Mutant p53-induced Up-regulation of Mitogen-activated Protein Kinase Kinase 3 Contributes to Gain of Function

Abstract

Mitogen-activated protein kinase kinase 3 (MAP2K3) is a member of the dual specificity kinase group. Growing evidence links MAP2K3 to invasion and tumor progression. Here, we identify MAP2K3 as a transcriptional target of endogenous gain-of-function p53 mutants R273H, R175H, and R280K. We show that MAP2K3 modulation occurred at the mRNA and protein levels and that endogenous mutant p53 proteins are capable of binding to and activate the MAP2K3 promoter. In addition, we found that the studied p53 mutants regulate MAP2K3 gene expression through the involvement of the transcriptional cofactors NF-Y and NF-B. Finally, functional studies showed that endogenous MAP2K3 knockdown inhibits proliferation and survival of human tumor cells, whereas the ectopic expression of MAP2K3 can rescue the proliferative defect induced by mutant p53 knockdown. Taken together, our findings define a novel player through which mutant p53 exerts its gain-of-function activity in cancer cells.TP53 gene mutations are the most frequent genetic alterations in human cancers; Ͼ50% of all human cancer cases carry mutations within the TP53 locus (1). Most of these are missense point mutations and are localized in the core DNA-binding domain (2). These alterations disrupt the normal transcriptional capacity of p53 and compromise its tumor suppressor properties by abrogating its transcriptional activity on genes connected with cell cycle arrest, apoptosis, or DNA repair, in response to a variety of stress signals (3, 4). Recent studies have shown that mutations of the TP53 gene can confer additional functions (gain of function, GOF) 2 that are exerted in a variety of ways, ranging from enhanced proliferation in culture, increased tumorigenicity in vivo, and enhanced resistance to a variety of commonly used anti-cancer drugs (5, 6). The GOF hypothesis has recently been reinforced by studies employing mutant p53 (mutp53) "knock-in" mice, which show a higher frequency of tumor development and increased metastatic potential, compared with p53-deficient mice (7,8). Furthermore, RNA interference (RNAi) studies demonstrated that depletion of mutp53 renders cancer cells more sensitive to DNA-damaging chemotherapeutic agents in vitro (9, 10) and reduces tumor malignancy both in vitro and in vivo (10). In agreement with these results, tumor growth delay studies, performed in the HT29 xenograft model, showed that conditional silencing of mutp53 does not only impact on tumor growth but leads to tumor architecture modifications, with consistent reduction in stromal invasion and tumor angiogenesis (11). At the molecular level, these GOF effects were shown to be linked to the ability of mutp53 to modulate the expression of several genes, such as MDR1 (12), c-MYC (13), CD95 (Fas/APO-1) (14), EGR1 (9), MSP/MST-1 (15), GEF-H1 (16), ID2 (17), GRO1 (18), PPARGC1A, FRMD5 (19), and ID4 (20), supporting the hypothesis that mutp53-specific transcriptional activity is required for at least some of the mutp53 GOF effects. However, the molecular mechanisms underlying the GOF o...

Understanding wild-type and mutant p53 activities in human cancer: new landmarks on the way to targeted therapies

“…mitogen-activated protein kinase-kinase 3-depleted cells showed attenuated growth, although overexpression enhanced cell growth. 45 These findings suggest upregulation of mitogen-activated protein kinasekinase 3 expression represent a mechanism for mutant p53 GOF.…”

Expression of Slug Is Regulated by c-Myb and Is Required for Invasion and Bone Marrow Homing of Cancer Cells of Different Origin

“…For ChIP experiments performed in K562 cells, samples were prepared as described previously (28). Immunoprecipitations were carried out using 6 g of anti-c-Myb (clone 1-1 from Millipore), anti-p53 (DO-1 from Santa Cruz), or no antibody.…”

YAP enhances the pro‐proliferative transcriptional activity of mutant p53 proteins

“…NF-Y is a heterotrimeric transcription factor with a high binding affinity for the CCAAT consensus and consisting of three subunits, NF-YA, NF-YB, and NF-YC, all required for binding the CCAAT motif that is present in 30% of all eukaryotic promoters [28]. It has been demonstrated that human p53 mutants physically interact with NF-Y and that this interaction specifically occurs on the CCAAT boxes of several cell cycle gene promoters.…”