Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention AACR Conference on Molecular Diagnostics - 2008
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[Cancer Research 66, 6319-6326, June 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Mutant p53 Induces the GEF-H1 Oncogene, a Guanine Nucleotide Exchange Factor-H1 for RhoA, Resulting in Accelerated Cell Proliferation in Tumor Cells

Shinji Mizuarai, Kazunori Yamanaka and Hidehito Kotani

Functional Genomics, Banyu Tsukuba Research Institute, Merck Research Laboratory, Tsukuba, Ibaraki, Japan

Requests for reprints: Hidehito Kotani, Functional Genomics, Banyu Tsukuba Research Institute, Merck Research Laboratory, Tsukuba, Ibaraki 300-2611, Japan. Phone: 81-29-877-2202; Fax: 81-29-877-2027; E-mail: hidehito_kotani{at}merck.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tumor suppressor gene p53 is known to induce G1-S and G2-M cell cycle arrest and apoptosis by transactivating various wild-type (WT) p53 regulatory genes. Mutational inactivation of p53 is detected in more than half of human cancers, depriving the p53 protein of its tumor-suppressive functions. Recent studies have shown that mutant p53 provides tumor cells with gain-of-function properties, such as accelerated cell proliferation, increased metastasis, and apoptosis resistance. However, the mechanism underlying the elevated tumorigenicity by p53 mutation remains to be elucidated. In the present study, we showed that GEF-H1, a guanine exchange factor-H1 for RhoA, is transcriptionally activated by the induction of mutant p53 proteins, thereby accelerating tumor cell proliferation. Osteosarcoma U2OS cell lines, which express inducible p53 mutants (V157F, R175H, and R248Q), were established, and the expression profiles of each cell line were then analyzed to detect genes specifically induced by mutant p53. We identified GEF-H1 as one of the consensus genes whose expression was significantly induced by the three mutants. The GEF-H1 expression level strongly correlated with p53 status in a panel of 32 cancer cell lines, and GEF-H1 induction caused activation of RhoA. Furthermore, growth of mutant p53 cells was dependent on GEF-H1 expression, whereas that of WT p53 cells was not. These results suggest that increased GEF-H1 expression contributes to the tumor progression phenotype associated with the p53 mutation. (Cancer Res 2006; 66(12): 6319-26)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mutation of p53 is the most frequent event thus far identified in human cancers (1, 2). Germ-line mutations of p53 are causative events for Li-Fraumeni syndrome, which predisposes its patients to cancer development (3). Inactivation of p53 is caused by several distinct mechanisms, such as chromosome deletion, point mutation, degradation by up-regulated MDM2, and aberrant splicing, although the mechanisms by which the p53 locus is inactivated differs among tumor types. In lung tumor, 70% of p53 inactivation results from point mutations, whereas, in colon tumors, 60% are associated with p53 point mutations. The mutational spectrum of the p53 gene, analyzed in >10,000 human cancers from a variety of tissues and cell lines, shows that ~80% of mutation localizes in core DNA-binding regions (4). These mutations disrupt the transcriptional activation ability of wild-type (WT) p53 protein, including induction of genes connected with cell cycle arrest, apoptosis, and DNA repair, which play pivotal roles in normal cells to prevent cancer development (5, 6).

The p53 tumor suppressor gene is activated by various cellular stresses, such as DNA-damaging drug treatment, UV irradiation, or hypoxia, and is a central factor in regulating cell cycle arrest and apoptosis on these stresses by transactivation of p53 regulatory genes (6, 7). In G1-S cell cycle arrest, for example, p53 induces CDKN1A (p21Cip), leading to the inactivation of G1-S regulatory cyclin-dependent kinases (CDK) 4/6 (8) followed by retinoblastoma dephosphorylation and subsequent E2F inactivation. In the G2-M phase, p53 induces 14-3-3{sigma} or GADD45 expression, which causes inactivation and translocation of the G2-M regulatory CDK CDC2/cyclin B complex out of the nucleus (9). When cell damage is irreversible and severe, the cells execute p53-dependent apoptosis through p53 pathway activation. The activated p53 induces expression of various apoptosis regulatory genes, including Bax, Apaf-1, and CD95 (10–12), subsequently resulting in apoptotic events characterized by cytochrome c release, caspase-3 activation, and DNA fragmentation (6, 7).

