Cancer Research Landon Prizes for Basic and Translational Cancer Research AACR Conference on Molecular Diagnostics - 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 2396-2401, March 15, 2007. doi: 10.1158/0008-5472.CAN-06-2425
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Weisz, L.
Right arrow Articles by Oren, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weisz, L.
Right arrow Articles by Oren, M.

Priority Reports

Mutant p53 Enhances Nuclear Factor {kappa}B Activation by Tumor Necrosis Factor {alpha} in Cancer Cells

Lilach Weisz1, Alexander Damalas1,2,5, Michalis Liontos5, Panagiotis Karakaidos5, Giulia Fontemaggi2,3, Revital Maor-Aloni1, Marina Kalis1, Massimo Levrero2,3,4, Sabrina Strano2, Vassilis G. Gorgoulis5, Varda Rotter1, Giovanni Blandino2,3 and Moshe Oren1

1 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; 2 Department of Experimental Oncology, Regina Elena Cancer Institute, 3 Rome Oncogenomic Center, Via delle Messi d'oro and 4 Fondazione Andrea Cesalpino, University La Sapienza, Rome, Italy; and 5 Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, University of Athens, Greece

Requests for reprints: Moshe Oren, Molecular Cell Biology, Weizmann Institute, Rehovot 76100, Israel. Phone: 972-8-9342358; Fax: 972-8-9346004; E-mail: moshe.oren{at}weizmann.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutations in the p53 tumor suppressor are very frequent in human cancer. Often, such mutations lead to the constitutive overproduction of mutant p53 proteins, which may exert a cancer-promoting gain of function. We now report that cancer-associated mutant p53 can augment the induction of nuclear factor {kappa}B (NF{kappa}B) transcriptional activity in response to the cytokine tumor necrosis factor {alpha} (TNF{alpha}). Conversely, down-regulation of endogenous mutant p53 sensitizes cancer cells to the apoptotic effects of TNF{alpha}. Analysis of human head and neck tumors and lung tumors reveals a close correlation between the presence of abundant mutant p53 proteins and the constitutive activation of NF{kappa}B. Together, these findings suggest that p53 mutations may promote cancer progression by augmenting NF{kappa}B activation in the context of chronic inflammation. [Cancer Res 2007;67(6):2396–401]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The p53 tumor suppressor is a target for frequent mutations in human cancer. In its wild-type (wt) form, p53 functions as a transcriptional regulator and exerts potent antitumor effects. In response to a variety of stress signals, many of which are associated with cancer initiation or progression, p53 is activated; this promotes outcomes such as growth arrest, replicative senescence, or apoptosis (reviewed in refs. 1, 2).

Although the frequent p53 mutations serve primarily to abrogate the tumor suppressor function of wt p53, there is mounting evidence that the resultant mutant p53 proteins, often produced copiously in cancer cells, may also contribute actively to carcinogenesis through gain of function (3, 4). This notion was reinforced by recent studies employing mutant p53 "knock-in" mice, which display broader tumor spectrum and increased aggressiveness and metastatic potential (5, 6). In cultured cells, overexpression of tumor-associated p53 mutants was shown to attenuate apoptosis induced by a variety of agents (reviewed in refs. 3, 4). Furthermore, down-regulation of endogenous mutant p53 by RNA interference (RNAi) renders cancer cells more sensitive to killing by DNA-damaging chemotherapeutic agents in vitro (7) and in vivo (8).

Like p53, the transcription factor nuclear factor {kappa}B (NF{kappa}B) has also been extensively implicated in the regulation of apoptosis and modulation of cancer therapy responses. The NF{kappa}B family consists of several proteins, including RelA (commonly known as p65), RelB, c-Rel, p50, and p52, operating as heterodimers or sometimes homodimers (for recent reviews, see refs. 9, 10). By modulating the expression of its target genes, NF{kappa}B regulates critical biological processes, including immune and inflammatory responses. Moreover, aberrant NF{kappa}B activity has been implicated in carcinogenesis and in the control of the cellular response to anticancer agents (reviewed in refs. 9, 10). In many, although not all, cases, NF{kappa}B activation inhibits apoptosis and favors cell proliferation. Conversely, inhibition of NF{kappa}B can facilitate cell killing. Importantly, constitutive NF{kappa}B activation, associated with chronic inflammatory processes, can promote tumor development.

