p53 mutants induce transcription of NF-κB2 in H1299 cells through CBP and STAT binding on the NF-κB2 promoter and gain of function activity

Abstract

Cancer cells with p53 mutations, in general, grow more aggressively than those with wild-type p53 and show “gain of function”(GOF) phenotypes such as increased growth rate, enhanced resistance to chemotherapeutic drugs, increased cell motility and tumorigenicity; although the mechanism for this function remains unknown. In this communication we report that p53-mediated NF-κB2 up-regulation significantly contributes to the aggressive oncogenic behavior of cancer cells. Lowering the level of mutant p53 in a number of cancer cell lines resulted in a loss of GOF phenotypes directly implicating p53 mutants in the process. RNAi against NF-κB2 in naturally occurring cancer cell lines also lowers GOF activities. In H1299 cells expressing mutant p53, chromatin immunoprecipitation (ChIP) assays indicate that mutant p53 induces histone acetylation at specific sites on the regulatory regions of its target genes. ChIP assays using antibodies against transcription factors putatively capable of interacting with the NF-κB2 promoter show increased interaction of CBP and STAT2 in the presence of mutant p53. Thus, we propose that in H1299 cells, mutant p53 elevates expression of genes capable of enhancing cell proliferation, motility, and tumorigenicity by inducing acetylation of histones via recruitment of CBP and STAT2 on the promoters causing CBP-mediated histone acetylation.

Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication

“…Several laboratories have demonstrated that GOF p53 accelerates cell proliferation and tumor growth in comparison with p53-null or GOF p53-knockdown cells (12,15,16). Since the data presented above indicate that GOF p53 requires its TADs to increase origin firing and to prevent replication fork col- mental Figure 6).…”

“…Cells were partially synchronized by density arrest and replating and sequentially labeled with IdU and chlorodeoxyuridine (CldU) at early S phase. Cellular genomic DNA was then spread on slides, and replicating DNA fibers were detected by immunostaining of incorporated IdU and CldU using with p53-null or -knockdown cells (15,16) and upregulates expression of cell-proliferating genes (27). Therefore, experiments were carried out to determine whether GOF p53 hastens the time of S phase entry compared with p53-null cells.…”

“…The ability of GOF p53 mutants to increase origin firing in a human lung cancer cell line that naturally expresses GOF p53 was also determined. For this purpose, endogenous p53R273C expression in the human SCLC lung cancer cell line H1048 or R175H expression in the human non-small cell lung cancer (NSCLC) VMRC-LCD cell line (http://p53.iarc.fr/ CellLines.aspx) was stably knocked down by expressing shRNA against p53 (shp53) or a control shRNA against GFP (shGFP) from a lentiviral expression vector (16). H1048 and VMRC cells stably expressing shGFP or shp53 were synchronized by density arrest and replating.…”

“…In addition to loss of WT p53 function, the ability of GOF p53 to activate transcription of proliferative genes (11)(12)(13) or to deregulate signaling pathways (14) has been connected to its oncogenic properties. Inhibition of tumor formation by knockdown of endogenous mutant p53 has been demonstrated in human lung cancer cells using RNAi and knockin (KI) mouse models (15,16). A recent study has reported that inactivation or destabilization of GOF p53 reduces tumor growth in mice, extending their survival (17).…”

Gain-of-Function Activity of Mutant p53 in Lung Cancer through Up-Regulation of Receptor Protein Tyrosine Kinase Axl

“…Both transfection and luciferase assays were as described previously in triplicate. 57 Chromatin immunoprecipitation. ChIP was performed as described.…”