In addition to the loss of function of p53, recent studies have shown that mutation of the p53 gene confers additional functions on p53 (gain-of-function) and confer advantageous growth characteristics on malignant cells. For example, overexpression of mutant p53 in null p53 cells has been shown to enhance plating efficiency in agar cell culture (13). Transgenic mice expressing exogenous mutant p53 in a p53+/– background showed accelerated and more spontaneous tumor development compared with p53-deficient mice (14, 15). Very recently, two groups, Lang et al. (16) and Olive et al. (17), established mice harboring mutant p53 in an endogenous locus to accurately model physiologic tumorigenesis and showed that the mice with endogenous p53 mutation had a high frequency of tumor development or metastasis compared with p53-deficient mice. These data all point to additional roles for p53 mutations compared with null p53 deletions, and the gain-of-function properties could be ascribed to the ability of mutant p53 to transactivate distinctive sets of genes that are not induced by WT p53. Such genes have been identified and include the drug-resistant gene (MDR-1), transcription factor (EGR1), receptor tyrosine kinase (EGF-R), cell cycle regulation (MAD1), telomerase, asparagines synthase, and gene sets from microarray experiments (18–22). However, the roles of these genes in mutant p53-mediated cell proliferation are largely unclear.

Guanine nucleotide exchange factor-H1 (GEF-H1) activates a small G-protein RhoA oncogene by increasing the GTP-bound form of RhoA (23–25). Activated RhoA transduces various signals into downstream signaling cascades, such as cytoskeleton reorganization, cellular invasion, and cell proliferation, all of which contribute to cancer progression (26, 27). A recent study reported that GEF-H1 is bound to microtubules, indicating that its activity is regulated by a cycle of microtubule binding and release (23). In GEF-H1-overexpressed cells, the COOH-terminal region of GEF-H1 is bound to microtubules, and the cells become resistant to the microtubule-disrupting anticancer agent nocodazole. By transfecting GEF-H1 into mouse fibroblasts, it was also shown that GEF-H1 itself has transformation ability without further alteration of genes, such as Ras or p53 (28). A cell line transformed by GEF-H1 transfection can induce tumor development after injection into nude mice. Microarray analysis to identify the response markers of Gleevec in gastrointestinal stromal tumors showed that GEF-H1 is one of six genes down-regulated after durable Gleevec treatment (29). Furthermore, other GEF family proteins are mutated or translocated in several cancers, such as Tiam1 in renal carcinoma (30) or the Bcr part of Bcr-Abl caused by the Philadelphia chromosome in leukemia (31). Although an increasing number of experiments suggest that GEF-H1 is involved in cancer progression, it remains to be clarified how GEF-H1 expression and activity is regulated during the multistep transformation process of oncogene activation and tumor suppressor gene inactivation.

In the present study, we revealed by microarray analysis that overexpression of mutant p53s rapidly induces mRNA expression of GEF-H1 in osteosarcoma U2OS cells. We showed that GEF-H1 expression is strongly correlated to p53 status in a panel of 32 cancer cell lines. Activation of RhoA was significantly correlated with GEF-H1 induction by mutant p53. Finally, we showed that increased GEF-H1 expression in mutant p53 cancer cell lines confers advantageous cell proliferation to those mutant cell lines. Our findings indicate that p53 mediates gene expression in tumor cells, which is characterized by the gain-of-function of GEF-H1 oncogene functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Establishment of U2OS stable clones expressing WT and mutant p53. Total RNA of U2OS cells was extracted using a RNA purification kit (RNeasy, Qiagen, Hilden, Germany), and cDNA for WT p53 was amplified by PCR. Mutant V157F, R175H, and R248Q p53 were constructed by PCR mutagenesis, and sequences were confirmed using a DNA sequencer. WT and mutant V157F, R175H, and R248Q p53 genes were subcloned into the BamHI-NheI site of the pTRE-Hyg vector (Clontech, Mountain View, CA) under the control of a doxycycline-inducible promoter. U2OS (Tet-On) cells (Clontech) were transfected with the WT and mutant p53 expression vectors, and stable cell lines that expressed almost the same level of p53 expression among the clones were selected with 400 ng/mL of hygromycin B.

