ΔNP63 MULTIFUNCTIONALITY IN TRIPLE NEGATIVE BREAST CANCER – ROLE IN CELL ADHESION AND POTENTIAL INTERACTING PARTNERS

Background:

 Protein p63 is essential for the development and differentiation of stratified squamous epithelium. It belongs to the p53 protein family, which consists of p53, p63 and p73 proteins. At least six isoforms of this protein are expressed and it can act as both tumour supressor or oncogene. We can distinguish full-length isoforms (TAp63) and DN isoforms with shortened N-terminal transactivation domain due to the use of alternative promoters. In breast carcinomas, p63 expression is usually lost, except of subgroup of triple-negative breast carcinomas (TNBC). Our goal was to investigate p63 role in carcinogenesis of TNBC.

Materials and Methods:

We developed a tetracycline-inducible model system to study the role of p63 in breast cancer cells (MDA MB 468). We analysed the impact of p63 expression on cellular proliferation, migration, adhesion, gene expression profiling and signalling pathway activation by xCELLigence real-time cell analysis, trypsynization test and real-time PCR. Potential p63 interacting partners were co-immunoprecipitated with DNp63 specific antibody and analysed by mass spectrometry. Furthermore, we have prepared a knock-out p63 cell line (CRL 2335) by CRISPR-Cas system to observe changes in the cell phenotype and signalling pathways after eliminating p63 expression.

Results:

 DNp63 expression in breast cancer cells caused morphological changes and the loss of cellular adhesion. We supposed that reduced cell adhesion is caused by up-regulation of phosphorylated form of EGFR. Additionally, elimination of p63 expression seems to be important in maintaining a basal phenotype of cancer cells, as evidenced by changes in specific markers. Using mass spectrometry method we were able to identify many potential interacting partners, which play role in cancer development and progression like: peroxiredoxin 2, stathmin 1, PDLIM 1 protein. Now our aim is to investigate the influence of these interactions on breast cancer cell.

Conclusion:

 Based on results obtained with our new model cell lines (with p63 overexpression and p63 knock-out) we suggest that DNp63 isoform plays an important role in regulation of cell adhesion through the EGFR pathway activation and is also needed for maintaining a basal phenotype of breast cancer cells.

The work was supported by European Regional Development Fund, the state budget of the Czech Republic for Regional Centre for Applied Molecular Oncology – RECAMO (CZ.1.05/ 2.1.00/ 03.0101) and by IGA NT/ 14602-3/ 2013, by MH CZ – DRO (MMCI, 00209805) and the state budget of the Czech Republic (LO1413).