Disrupting FAK and p53 interaction with peptides as a novel cancer therapy approach. Free

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It is known that p53 alterations are commonly found in tumors cells. Another marker of tumorigenesis is Focal Adhesion Kinase (FAK), a non-receptor kinase that is overexpressed in many types of tumors and associates with multiple cell surface receptors and intracellular signaling proteins through which it can play an important role in survival signaling. In our previous report we have determined that the N-terminal domain of FAK physically interacted with the N-terminal transactivation domain of p53, as demonstrated by pull-down, immunoprecipitation and co-localization analyses (JBC, 280, 25008-250021, 2005).

In the present study we performed phage display assay with the N-terminal fragment of FAK (1-423 a.a.) and identified a 7 amino-acid peptide that is highly homologous to a peptide from the N-terminal fragment of p53 of Woodchuck (Marmota monax) that corresponds to the human p53 peptide. We performed site-directed mutagenesis to mutate the 7 amino-acids by deleting the first four amino-acids and changing the next 3 amino-acids in the human GST-p53 and GST-N-p53. Then, we performed pull-down assays with human full length FAK and demonstrated that mutant GST-p53 proteins had significantly lower background level of binding with FAK than wild type p53 proteins. We synthesized TAT-conjugated peptides containing the wild type p53 site and the mutant site to cause apoptosis in cancer cells.

Thus, we localized the site of FAK binding to a 7 amino-acid region in the N-terminal proline-rich domain of p53. We conjugated these peptides (wild type and mutant) to TAT peptide sequences to penetrate into the cells, and demonstrated that the wild type p53 peptide inhibited cell viability of HCT116 p53+/+ cells in the presence of doxorubicin (0.5 μg/ml) more significantly than mutant p53 peptide. Furthermore, TAT-p53 peptide decreased viability of MCF-7 cells, while the mutant peptide, containing the deleted 7 amino-acid binding site with FAK, did not cause this effect. In addition, FAK-inhibited p53 transcriptional activity of p21 was abrogated by the introduction of mutant p53 with the deleted binding site into HCTp53-/- cells.

Thus, the disruption of FAK and p53 binding with peptide, containing the binding site, can be used in cancer therapy. This will allow for the finding of apoptotic small molecule drug inhibitors using computer modeling approaches.