Notably, it was recently reported that excess mutant p53 can elevate NF{kappa}B activity via transactivation of the NF{kappa}B2 gene, and that mutant p53 expression correlates positively with NF{kappa}B activity in cultured cancer cells (11, 12). Because the activation of NF{kappa}B by the cytokine tumor necrosis factor {alpha} (TNF{alpha}) can drive cancer progression in the context of chronic inflammation (13, 14), we investigated the effect of mutant p53 on the NF{kappa}B response to TNF{alpha}. We report that mutant p53 augments the activation of NF{kappa}B by TNF{alpha}, and that down-regulation of endogenous mutant p53 sensitizes cancer cells to killing by the cytokine. Remarkably, elevated mutant p53 protein is closely correlated with increased NF{kappa}B activation in human premalignant and malignant lesions. These findings suggest a role for mutant p53/NF{kappa}B cooperation in human cancer, especially under conditions of chronic exposure to inflammatory cytokines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and plasmids. H1299 p53R175H-inducible cells were maintained in RPMI-1640 (Sigma, St. Louis, MO) supplemented with 10% FCS (Sigma). To induce p53 expression from the metallothionein promoter, ZnCl2 was added to a final concentration of 100 µmol/L. SKBR3 cells were maintained in McCoy's medium (Sigma) supplemented with 10% FCS. pSuper p53 (p53 RNAi) and pSuper LacZ (LacZi) plasmids were kindly provided by R. Agami (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Small interfering RNA (siRNA) oligonucleotides were purchased from Dharmacon (Chicago, IL). LacZi sense sequence: GUG ACC AGC GAA UAC CUG U dT dT; and p53i sense sequence: GCA UGA ACC GGA GGC CCA U dT were used. Recombinant human TNF{alpha} was purchased from R&D Systems (Minneapolis, MN).

Transfections. Transfections, including synthetic siRNA oligonucleotides (100 pmol/35-mm dish), were done with Dharmafect 3 (Dharmacon). Unless otherwise stated, transfections employing plasmid DNA only were done with Maxfect (Molecula, Columbia, MD).

Apoptosis assays. Analysis of apoptosis by fluorescence-activated cell sorting was as described (7). WST1 kit was purchased from Roche Diagnostics (Basel, Switzerland). Caspase 3 kit was purchased from Promega (Madison, WI).

Luciferase assays. Luciferase assays, using the dual luciferase system (Promega), were essentially as described (7).

Immunohistochemistry. Immunohistochemical staining of tumor sections used the indirect streptavidin-biotin-peroxidase protocol (15). Protein expression in cultured cells was visualized by indirect immunofluorescence.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutant p53 enhances induction of NF{kappa}B activity by TNF{alpha}. To assess the impact of tumor-associated mutant p53 proteins on the transcriptional activity of NF{kappa}B upon exposure to TNF{alpha}, we used p53-null human large cell lung cancer H1299 cells, stably expressing the hotspot mutant p53R175H under the metallothionein promoter. Upon exposure to zinc, expression of mutant p53 is readily induced (see Fig. 3A). Treatment with TNF{alpha} in the absence of zinc stimulated NF{kappa}B activity, recorded with a luciferase reporter plasmid driven by five consecutive NF{kappa}B binding sites (Fig. 1A ). NF{kappa}B activity peaked early and did not increase any further beyond 6 h of TNF{alpha} exposure. In contrast, induction of mutant p53 expression by addition of zinc led to a continuous increase in NF{kappa}B activity in response to TNF{alpha} treatment. Induction of mutant p53 alone did not increase NF{kappa}B activity (Supplementary Fig. S1, top), nor did zinc affect NF{kappa}B activation in parental p53-null H1299 cells (Supplementary Fig. S1, bottom). Hence, mutant p53 overexpression selectively augments cytokine-triggered NF{kappa}B activation.


