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. 2012 Feb 1;518(1):79-88.
doi: 10.1016/j.abb.2011.12.006. Epub 2011 Dec 16.

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

Catherine A Vaughan  1 , Shilpa Singh, Brad Windle, Heidi M Sankala, Paul R Graves, W Andrew Yeudall, Swati P Deb, Sumitra Deb
Affiliations

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

Catherine A Vaughan et al. Arch Biochem Biophys. .

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.

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Figures

Figure 1
Figure 1. Reducing p53 levels in cancer cells with mutant p53 by RNAi lowers chemoresistance
The indicated cells were transfected with control (si-luc or si-scrambled) or p53-specific siRNA, treated with appropriate concentrations of etoposide for 48h depending on the sensitivity of the cell line to the drug, and colony formation determined (upper panels) as described in Materials and methods. Immunoblots show the efficacy of p53 siRNA treatment. Erk2, GAPDH, or actin was used as a loading control (lower panels). Percent survival is shown in the figures with error bars representing standard deviation from the average number of colonies. The percent survival was calculated by dividing the average number of drug-treated colonies by the average number of DMSO-treated colonies and multiplying by 100. Experiments were done in triplicate and data shown are representative of multiple independent repeats.
Figure 2
Figure 2. p53 knock down causes decrease in growth rate in cancer cell lines
A–C Mutant p53 levels were knocked down in three cancer cell lines by lentivirus expressing p53 shRNA as indicated and their growth rates measured in comparison to the corresponding cell lines generated after infecting with lentivirus expressing GFP shRNA as described in Materials and methods. Cells were harvested every other day and cell numbers were determined by Coulter Counter. Error bars represent standard deviation between the average cell counts. Experiments were done in triplicate, and data are representative of multiple independent repeats. Immunoblots show the efficacy of p53 knock down. ERK2 was used as a loading control. D. For the H1048 cells, we performed a PARP cleavage assay to check for caspase activation using PARP antibody from Cell Signaling. E. Apoptosis DNA-Ladder assay of H1048 cells stably transfected with shp53 (1) or shGFP (2). U937 cells after treatment with CAM (3) were used as a positive control for apoptosis.
Figure 3
Figure 3. p53 knock down in cancer cell lines with naturally occurring p53 mutations causes a decrease in rate of motility and tumor growth
A. The indicated cell lines were cultured to confluence, a scratch made in the monolayer, and the distance measured as described in Methods. p>0.001 for both. Two mutant p53 knocked down clones (confirmed by Western blot, as indicated) were used for each cell line with the average migration between the clones shown. Error bars represent standard deviation from the mean migration rate. B. Mutant p53 levels were knocked down in the lung cancer cell line H1048 (p53-R273C) by lentivirus expressing p53 shRNA or GFP shRNA (used as our control cell line) as indicated and its tumorigenic ability measured after subcutaneous injection into nude mice. For all injections, 1x107 cells/250μl media were used. Mice were injected subcutaneously and tumors allowed to grow to a maximum size of 1cm, measuring periodically as described before (17, 18). Three different clones of H1048 cells with mutant p53 levels knocked down by shRNA were used in comparison to two GFP shRNA control cell lines to eliminate clonal variations. Average tumor size was calculated by taking the average of the width and length of each tumor, then taking the average of all tumors from the particular cell line.
Figure 4
Figure 4. Reduction of NF-κB2 causes reduction of drug sensitivity, growth rate and rate of and motility
A. Chemoresistance of MDA-MB-435 cells depends on the NF-κB2 level. The indicated cells were transfected with control or NF-κB2-specific siRNA, treated with etoposide, and colony formation determined as described in Materials and methods (upper panels). Percent survival is shown in the figure with error bars representing standard deviation from the average number of colonies from triplicate samples. B. Motility of MDA-MB-435 cells depends on the NF-κB2 level. MDA-MB-435 cells were transfected as above. 48h later, standard Transwell migration assays were carried out, and migrating cells stained and counted as described [52]. Bar = +/− 1SD from the mean of 18 samples. C. NF-κB2 and Erk2 levels were determined by immunoblotting for MDA-MB-435 cells used in A and B. D. H1299 cells expressing an empty vector (HC5) or p53-R273H were treated with control (si-scrambled) or RNAi against NF-κB2, treated with paclitaxel, and colony formation determined as described in Materials and methods. E. Western analysis of NF-κB2 knock down by siRNA in H1299 cells expressing an empty vector or p53-R273H.
Figure 5
Figure 5. Promoter deletion analysis by transient transcription analysis does not indicate a mutant p53 response element
A number of NF-κB2 [8] promoter deletion mutants were generated by PCR, and their promoter activity was measured by transient transcriptional analysis using H1299 p53-null lung cancer cell lines transfected with empty vector as control, or p53-R273H (left panel) or p53-D281G (right panel). Forty-eight hours after transfection, cells were harvested and luciferase assays were performed using equal amounts of protein. The figure shows a representative example of multiple experiments. Relative fold of activation in luciferase activity has been plotted compared to that obtained by vector alone. Error bars indicate deviations in luciferase readings relative to vector transfected cells. All experiments were done in triplicate.
Figure 6
Figure 6. ChIP analysis indicates acetylation of histones on the NF-κB2 promoter
ChIP analysis was performed on H1299 cells stably transfected with vector (HC5) or mutant p53-R273H (R273H) to test whether histones are preferentially acetylated on the NF-κB2 promoter, and if different transcription factors and mutant p53 are nucleated on the promoter. As described in Materials and methods, cells were crosslinked with formaldehyde, harvested and DNA sonicated to generate 500 bp-2 Kb fragments. Designated proteins were immunoprecipitated using respective antibodies. Cross-linking was reversed and the DNA isolated. QPCR was performed using gene specific primers. Normalized values represent QPCR values normalized to the GAPDH gene whose expression is not significantly affected by mutant p53. The promoter specific primers are located within 1000 bp of the transcriptional start site. A. ChIP performed with antibodies directed against acetylated histone H3 (acetylated at K9 and K14) and H4 (acetylated at K16). B. ChIP performed with antibodies directed against CREB, NF-κB p65, and STAT. C. ChIP performed with antibodies directed against CBP, cRel and p53 DO1. D. ChIP performed with an antibody against the total p53 protein.
Figure 7
Figure 7. ChIP analysis indicates increased acetylation of histone H3 on multiple mutant p53 _target gene promoters
Promoter regulatory regions of mutant p53 _target genes MCM6, EBAG9, Cyclin B2 and Integrin alpha 6 were tested by ChIP assay to determine whether histone H3 becomes preferentially acetylated in the presence of mutant p53, as described in the legend to Figure 6. The primer sequences have been described in Materials and Methods and are based on sequence information obtained at NCBI. The primer sequences are within 1000 bp of the predicted transcriptional start site. Values represent QPCR measurements normalized to the GAPDH gene whose expression is not significantly affected by mutant p53. All these experiments have been performed multiple times on independent biological replicates to verify their authenticity.
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