Microarray analysis. At 12 and 15 hours after doxycycline treatment (0.5 µg/mL), U2OS cells expressing WT and mutant p53 were harvested, and total RNA was extracted with a RNA purification kit (RNeasy). Total RNA (~10 µg) was reverse transcribed to cDNA and subjected to microarray analysis. The HG-U133A chip (Affymetrix, Santa Clara, CA), which contains human probes for ~14,000 genes, was used for the hybridizations. Hybridization and the array wash procedure were done according to the Affymetrix fluidics station protocols.

Each gene is represented by a set of 16 probes, and each probe has 25mer oligonucleotides on the GeneChip. The expression levels of genes were measured by comparing the signal intensities of these probe sets with the one-base mismatch oligonucleotide probes using Affymetrix Microarray Suite version 5.0 (GeneChip software MAS 5.0). GeneChip software provides "signal" values and "detection" calls, "present" (P), "marginal" (M), or "absent" (A), for each probe set. Gene expression was scored as "P" when the perfect match signal exceeded its mismatch counterpart with P < 0.04, "M" with P = 0.04 to 0.06, and "A" with P > 0.06. Genes scored as "P" were analyzed for the subsequent comparison analysis. GeneSpring 6.0 software (Silicon Genetics, Redwood City, CA) was used to do the calculations for hierarchical clustering and to display the results graphically. We used the value of log2 ratio, and cosine correlation was applied for clustering of genes.

Quantitative real-time reverse transcription-PCR. Total RNA was extracted from 5 x 105 cells using the RNeasy kit. Reverse transcription was done for 500 ng total RNA, and the cDNA obtained was applied to Taqman PCR for quantification of mRNA expression. Data were collected and analyzed using an ABI PRISM 7700 sequence detector system (Applied Biosystems, Warrington, United Kingdom). The relative mRNA expression data were normalized to ß-actin. The sequences of the primers and Taqman probes were as follows: Taqman probe for p21Cip, 5'-CGGCGGCAGACCAGCATGAC-3'; PCR forward and reverse primers for p21Cip, 5'-TGGAGACTCTCAGGGTCGAAA-3' and 5'-GGCGTTTGGAGTGGTAGAAATC-3'; Taqman probe for p53, 5'-CTGTCCCCCTTGCCGTCCCA-3'; PCR forward and reverse primers for p53, 5'-CCTATGGAAACTACTTCCTGAAAACAA-3' and 5'-ACAGCATCAAATCATCCATTGC-3'; Taqman probe for GEF-H1, 5'-CCGGGATGACCGGAGCACCTG-3'; PCR forward and reverse primers for GEF-H1, 5'-TGAGATGTACGAGGTGCACACA-3' and 5'-CGCACGCTCTGCTGAATG-3'; Taqman probe for Bax, 5'-CCCGAGAGGTCTTTTTCCGAGTGGC-3'; and PCR forward and reverse primers for Bax, 5'-CCGCCGTGGACACAGACT-3' and 5'-TTGCCGTCAGAAAACATGTCA-3'. Predeveloped Taqman assay reagents (Applied Biosystems) were used for the Taqman probe and primers for ß-actin. Cell lines used for the analysis of GEF-H1 expression in Fig. 4A were as follows: WT p53, HCT116, RT4, A427, PA1, HepG2, MKN74, MCF7, A549, and U2OS; mutant p53, UM-UC-3 (F113C), T24 (Y126, deletion), ScaBER (R110L), J82 (E271K, V274F, K320N), DLD1 (S241F), Cal27 (H193L), SCC25 (R290, frameshift), PC13 (G334V), H211 (R248Q), LX1 (R273H), Lu135 (G244C), OVCAR3 (R248Q), MDAH2774 (R273H), SK-OV3 (H179R), ES-2 (S241F), NCI-N87 (R248Q), MKN1 (V143A), U-118MG (R213Q), and BxPC3 (Y220C); and p53-deficient, KATOIII, SaoS2, NCI-H69, and HeLa (p53 inactivation by E6).