Figure 3
View larger version (32K):
[in this window]
[in a new window]

  		Figure 3. Mutant p53 protects cancer cells against killing by TNF{alpha}. A, H1299 cells stably expressing mutant p53R175H under the metallothionein promoter were treated with ZnCl2. Four hours later, cells were treated with 50 ng/mL TNF{alpha} for 48 h or left untreated. Cell death was assessed by staining of fixed, permeabilized cells with propidium iodide, followed by fluorescence-activated cell sorting analysis. Numbers indicate the percentage of cells with sub-G1 DNA content, indicative of apoptosis (top). Mutant p53 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were assessed by Western blot analysis (bottom) employing sequential probing with a mixture of the p53-specific monoclonal antibodies PAb1801 and DO-1 followed by anti-GAPDH antibody (Chemicon). B, SKBR3 cells were transfected with synthetic p53 siRNA or LacZ siRNA (LacZi) as a control. Twenty-four hours later, cells were treated with 50 ng/mL TNF{alpha} or left untreated. Cell death and protein analysis were done as in (A). C, SKBR3 cells were transiently transfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) as a control. One day later, cells were transfected with an siRNA-resistant plasmid encoding p53R175H (m175) or pCDNA3 as a control. Forty-eight hours later, cells were treated with 200 ng/mL TNF{alpha} for an additional 48 h, at which time the cultures were trypsinized and the cells counted. A total of 20,000 cells of each sample were subjected to analysis of caspase-3/7 activity (Apo-ONE kit, Promega). Right, Western blot analysis for p53, with vinculin as a loading control, done on samples not treated with TNF{alpha}.

 

Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		Figure 1. Mutant p53 enhances the activation of NF{kappa}B by TNF{alpha} in cancer cells. A, H1299 cells expressing mutant p53R175H under transcriptional control of the metallothionein promoter were transiently transfected with 200 ng firefly luciferase reporter plasmid driven by five consecutive artificial NF{kappa}B binding sites (NF{kappa}B-luc), as well as 5 ng cytomegalovirus Renilla luciferase. Twenty-four hours later, ZnCl2 was added where indicated to induce mutant p53 expression. Four hours later, 50 ng/mL TNF{alpha} was added for the indicated time periods, after which cells were extracted and subjected to dual luciferase analysis. Firefly luciferase readings were normalized for Renilla luciferase. NT, nontreated. Columns, means; bars, SD. B, SKBR3 cells were transiently transfected with NF{kappa}B-luc (200 ng) and CMV Renilla luciferase (5 ng), together with plasmids (400 ng) expressing p53 shRNA (p53i) or LacZ shRNA (LacZi). Twenty-four hours later, cells were treated with 50 ng/mL TNF{alpha} for the indicated time periods and subjected to luciferase analysis as in A (top). Columns, means; bars, SD. Western blot analysis for p53, with vinculin (Vinc) as a loading control, is shown in the bottom. C, SKBR3 cells were transiently transfected with a plasmid encoding mutant p53R175H or control vector, each at 250 ng/35 mm dish, together with 100 ng NF{kappa}B-luc and 5 ng CMV-Renilla luciferase. Transfection employed the JetPEI reagent (Polyplus). Twenty-four hours later, TNF{alpha} was added to 50 ng/mL where indicated. Luciferase analysis was done after an additional 48 h. Columns, means; bars, SD. D, SKBR3 cells were transiently transfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) as a control. One day later, cells were transfected with NF{kappa}B-luc (200 ng) and CMV Renilla luciferase (5 ng), together with a plasmid encoding p53R175H, made substantially siRNA resistant by the introduction of multiple mismatches within the siRNA-complementary sequence (m175), or pCDNA3 as a control. Forty-eight hours later, cells were treated with 50 ng/mL TNF{alpha} for 48 h and subjected to luciferase analysis as in A (top). The bottom shows Western blot analysis for p53, with vinculin as a loading control, done on samples not treated with TNF{alpha}. The sequence of the siRNA was GCAUGAACCGGAGGCCCAUdT, derived from positions 733 to 751 of the p53 open reading frame, whereas in the sequence of the corresponding p53 region, the m175 plasmid was GCATGAATCGAAGACCGAT.

