Abstract 2239: A peptide mimicking a region in proliferating cell nuclear antigen (PCNA) specific to key protein interactions is cytotoxic in cancer cells.

Abstract

Abstract A common theory in cancer progression is the “mutator phenotype” hypothesis. It is thought that as DNA begins to replicate with less fidelity in certain cells, errors accumulate,eventually leading to the incidence of cancer. Although the actual driving force behind this loss of DNA replication fidelity has yet to be determined, it is very possible that a malfunction within the replication machinery itself is propagating the mutations. An important factor in DNA replication and repair is a highly conserved 29 kDa protein, proliferating cell nuclear antigen (PCNA). PCNA forms a homotrimer that encircles the DNA strand during replication, slides down the DNA strand, and loads numerous proteins and enzymes that act in the proper replication of DNA. Molecules requiring PCNA for activity in cellular processes include, but are not limited to: polymerase δ and ε, flap endonuclease -1 (FEN-1), and xeroderma pigmentosum G (XPG). Because PCNA is so essential, if its function within the cell is altered, this could potentially be a contributing factor to the accumulation of errors in cancer cells. Prior work in the lab has shown that cancer cells harbor two distinct isoforms of PCNA: the basic isoform identical to normal cells and an acidic isoform specific to cancer cells (caPCNA). The caPCNA isoform is the result of post-translational modifications by methyl esterification on specific glutamic and aspartic acid residues. A rabbit polyclonal antibody (caPCNAab) has been developed against a peptide fragment representing the cancer associated domain of caPCNA. This antibody has been tested against commercially available antibodies (PC10 and C20), and displays the ability to distinguish between the PCNA isoforms. In preliminary testing, caPCNAab exhibited the ability to inhibit both DNA polymerase δ and SV40 DNA replication activity using in vitro assays with breast cancer cell extract. By using this information, a peptide was then developed to mimic the cancer-associated sequence and compete for binding of accessory proteins to caPCNA, thereby targeting cells containing caPCNA and inhibiting DNA replication. In the present study, we characterize the specificity of the caPCNA antibody by scanning the epitope specific to caPCNA using immunohistochemical staining of progressive breast cancer tissue samples. We have also developed a novel peptide (caPeptide) that specifically mimics this domain specific to caPCNA, competes with full-length caPCNA for its binding partners, and creates cytotoxicity within cancer cells. The caPeptide exhibits cytotoxic activity in cancer cells using in vivo studies that include: MTT, flow cytometry, and colony formation assays. The caPeptide has also shown to specifically block in vitro DNA replication using the SV40 DNA replication assay, along with polymerase α and δ activity assays and surface plasmon resonance (SPR) binding studies. Citation Format: Shanna Smith, Linda Malkas, Robert Hickey, Robert Lingeman, Caroline Li. A peptide mimicking a region in proliferating cell nuclear antigen (PCNA) specific to key protein interactions is cytotoxic in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2239. doi:10.1158/1538-7445.AM2013-2239