Figure 4
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  		Figure 4. GEF-H1 mRNA expression is correlated with p53 status in cancer cells. A, GEF-H1 expression in U2OS cells expressing WT or mutant p53. U2OS stable clones with WT or mutant p53 were treated with doxycycline to induce p53. GEF-H1 mRNA expression was measured at each indicated time by real-time RT-PCR. B, correlation of GEF-H1 mRNA expression and p53 genetic statuses in cancer cell lines. Relative GEF-H1 mRNA expression levels were quantified with real-time RT-PCR in 32 cancer cell lines with WT, naturally occurring mutant, or deficient p53 genes. C, correlation of GEF-H1 and mutant p53 mRNA expression in cancer cell lines. In mutant cancer cell lines, both GEF-H1 and mutant p53 expression were measured by real-time RT-PCR. R, Pearson correlation coefficient. D, GEF-H1 expression is not induced by silencing WT p53 expression. U2OS cells were transfected with p53 siRNA, control luciferase siRNA, or R248Q expression vector. At 48 hours after transfection, mRNA expression was measured by real-time RT-PCR. E, GEF-H1 mRNA expression is reduced by mutant p53 silencing. U2OS cells overexpressing mutant p53 (R248Q) or ScaBER were treated with siRNA for p53 to suppress mutant p53. After 48 hours, GEF-H1 expression was quantified by real-time RT-PCR.

 
Immunoblotting. For immunoblotting of p53 and total RhoA, total cell lysate was extracted from 2 x 106 cells with Mammalian Protein Extraction Reagent buffer (Pierce, Rockford, IL), and 20 µg total protein was subjected to 10% SDS-PAGE analysis. Proteins were visualized by enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ) using primary antibody specific to human p53 (SC263, Santa Cruz Biotechnology, Santa Cruz, CA) and RhoA. For the detection of the GTP-bound form of RhoA, a Rho activation kit (Pierce) was used according to the manufacturer's instructions. Briefly, cell lysates were extracted from 3 x 107 cells and immunoprecipitated with glutathione S-transferase–Rhotekin, a RhoA effector. The immunoprecipitated samples were subjected to Western blotting using a primary antibody for RhoA.

Gene silencing by small interfering RNA. For small interfering RNA (siRNA) transfection, WT or mutant p53 cells were seeded at a confluency of 5% 1 day before transfection and transfected with siRNA for GEF-H1 or p53 using siLentFect (Bio-Rad, Hercules, CA). The absolute amount of GEF-H1 mRNA in the cell lines estimated by the result of real-time reverse transcription-PCR (RT-PCR) was as follows: WT p53 cells, A549, 207 copies per cell; MCF7, 185 copies per cell; RT4, 165 copies per cell; and A427, 133 copies per cell and mutant p53 cells, SK-OV3, 469 copies per cell; UM-UC-3, 459 copies per cell; ScaBER, 447 copies per cell; and ES-2, 420 copies per cell. The siRNA for GEF-H1 was a pool of four sequences, which were purchased from Dharmacon (Lafayette, CO). The p53 siRNA was purchased from Ambion (Austin, TX). As a negative control, we used siRNA for luciferase, which does not interfere with any mammalian mRNA (Dharmacon). For transfection using a 96-well plate, siRNA solution (12.5-50 nmol/L) was complexed with siLentFect in 10 µL Opti-MEM (Invitrogen, Carlsbad, CA). After incubating the complex for 20 minutes, cells were treated with the lipid/siRNA solution. At 6 hours after transfection, 100 µL growth medium was added to the transfected cells. Two days after the transfection, mRNA expressions were quantified by real-time RT-PCR. Three days after the transfection, cell viability was measured using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) reagent (Kishida Chemical, Osaka, Japan). Briefly, 20 µL WST-8 solution was added to cells containing 200 µL medium, and the mix was incubated for 2 hours at 37°C. Absorbance was then measured at 450 nm using a plate reader. In this system, the amount of formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of WT and mutant p53 stable cell lines. Osteosarcoma U2OS cells were transfected with WT and mutant p53 expression vectors (V157F, R175H, and R248Q; Fig. 1A ), and stable cell lines were established and then used to systematically analyze common mutant p53-inducible signature sets by DNA microarray analysis. Both R175H and R248Q mutants are one of five ‘hotspot’ mutants (32) detected in various cancers (breast, colon, and bladder), whereas the V157F mutation is frequently detected in lung cancer. All three mutations are located in the DNA-binding domain of p53 protein and cause loss of WT p53 transcriptional activation activities. All transfected p53 variants were under the control of an inducible promoter, and p53 expression was regulated by the addition of doxycycline. We selected stable clones, in which p53 induction levels are almost the same among the WT and mutant p53 clones. Figure 1B and C shows the p53 mRNA and protein expression levels, respectively, which were analyzed by quantitative real-time RT-PCR and Western blotting in the established cell lines after p53 induction. At 24 hours after p53 induction, a >10-fold increase in both WT and mutant p53 expression was seen in each cell line, at both mRNA and protein level, compared with control cells. In established mutant p53 cells, the V157F, R175H, and R248Q mutants were expected to lose the transactivation function of p53 regulatory genes. To confirm the loss of transactivation ability by mutant p53, the mRNA expression levels of p53 regulatory p21Cip and Bax were measured after the induction of p53. In WT p53 cells, both p21Cip and Bax were increased in an induction time-dependent manner, whereas no induction was observed in any of the three mutant p53 lines or control cells (Fig. 2A and B ). After p53 induction, apoptosis was observed in WT p53 cells, whereas no apoptotic induction was observed in mutant p53 cells (data not shown).