 
To explore this notion in a more relevant context, we employed SKBR3 human breast cancer cells, harboring endogenous p53R175H. SKBR3 cells were transiently transfected with a plasmid expressing p53-specific small hairpin RNA (shRNA; ref. 16) to knockdown endogenous p53 and then challenged with TNF{alpha}. Cells transfected with a control lacZ shRNA plasmid mounted a robust and long-lasting NF{kappa}B response when treated with TNF{alpha} (Fig. 1B). In contrast, knockdown of endogenous mutant p53 severely blunted the extent and duration of NF{kappa}B activation. Increasing the levels of p53R175H further augmented NF{kappa}B activation (Fig. 1C). Similar results were obtained when endogenous mutant p53 was knocked down with a synthetic siRNA (Fig. 1D). Importantly, transfection of a plasmid encoding p53R175H, made substantially siRNA resistant by the introduction of multiple mismatches within the siRNA-complementary sequence (m175, Fig. 1D), overcame efficiently the inhibitory effect of the siRNA. Thus, endogenous mutant p53 in cultured human cancer cells enables stronger and longer NF{kappa}B activation by TNF{alpha}.

Mutant p53 enhances nuclear accumulation of NF{kappa}B in response to TNF{alpha}. Exposure of cells to TNF{alpha} triggers rapid nuclear translocation of the p50/p65 NF{kappa}B dimer, enabling its transcriptional effects. We therefore employed a synthetic p53 siRNA to knock down endogenous SKBR3 mutant p53 and monitored p65 localization in response to TNF{alpha}. As seen in Fig. 2A (red) , the extent of p53 knockdown was not uniform throughout the culture: whereas many cells hardly exhibited any nuclear p53, consistent with an efficient knockdown, a minority retained easily detectable residual nuclear p53.


Figure 2
View larger version (56K):
[in this window]
[in a new window]

  		Figure 2. Nuclear p65 accumulation upon TNFa treatment is enhanced in the presence of mutant p53. A, SKBR3 cells were seeded on glass coverslips and transfected with synthetic p53 siRNA. Twenty-four hours later, cells were treated with 50 ng/mL TNF{alpha} for 30 min (TNF) or left untreated (NT). Slides were then fixed in 4% paraformaldehyde in PBS for 10 min, rinsed 3x with PBS, blocked for 30 min with PBS containing 0.1% Triton X-100, 5% goat serum, and 0.2% bovine serum albumin, and incubated overnight with either anti-p65 C-20 (Santa Cruz Biotechnology, Santa Cruz, CA) or a mixture of the p53-specific monoclonal antibodies PAb1801 and DO-1. Slides were washed 3x with PBS, incubated with secondary antibodies conjugated to Alexa 466 (Molecular Probes, Eugene, OR) or Cy3 (Chemicon, Temecula, CA) for 1 h, and visualized under a fluorescence microscope. B, SKBR3 cells were treated as in (A). Multiple fields were observed by fluorescence microscopy, and relative p53 and nuclear p65 levels were scored in parallel for each individual cell. Cells were segregated into two categories, based on residual p53 levels after knockdown. The distribution of relative nuclear p65 levels among cells of each category is shown. The data are based on counting a total of 324 cells, in four groups. Columns, means; bars, SD. C, cells from the same experiment as in (A) were fixed and stained after 3 h of treatment with TNF{alpha}. D, SKBR3 cells were treated as in (C). Multiple fields were observed by fluorescence microscopy, and relative p53 and nuclear p65 levels were scored in parallel for each individual cell. Cells were segregated in two categories, based on residual p53 levels after knockdown. The distribution of relative nuclear p65 levels among cells of each category is shown. The data are based on counting a total of 242 cells in four groups. Columns, means; bars, SD.

