Abstract 5770: MDM2 protein variants expression in the plasma membrane of cancer cells: A target for anticancer peptide PNC-27

Abstract

Abstract PNC-27 anti-cancer peptide, derived from the MDM2 binding site of p53 and linked to a membrane residency peptide (MRP), has been shown to cause necrosis of cancer cells without affecting untransformed cells. PNC-27 was also able to eradicate pancreatic tumor xenografts in mice (Michl et al, Int. J of Cancer, 2006). Recently, we have identified the mechanism of action of this peptide as due to formation of oligomeric pores in the plasma membrane (PM) of cancer cells but not in the PM of untransformed cells. The mechanism of pore-formation by PNC-27 closely resembles the pore-formation process by streptolysin-O, melittin and similar pore-forming agents. We have shown MDM2 as a targeting molecule that leads to PNC-27 selectivity towards cancer cells by its mis-localization to cancer cell PM (Sarafraz-Yazdi et al, PNAS, in press). Examining purified PM of a variety of cancer cells by immunoblotting, we now provide evidence for multiple MDM2 protein variants, and that were absent in the PM of normal untransformed cells. To confirm the purity of the isolated PM, fractions were also immunoblotted for the PM markers Na+/K+-ATPase, E-Cadherin and ß-Catenin, all of which were enriched in the PM fractions. In contrast, Abs against membrane markers specific for intracellular organelles, COX IV and Cytochrome C for mitochondrial membrane, showed no reaction in the PM fractions while they reacted with total membrane fractions. Three protein variants of MDM2 with MW of 27kD, 40kD and 57kD were consistently expressed in the PM of human and rat pancreatic cancer cells, human melanoma, breast cancer. We also confirmed these observations in isolated PM from freshly obtained primary ovarian tumors from human patients with aggressive tumor. Remarkably, no MDM2 variant was detectable in the PM fractions of primary human fibroblasts and untransformed pancreatic HPNE cells. Our finding of differently sized MDM2 variants complies with other, previously reported MDM2 protein variants. Of the different MDM2 mRNA splice variants published, 5 have been shown to be translated into protein including proteins of 27kD, 40kD and 57kD. Our present study demonstrates the localization of the 3 variant proteins in the PM of cancer cells, suggesting a possible role for these membrane-localized MDM2 variant in the action of PNC-27. The notion of this specific MDM2-PNC-27 interaction at the PM level was strongly supported by the effective competition by a monoclonal MDM2-specific Ab, reducing PNC-27-mediated cytotoxicity by >80% as measured by LDH cytotoxicity assay and propidium iodide staining of the nuclei of dead cells in real-time spinning disc confocal microscopy. These findings for the first time demonstrate not only the novel localization of different MDM2 variants in PM of different cancer cells but they also suggest a potentially wider applicability of PNC-27 as a novel selective anti-cancer drug for clinical trials. 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 5770.