Figure 1
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  		Figure 1. Establishment of U2OS cells expressing inducible WT and mutant p53. A, schematic diagram of p53 protein showing the characteristics of mutants V157F, R175H, and R248Q. %, mutational frequency in each domain. B, mRNA induction of WT and mutant p53 in U2OS cells. U2OS cells were transfected with inducible WT and mutant p53 genes (V157F, R175H, and R248Q), and stable cell lines were established. mRNA expression of p53 in each clone was measured by real-time RT-PCR at the indicated hour after doxycycline addition. C, p53 protein induction in U2OS cells. U2OS stable clones were treated with or without doxycycline (DOX) for 24 hours, and whole protein was then extracted. Subsequently, p53 protein was detected with immunoblotting.

 

Figure 2
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  		Figure 2. WT p53 functions of gene transactivation are lost in mutant p53 cells. Relative mRNA expression of p21Cip (A) and Bax (B) in WT and mutant p53-expressing cells. After the induction of WT and mutant p53 expression in U2OS cells, total RNA was extracted at the indicated time, and the mRNA expression levels of p21Cip and Bax were measured by real-time RT-PCR analysis.

 
Induction of GEF-H1 by mutant p53 expression. To identify genes induced by mutant p53 expression, we compared the expression profiles of U2OS cells expressing WT and mutant p53s (V157F, R175H, and R248Q). To reduce the secondary effects of p53 induction, RNA was isolated from each cell line at 12 and 15 hours after p53 induction. The expression profiles of ~14,000 genes were analyzed using the Affymetrix oligonucleotide microarray HG-U133A GeneChip. We focused on genes whose expression was statistically increased (P < 0.005) in all three mutant p53 cell lines after 15-hour induction and for which the fold increase was >1.5-fold in at least one mutant p53. Next, we eliminated genes whose expression was statistically increased and for which the fold increase was >1.2-fold in WT p53 cells at either 12 or 15 hours after induction. Based on these criteria, all the known p53 regulatory genes were eliminated. Eleven genes were identified to be specifically increased by mutant p53s, and Fig. 3 shows the heat map of the expression pattern for the extracted genes, showing the time course induction of these 11 genes. Among the genes, expression of a Rho guanine exchange factor, GEF-H1, showed the greatest statistically significant increase. Based on the reported oncogenic functions on GEF-H1, we focused on GEF-H1 to further analyze the correlation of GEF-H1 expression with p53 status and GEF-H1 involvement in cancer progression. GEF-H1 has transforming ability in mouse fibroblasts and is a known activator of the RhoA oncogene, and overexpression is expected to cause drug resistance to microtubule-targeted agents.


Figure 3
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  		Figure 3. Heat map of expression of mutant (mt) p53-inducible genes. Expression profile of U2OS cells expressing inducible WT and mutant p53 was analyzed using the Affymetrix oligonucleotide microarray. Genes for which expression was specifically increased in mutant V157F, R175H, and R248Q p53 cells, but not in WT p53 cells, were extracted. The fold increase in extracted genes against control cells as the log2 ratio and as a heat map. The degrees of redness and greenness represent induction and repression, respectively.