 
As expected, p65 in untreated cells was predominantly cytoplasmic (Fig. 2A, NT, top), indicating that NF{kappa}B was largely inactive. In contrast, 30 min of TNF{alpha} treatment resulted in nuclear translocation of p65, indicative of NF{kappa}B activation (Fig. 2A, bottom). Remarkably, although some p65 nuclear translocation was evident in all cells, those harboring higher residual nuclear mutant p53 levels consistently displayed also more intense nuclear p65 staining (Fig. 2A, bottom). Visual quantification of multiple cells confirmed a nonrandom association between higher mutant p53 concentration and more pronounced nuclear p65 accumulation (Fig. 2B).

After 3 h of TNF{alpha} treatment, some cells already displayed very little nuclear p65, indicative of shutoff of the transcriptional activity of NF{kappa}B, whereas others still retained varying amounts of nuclear p65 (Fig. 2C). Remarkably, cells with higher nuclear mutant p53 retained preferentially also nuclear p65, which was rarely seen in cells with low p53 (Fig. 2C; quantified in Fig. 2D).

Collectively, these observations confirm that the amount of mutant p53 correlates closely with the extent of NF{kappa}B activation, arguing that mutant p53 can promote more efficient and more persistent NF{kappa}B activation by TNF{alpha} in cancer cells.

Down-regulation of mutant p53 sensitizes tumor cells to killing by TNF{alpha}. By engaging the extrinsic death pathway, TNF{alpha} can trigger apoptosis in target cells, which may be prevented by concurrent activation of the NF{kappa}B pathway. We therefore determined whether the ability of mutant p53 to augment NF{kappa}B activation could counteract TNF{alpha}-induced apoptosis. Indeed, expression of Zn2+-inducible p53R175H in H1299 cells, which elevates NF{kappa}B activity in cytokine-treated cells (see Fig. 1A), partially compromised apoptotic death upon TNF{alpha} treatment (Fig. 3A ); Zn2+ alone did not protect parental H1299 cells (Supplementary Fig. S2).

To assess the impact of endogenous mutant p53 on TNF{alpha}-induced apoptosis, synthetic siRNA was employed to knock down p53 in SKBR3 cells. As seen in Fig. 3B, down-regulation of endogenous mutant p53 resulted in significantly enhanced apoptosis. A similar effect was seen when a p53 shRNA expression plasmid was used instead of synthetic RNA (Supplementary Fig. S3A). Furthermore, mutant p53 knockdown resulted in elevated caspase activation (Supplementary Fig. S3B), indicative of increased apoptosis. Sensitization of mutant p53-depleted cells to the adverse effects of TNF{alpha} was also seen with the WST1 assay, which monitors viable, metabolically active cells (Supplementary Fig. S3C). Interestingly, in control cells (LacZi), TNF{alpha} sometimes stimulated proliferation mildly. Importantly, transfection of a siRNA-resistant plasmid encoding p53R175H overcame efficiently the proapoptotic effect of p53 siRNA, as assessed by caspase activity or annexin V staining (Fig. 3C and Supplementary Fig. S3D, respectively; compare p53i-Cont to p53i-m175).

Hence, mutant p53 can offer increased protection against TNF{alpha}-induced cell death. Acquisition of p53 mutations is therefore expected to provide incipient tumor cells with a selective survival advantage in a microenvironment involving chronic exposure to this proinflammatory cytokine.

Nuclear NF{kappa}B and reduced apoptosis correlate with mutant p53 in premalignant and malignant lesions. To assess the relevance of our in vitro observations to human cancer, we asked whether the presence of mutant p53 correlated with NF{kappa}B activity in tumors. To that end, we analyzed the expression of p65 and mutant p53 in head and neck squamous cell carcinoma. In all tumors with mutant p53, increased nuclear p65 staining was present in areas of intense p53 staining (Fig. 4A ). Moreover, when comparing individual tumors across the series, a clear correlation was found between p65 staining intensity and presence of p53 mutations (Supplementary Fig. S4). Interestingly, this was also evident in dysplastic epithelium (Fig. 4A), suggesting that this correlation is established already very early in cancer development. Nuclear p65 was not present in immediately adjacent hyperplastic epithelium.


