Abstract 746: Functional crosstalk between the p53 and NF-kB transcription factors.

Abstract

Abstract The tumor suppressor p53 and NF-kB are sequence-specific transcription factors (TFs) playing crucial roles in controlling cell proliferation and cell survival with an undeniable impact on cancer progression. In response to cellular stresses they induce the expression of a large variety of genes. Synergistic or antagonistic interactions between the p53 and NF-kB proteins may modulate the expression of common targets and potentially lead to a modified cellular response. To specifically investigate cell-autonomous, cooperative interactions between NF-kB and p53 on gene expression changes, we performed a genome-wide transcriptome analysis in the breast-cancer derived MCF-7 cells following single or combinatorial treatments with the chemotherapeutic drug doxorubicin (DXR) and the NF-kB inducer TNF-alpha (TNF). The microarray results revealed 720 upregulated (log2FC>1.5) and 926 repressed genes for the DXR+TNF treatment (log2FC 0.1} and 188 repressed genes {log2[FCDXR+TNF] - (log2[FCDXR] + log2[FCTNF]) < -0.1}. Notably, gene ontology analysis applied to these gene groups indicated an apparent enrichment for epithelial mesenchymal transition and cell migration functions. We focused the validation experiments on 15 up-regulated genes to investigate the direct involvement of p53 and NF-kB on their expression using RT-qPCR assays coupled to p53 activation by DXR, 5-fluorouracil or Nutlin, p53 RNAi and/or NF-kB chemical inhibition. In silico analysis of the potential target genes’ promoter sequence identified both p53 and NF-kB responsive regions where cis-mediated interactions of these TFs may occur, a result that has been followed up by mining of available ChIP-seq data. Among the 15 chosen targets, we uncovered five (LAMP3, ETV7, UNC5B, NTN1 and PLK3) that were induced synergistically after DXR + TNF treatment in MCF-7 breast cancer cells. Over-expression of LAMP3 has already been associated with an increased metastatic potential for particular cancers, and ETV7 can act as a regulator of cell proliferation. To study the migratory phenotype associated with the DXR+TNF combinatorial treatment, we performed migration assays using three different approaches: trans-well migration assay, Xcelligence Migration Assay and wound healing. The results consistently showed an increase in migration potential upon DXR+TNF compared to mock or to single treatments. We propose that the functional crosstalk between the p53 and NF-kB TFs can lead to the activation of specific gene expression programs that may impact on cancer phenotypes. Given that most tumors carry an activated NF-kB pathway, and a significant proportion of breast cancers, particularly the luminal subtype, maintains wild type p53, the uncovered crosstalk could potentially modify the efficiency of cancer therapy. Citation Format: Alessandra Bisio, Judit Zámborszky, Sara Zaccara, Mattia Lion, Toma Tebaldi, Yari Ciribilli, Alberto Inga. Functional crosstalk between the p53 and NF-kB transcription factors. [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 746. doi:10.1158/1538-7445.AM2013-746