 
To confirm the microarray data of GEF-H1 induction, we did real-time RT-PCR and quantified the mRNA induction level of GEF-H1 on mutant p53 expression. Figure 4A shows that GEF-H1 mRNA expression is induced in a time-dependent manner only in mutant p53 cells. After 48 hours of induction, 5- to 10-fold increased expression was observed in mutant p53 clones, whereas no induction was observed in the control or WT p53 cells.

Next, we investigated whether the GEF-H1 mRNA expression level is higher in cancer cell lines with spontaneous p53 mutations compared with WT p53. First, cell lines with different p53 status were collected. The composition of p53 status in the 32 cell lines was as follows: WT p53, HCT116, RT4, A427, PA1, HepG2, MKN74, MCF7, A549, and U2OS; mutant p53, UM-UC-3, T24, ScaBER, J82, DLD1, Cal27, SCC25, PC13, H211, LX1, Lu135, OVCAR3, MDAH2774, SK-OV3, ES-2, NCI-N87, MKN1, U-118MG, and BxPC3; and p53 deficient, KATOIII, Saos2, NCI-H69, and HeLa (p53 inactivation by E6). Next, GEF-H1 mRNA expression was quantified by real-time RT-PCR analysis. The average expression level of GEF-H1 in mutant p53 cell lines was ~2-fold higher than in WT or deficient p53 cell lines (Fig. 4B). Moreover, in mutant p53 cell lines, the GEF-H1 expression level showed significant correlation with mutant p53 mRNA expression (correlation coefficient; R = 0.655, P < 0.01), indicating that GEF-H1 is induced by mutant p53 under physiologic conditions (Fig. 4C).

Several genes, such as blc-2 and MAP4, are transcriptionally repressed by WT p53. Because parental U2OS cells express a low level of WT p53, the possibility exists that dominant-negative mutant p53 (V157F, R175H, and R248Q) antagonizes the transrepressional effect of WT p53 on the GEF-H1 promoter rather than the induction of GEF-H1 by mutant p53. To rule out this possibility, we examined whether p53 silencing would induce GEF-H1 expression as for mutant p53 induction. In U2OS cells transfected with p53 siRNA, p53 mRNA expression was repressed by 80%. However, GEF-H1 was not induced by p53 silencing compared with control siRNA-treated cells (Fig. 4D), whereas a positive control of transient R248Q expression increased GEF-H1 expression similar to mutant p53 stable clones. To further confirm the relationship between the expression of mutant p53 and GEF-H1, U2OS (R248Q) and ScaBER (R110L) cells were treated with siRNA for p53 to repress mutant p53 expression, and mRNA expression of GEF-H1 was then measured by real-time RT-PCR. The mutant p53 silencing reduced GEF-H1 mRNA expression in a siRNA concentration-dependent manner (Fig. 4E), whereas control siRNA treatment did not change expression.

RhoA is activated by induction of GEF-H1. GEF-H1 is a guanine exchange factor for small G-protein RhoA; induction of GEF-H1 expression by mutant p53 is expected to increase the GTP-bound (activated) form of RhoA. We measured the activation level of RhoA in the established U2OS cells via p53 induction (Fig. 5 ). After 24 and 48 hours of WT or mutant p53 induction, cell lysate was extracted, and the amount of total and GTP-bound form of RhoA was measured in each cell line by immunoblotting. The activated form of RhoA was detected by immunoprecipitating with the Rho-binding domain of Rhotekin, a GTP-Rho-specific binding protein, and subsequent immunoblotting with RhoA antibody. In empty vector control cells, RhoA was not activated by the addition of doxycycline. In contrast, induction of any of the three p53 mutants significantly increased the expression of the activated form of RhoA (Fig. 5). Densitometric measurement of the immunoblot showed that an ~4-fold increase of RhoA was observed at 48 hours after mutant p53 induction. In WT p53 cells, RhoA was slightly inactivated compared with the control probably due to decreased cell viability caused by WT p53 expression. The total amount of RhoA, including both activated and inactivated forms, was almost constant among the control, WT, and mutant p53 clones. In addition, the mRNA expression level of RhoA did not change by mutant p53 induction (data not shown).


Figure 5
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  		Figure 5. RhoA is activated by GEF-H1 in mutant p53 cells. U2OS stable clones were treated with doxycycline to induce WT and mutant V157F, R175H, and R248Q p53. Total and activated forms of RhoA were detected with immunoblotting in each clone. The activated GTP-bound form of RhoA was immunoprecipitated with Rhotekin, a GTP-RhoA effector, and detected with subsequent Western blotting with anti-RhoA antibody.