Figure 4
View larger version (141K):
[in this window]
[in a new window]

  		Figure 4. Mutant p53 overexpression coincides with NF{kappa}B activation in human malignant and premalignant head and neck lesions. A, representative immunohistochemical results of NF{kappa}B p65 and p53 staining, on serial sections of paraffin-embedded specimens derived from human head and neck premalignant and malignant lesions. Formalin-fixed paraffin-embedded material, surgically resected from 15 patients with head and neck squamous cell carcinomas, some also containing adjacent hyperplastic and dysplastic lesions of the same patient on a single slide, was from a previously published series (20); the p53 status of these cases has been previously described (20). This group was supplemented with 10 new tumor specimens. The presence of mutant p53 was concluded on the basis of positive immunohistochemical staining for p53. In 12 of those cases, the presence of p53 mutations was confirmed by single-strand conformational polymorphism analysis (SCCP) and in three of those cases also by direct sequencing. Five of the immunohistochemistry-negative tumors were also subjected to SCCP analysis, and all five generated a pattern consistent with the presence of wt p53 only. Anti-p53 (DO-7; DAKO, Carpinteria, CA) and anti-NF{kappa}B p65 (F-6; Santa Cruz Biotechnology) monoclonal antibodies were employed on 5-µm paraffin sections using the indirect streptavidin-biotin-peroxidase protocol (19). Sections were incubated with the primary antibody at 1:100 dilution, at 4°C overnight. Haematoxylin was used as counterstain. N, normal tissue; T, tumor. Hyp and Dys, hyperplastic and dysplastic lesions, respectively. B, immunohistochemical analysis of Ser536-phosphorylated p65 (pp65) and p53 on serial sections of a head and neck squamous cell carcinomas harboring hyperplastic and tumor (T) tissue in the same section (middle). Direct DNA sequencing identified the p53 mutation in this tumor as Val175Phe (data not shown). Staining was essentially as in (A), except that a polyclonal anti-phospho-p65 NF{kappa}B (Ser536) antibody (Cell Signaling Technology, Danvers, MA) was employed to monitor NF{kappa}B activation. Sections were incubated with the primary antibody at 1:10 (phospho-p65) or 1:100 dilution (p53) at 4°C overnight. Higher magnification images of the dashed boxes are also shown. Arrows indicate areas of higher and lower phospho-p65 staining intensity within the tumor. Greater detail is provided in Supplementary Fig. S2.

 
NF{kappa}B activation in head and neck squamous cell carcinomas was also assessed with an antibody specific for Ser536-phosphorylated p65 (Fig. 4B). Strong NF{kappa}B activation (pp65) was observable in tumorous (T), but not adjacent hyperplastic regions. Of note, pp65 was most prominent in a subset of mutant p53-overexpressing cells, positioned in the periphery of the growing tumor. Staining for leukocyte common antigen confirmed the presence of infiltrating inflammatory cells in close proximity to the tumor (Supplementary Fig. S5). It is tempting to speculate that cytokines secreted by those cells drive the chronic activation of NF{kappa}B in adjacent tumor cells.

Essentially, similar observations were made in non–small cell lung cancers. All 20 cases with p53 mutations displayed moderate to intense cytoplasmic and often nuclear p65 staining (Supplementary Fig. S6C and D), whereas normal bronchial epithelium stained negative for both proteins (A and B). In contrast, only faint cytoplasmic p65 was observed in 15 tumors harboring wt p53 (E and F). Notably, the apoptotic index of cases with mutant p53 was significantly lower than in those with wt p53 (Supplementary Fig. S7).

Thus, within human tumors, mutant p53 overexpression correlates closely with increased NF{kappa}B activity and reduced apoptosis. Moreover, at least in some tumors, this correlation is already established early in tumor progression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study reveals that mutant p53 can promote NF{kappa}B activation in response to cytokine stimulation in cultured cancer cells and probably also within actual tumors. The molecular mechanism whereby this is achieved awaits further elucidation, although the data suggest that mutant p53 may affect both the strength and duration of NF{kappa}B activation. Moreover, it remains to be determined whether all, or only some, tumor-associated p53 mutations exert a similar effect.

