded by p53 target genes. Expression of TNF receptor was examined by flow cytometry. Results: In tumor slices treated ex vivo with TNF we observed apoptosis of not only endothelial, but also sarcoma cells. In vitro studies revealed that cell lines with active p53 were sensitive to TNF (80% and 40% of apoptosis), while 6 cell lines were resistant: 2 with wt p53 and Mdm2 amplification (2-4% of apoptosis), 2 with p53 mutation and 2 with p53 deletion (5-15% of apoptosis). TNFR I was expressed by all the cell lines. We next tested sensitivity to TNF after restoration of p53 activity. For this purpose we used either of two compounds: Nutlin-3a, an inhibitor of Mdm2, or CP-31398, a molecule restoring wild-type conformation to mutant TP53. In cell lines with Mdm2 amplification treatment with Nutlin alone resulted in decreased cell viability, while in combined treatment induced strong apoptosis synergistically with TNF. Similarly, treatment of cell lines expressing mutated p53 with CP-31398 led to a significant enhancement of sensitivity to TNF. Conclusions: The results of our ex vivo and in vitro studies suggest, that during ILP, TNF may directly kill sarcoma cells and its cytotoxic activity is correlated with the functionality of p53. Restoration of p53 activity may be advantageous not only in therapies based on DNA disruption, but also based on the use of TNFα. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1041.