 
GEF-H1 is necessary for growth in mutant p53 cancer cells. GEF-H1 has been shown to have transforming activity, and the induction of RhoA is also known to increase the oncogenic potential in tumor cell lines. Hence, we hypothesized that cancer cell lines with mutant p53 are more dependent on GEF-H1 expression for their proliferation compared with cells with WT p53. To investigate GEF-H1 function in p53 mutant cell lines, we examined the effect of GEF-H1 silencing by siRNA on the growth of various cancer cell lines with naturally occurring WT or mutant p53. We used RT4, A427, MCF7, and A549 for the WT p53 cell lines and SK-OV3, UM-UC-3, ScaBER, and ES-2 for the mutant p53 cell lines. To disrupt GEF-H1 expression, increasing concentrations of GEF-H1 siRNA were transfected into the various cell lines by lipofection. As a negative control, siRNA for luciferase was also transfected into the cells. We measured mRNA expression of GEF-H1 by real-time RT-PCR and confirmed that >80% of mRNA suppression was achieved on the condition of 50 nmol/L siRNA treatment in each cell line (Fig. 6A and B ). Three days after GEF-H1 silencing, cell viability was measured in each cell line using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In the WT p53 cell line, little effect on cell proliferation was observed, with a maximal effect in RT4 cells with 20% of growth inhibition (Fig. 6C). In contrast, in mutant p53 cells, growth inhibition was much more sensitive to GEF-H1 silencing. Cell growth was inhibited in a siRNA dose-dependent manner, and viability was decreased by ~60% by GEF-H1 silencing with siRNA in all four mutant p53 cell lines (Fig. 6D), indicating that GEF-H1 expression has an advantageous effect on cell proliferation in mutant p53 cells.


Figure 6
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  		Figure 6. Silencing of GEF-H1 by siRNA is effective for cell growth inhibition in mutant p53 cells compared with WT p53. GEF-H1 mRNA expression was measured by real-time RT-PCR analysis 48 hours after siRNA for GEF-H1, or control was transfected into cancer cell lines: WT p53, RT4, A427, MCF7, and A549 (A) and mutant p53, UMUC3, SK-OV3, ES-2, and ScaBER (B). At 72 hours after transfection, the viability of WT p53 (C) and mutant p53 cells (D) was measured using a modified MTT assay. Figure 6, GEF-H1 siRNA-treated cells; {blacklozenge}, control siRNA-treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we tried to identify novel gain-of-function genes induced by tumor-derived mutant forms of p53 by comparing the expression profiles of U2OS cells expressing WT and mutant p53s. Several important observations on the mechanisms for gain-of-function arose from this study. We identified GEF-H1 as one of the consensus genes induced by three p53 mutants, V157F, R175H, and R248Q. Because the induction of GEF-H1 was observed in an exogenous mutant p53 expression system, we also examined GEF-H1 expression in various cell lines with naturally occurring p53 status and showed that GEF-H1 expression is significantly higher in mutant p53 cell lines than in WT p53 cell lines. In GEF-H1-overexpressed cells, we showed that the GTP-bound form of RhoA is increased, indicating that the RhoA pathway is activated in mutant p53 cells. Finally, we found that silencing of GEF-H1 decreases cell viability more effectively in mutant p53 cells compared with WT p53 cells.

GEF-H1 mRNA expression was rapidly induced by mutant p53 expression in U2OS cells. However, the detailed mechanism of GEF-H1 induction by mutant p53 remains to be elucidated. It is speculated that there are two mechanisms by which mutant p53 induces gene expression. One mechanism is related to the nonsequence-specific activation of transcription by mutant p53. The consensus sequence for the binding of WT p53 has been well characterized and consists of two tandem copies of 5'-PuPuPuCWWGPyPyPy-3', with a spacer of ~15-bp sequences (33). However, the consensus sequence is not observed in the promoter regions of genes induced by mutant p53 in most cases, although chromatin immunoprecipitation studies have shown that mutant p53 proteins physiologically interact with some of the promoter sequence of induced genes (22, 34). Because the consensus sequence of the p53-binding site was not found within 1,000 bp of the GEF-H1 promoter region by TRANSFAC software analysis (data not shown), nonconsensus sequence-specific transcriptional activation may explain GEF-H1 induction by these mutant p53 proteins (35). The other proposed mechanism of transactivation by mutant p53 is the dominant-negative effect of mutant p53 on the transrepression ability of WT p53. WT p53 is known to repress several genes, such as MAP4, IL-6, and blc-2 (36–38). It has been proposed that WT p53 carries out gene repression in cooperation with a well-known transcriptional repressor, histone deacetylase and its corepressor mSin3a (39). For GEF-H1, the possibility of a dominant-negative effect on transrepression of p53 seems low because we were unable to observe further repression of GEF-H1 by inducing WT p53 in cell lines, and WT p53 repression by RNA interference did not cause GEF-H1 induction (Fig. 4D).