Mutant p53 expression was found to correlate positively with NF{kappa}B activity in cultured cancer cells (11, 12), even without external triggers. Thus, mutant p53 may maintain higher basal NF{kappa}B activity, which is further elevated when a proper activation signal such as TNF{alpha} is delivered. In particular, basal NF{kappa}B activity can be augmented through the ability of mutant p53 to transactivate the NFKB2 gene, encoding the p100/p52 subunit of NF{kappa}B (12), as we also observed (data not shown). However, this feature of mutant p53 is unlikely to account for its effect on the TNF{alpha} response, which is mediated by p50/p65 heterodimers rather than by p52. Accordingly, we observed a pronounced effect of mutant p53 on nuclear accumulation and retention of p65 upon cytokine exposure, as well as a strong correlation between mutant p53 overexpression and nuclear p65 staining in tumors.

Unlike mutant p53, much more is known about the interrelationship between wt p53 and NF{kappa}B. Whereas the two transcription factors often antagonize each other biochemically and biologically (e.g., ref. 17), they can sometimes cooperate or otherwise exhibit an interdependence (e.g., refs. 18, 19). The factors that determine which way the wt p53-NF{kappa}B crosstalk goes remain unknown; however, it is conceivable that wt p53 may sometimes acquire properties resembling those exhibited constitutively by mutant p53. Such aberrant behavior of wt p53, favoring survival rather than apoptosis, may be particularly advantageous in tumor cells emerging in a stressful environment.

Our findings suggest that chronic exposure to inflammatory cytokines will enforce a selective pressure for p53 mutations. In addition to abrogating the proapoptotic function of wt p53, those mutations might allow incipient cancer cells to benefit, rather than suffer, from the effects of TNF{alpha} and similar cytokines. Consequently, the down-regulation of mutant p53 in tumors exhibiting constitutive NF{kappa}B activation may attenuate the latter's antiapoptotic effect, rendering the tumors more sensitive to killing by cytotoxic anticancer agents.


   Acknowledgments
 
Grant support: EC FP6 funding (contract 502983), grant R37 CA40099 from the National Cancer Institute, Center of Excellence grant from the Flight Attendant Medical Research Institute, Associazione Italiana per la Ricerca sul Cancro, Italian Health Office, and Fondo per gli Investimenti della Ricerca di Base. A. Damalas held a long-term European Molecular Biology Organization fellowship. This publication reflects the authors' views and not necessarily those of the European Community.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank S. Wilder and N. Goldfinger for assistance, C. Gelinas, G. Piaggio, A. Sacchi, A. Costanzo, G. Natoli, and P. Stambolsky for helpful discussions and R. Agami for plasmids.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

L. Weisz and A. Damalas are joint first coauthors.

Received 7/ 3/06. Revised 12/11/06. Accepted 1/17/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Harris SL, Levine AJ. The p53 pathway: positive and negative feedback loops. Oncogene 2005;24:2899–908.[CrossRef][Medline]
  2. Oren M. Decision making by p53: life, death and cancer. Cell Death Differ 2003;10:431–42.[CrossRef][Medline]
  3. Cadwell C, Zambetti GP. The effects of wild-type p53 tumor suppressor activity and mutant p53 gain-of-function on cell growth. Gene 2001;277:15–30.[CrossRef][Medline]
  4. Sigal A, Rotter V. Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome [in process citation]. Cancer Res 2000;60:6788–93.[Abstract/Free Full Text]
  5. Lang GA, Iwakuma T, Suh YA, et al. Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 2004;119:861–72.[CrossRef][Medline]
  6. Olive KP, Tuveson DA, Ruhe ZC, et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell 2004;119:847–60.[CrossRef][Medline]
  7. Weisz L, Zalcenstein A, Stambolsky P, et al. Transactivation of the EGR1 gene contributes to mutant p53 gain of function. Cancer Res 2004;64:8318–27.[Abstract/Free Full Text]
  8. Bossi G, Lapi E, Strano S, Rinaldo C, Blandino G, Sacchi A. Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression. Oncogene 2006;25:304–9.[Medline]
  9. Karin M. Nuclear factor-{kappa}B in cancer development and progression. Nature 2006;441:431–6.[CrossRef][Medline]
  10. Pikarsky E, Ben-Neriah Y. NF-{kappa}B inhibition: a double-edged sword in cancer? Eur J Cancer 2006;42:779–84.[CrossRef][Medline]
  11. Gulati AP, Yang YM, Harter D, et al. Mutant human tumor suppressor p53 modulates the activation of mitogen-activated protein kinase and nuclear factor-{kappa}B, but not c-Jun N-terminal kinase and activated protein-1. Mol Carcinog 2006;45:26–37.[CrossRef][Medline]
  12. Scian MJ, Stagliano KE, Anderson MA, et al. Tumor-derived p53 mutants induce NF-{kappa}B2 gene expression. Mol Cell Biol 2005;25:10097–110.[Abstract/Free Full Text]
  13. Luo JL, Maeda S, Hsu LC, Yagita H, Karin M. Inhibition of NF-{kappa}B in cancer cells converts inflammation-induced tumor growth mediated by TNF{alpha} to TRAIL-mediated tumor regression. Cancer Cell 2004;6:297–305.[CrossRef][Medline]
  14. Pikarsky E, Porat RM, Stein I, et al. NF-{kappa}B functions as a tumour promoter in inflammation-associated cancer. Nature 2004;431:461–6.[CrossRef][Medline]
  15. Karakaidos P, Taraviras S, Vassiliou LV, et al. Overexpression of the replication licensing regulators hCdt1 and hCdc6 characterizes a subset of non–small-cell lung carcinomas: synergistic effect with mutant p53 on tumor growth and chromosomal instability—evidence of E2F-1 transcriptional control over hCdt1. Am J pathol 2004;165:1351–65.[Abstract/Free Full Text]
  16. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550–3.[Abstract/Free Full Text]
  17. Gurova KV, Hill JE, Guo C, et al. Small molecules that reactivate p53 in renal cell carcinoma reveal a NF-{kappa}B–dependent mechanism of p53 suppression in tumors. Proc Natl Acad Sci U S A 2005;102:17448–53.[Abstract/Free Full Text]
  18. Benoit V, de Moraes E, Dar NA, et al. Transcriptional activation of cyclooxygenase-2 by tumor suppressor p53 requires nuclear factor-{kappa}B. Oncogene 2006;25:5708–18.[CrossRef][Medline]
  19. Ryan KM, Ernst MK, Rice NR, Vousden KH. Role of NF-{kappa}B in p53-mediated programmed cell death. Nature 2000;404:892–7.[CrossRef][Medline]
  20. Gorgoulis V, Zoumpourlis V, Rassidakis G, et al. Molecular analysis of p53 gene in laryngeal premalignant and malignant lesions. p53 protein immunohistochemical expression is positively related to proliferating cell nuclear antigen labelling index. Virchows Arch 1995;426:339–44.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
G. Dhar, S. Banerjee, K. Dhar, O. Tawfik, M. S. Mayo, P. J. VanVeldhuizen, and S. K. Banerjee
Gain of Oncogenic Function of p53 Mutants Induces Invasive Phenotypes in Human Breast Cancer Cells by Silencing CCN5/WISP-2
Cancer Res., June 15, 2008; 68(12): 4580 - 4587.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Li, F. Guessous, S. Kwon, M. Kumar, O. Ibidapo, L. Fuller, E. Johnson, B. Lal, I. Hussaini, Y. Bao, et al.
PTEN Has Tumor-Promoting Properties in the Setting of Gain-of-Function p53 Mutations
Cancer Res., March 15, 2008; 68(6): 1723 - 1731.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
P. Secchiero, F. Corallini, E. Rimondi, C. Chiaruttini, M. G. di Iasio, A. Rustighi, G. Del Sal, and G. Zauli
Activation of the p53 pathway down-regulates the osteoprotegerin expression and release by vascular endothelial cells
Blood, February 1, 2008; 111(3): 1287 - 1294.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Weisz, L.
Right arrow Articles by Oren, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weisz, L.
Right arrow Articles by Oren, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online