Increased expression of GEF-H1 in mutant p53 cells is expected to cause several advantageous effects on tumor development. Recently, GEF-H1 expression was reported to transform mouse fibroblasts without genetic alterations in other oncogenes and tumor suppressor genes (28). In addition, GEF-H1 is an activator of RhoA, which also works as an oncogene by transducting various downstream signals (23). Because RhoA modulates cell migration and polarity by reorganizing the actin cytoskeleton (26, 27), activation of RhoA by GEF-H1 overexpression in mutant p53 cells might enhance the invasion or metastatic properties of tumor cells. In addition to cell motility, RhoA is involved in cell proliferation. RhoA activation leads to the inactivation of the CDK inhibitors p21Cip and p27Kip and induction of cyclin D1 expression, which results in cell cycle acceleration (27). Such speculation is supported by the fact that the activated RhoA pathway in tumorigenic p53-deficient mouse fibroblasts produces accelerated cell proliferation and motility (40, 41). GEF-H1 overexpression also confers resistance to a microtubule-disrupting agent, nocodazole (23). GEF-H1, which interacts with microtubules, is expected to protect the cells from microtubule-disrupting agents. These results infer that mutant p53 cells with increased GEF-H1 expression might become more resistant to chemotherapy agents.

Previous studies have identified several genes up-regulated by mutant p53, such as MDR-1, EGF-R, and hsMAD1 (18–20). With different sets of criteria, MDR-1 was also statistically increased in mutant p53 cells in the present study; however, we did not observe the induction of those reported mutant p53-inducible genes in our study. One possible explanation is the difference in expression system of mutant p53. We used a mutant p53-inducible system and analyzed the expression profile immediately after p53 induction to eliminate the secondary effects of mutant p53 expression. Previous studies have used mutant p53 constitutive expression systems (20, 21). Another possibility is the difference in parental cell lines used in each study. Even in previous publications, mutant p53-inducible gene sets identified in the expression profile analysis are not consistent among reports, implying that induction might be dependent on the cell type used in each study. Hence, the significance of the induced genes by mutant p53 should be examined in cancer cell lines or clinical cancer samples with naturally occurring p53 status. Among the genes identified as mutant p53-inducible genes, expression of MDR-1 has been shown to correlate with mutant p53 expression in clinical tumor samples (42). For GEF-H1, the expression level correlated with p53 status in 32 cancer cell lines with naturally occurring p53 status. Further studies are required to examine whether GEF-H1 expression is increased in clinical cancer samples with mutant p53.

In the present study, we showed that GEF-H1 is induced by the overexpression of mutant p53. In addition, the expression of GEF-H1 is significantly increased in cell lines with mutant p53 rather than those with WT p53. We also showed that GEF-H1 silencing is more sensitive in mutant p53 cells than in WT p53 cells, indicating that GEF-H1 expression confers an advantageous effect on cell proliferation in mutant p53 cells. Although an increasing number of reports have indicated that GEF-H1 might be involved in tumorigenesis, how the expression and activity are regulated in the multistep oncogene or tumor suppressor mutations is unknown. The fact that mutant p53 induces GEF-H1, as revealed by this study, provides insight into the mechanism of tumor progression accompanying p53 mutation. It would also be interesting to examine GEF-H1 or its related genes in this pathway as new generation anticancer drug targets.


   Acknowledgments
 
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 K. Takahashi and T. Yamamoto for their expertise in microarray analysis and T. Eguchi for critical comments.

Received 12/27/05. Revised 3/16/06. Accepted 4/13/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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