Inhibits Tumor Growth by Inducing Apoptosis ...

Homeobox Msx1 Interacts with p53 Tumor Suppressor and Inhibits Tumor Growth by Inducing Apoptosis Kyoungsook Park, Kwangbae Kim, Seung Bae Rho, et al. Cancer Res 2005;65:749-757.

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Research Article

Homeobox Msx1 Interacts with p53 Tumor Suppressor and Inhibits Tumor Growth by Inducing Apoptosis Kyoungsook Park, Kwangbae Kim, Seung Bae Rho, Kyusam Choi, Dojin Kim, Sun-Hee Oh, Jinhee Park, Seung-Hoon Lee, and Je-Ho Lee Molecular Therapy Research Center, Sungkyunkwan University, Samsung Medical Center Annex 8F, Kangnam-ku, Seoul, Korea

occur in HPV-positive cervical cancer cells (9). Consistent with this, the expression of peptides that specifically bind to E6 results in p53 accumulation and apoptosis in HPV-positive cancer cells (10). The homeodomain is an evolutionarily conserved 60-amino-acid DNA-binding domain. Homeobox genes function as essential transcriptional regulators in a variety of developmental processes. Homeoproteins orchestrate gene expression and regulate cell growth, proliferation, differentiation, cell-to-cell communication, and the apoptotic pathway during pattern formation in embryogenesis (reviewed in refs. 11, 12). Recent findings suggest that deregulated expression of homeobox genes is involved in human oncogenesis (13–15). Homeobox genes that are down-regulated in cancer share several features including tissue specificity and epigenetic loss of function. They can be best described as tumor modulators (16). Msx1 encodes a homeoprotein that functions as a transcriptional repressor through interactions with components of the core transcription complex as well as other homeoproteins. Msx1 is expressed at sites where cellular proliferation and apoptosis occur during pattern formation in embryogenesis (reviewed in ref. 12). The biochemical properties of Msx1 protein and its fundamental role as an embryonic transcriptional regulator are well established. Evidence linking Msx1 to the regenerative ability as well as growth of hair and teeth has been well documented. However, its role as a regulator of tumor suppressor in adult tissues remains unknown. Here we show that Msx1 interacts with p53 and inhibits tumor growth by inducing apoptosis in vitro and in vivo.

Abstract The stability of wild-type p53 is critical for its apoptotic function. In some cancers, wild-type p53 is inactivated by interaction with viral and cellular proteins, and restoration of its activity has therapeutic potential. Here, we identify homeobox Msx1 as a p53-interacting protein and show its novel function as a p53 regulator. Overexpression of homeobox Msx1 induced apoptosis of cancer cells harboring nonfunctional wild-type p53 and suppressed growth of human tumor xenografts in nude mice. The homeodomain of Msx1 functions as a protein-protein interacting motif rather than a DNA-binding domain and is essential for stabilization, nuclear accumulation, and apoptotic function of wild-type p53. The identification of a novel function of Msx1 as a p53 regulator may open new avenues for developing improved molecular therapies for tumors with a nonmutational p53 inactivation mechanism. (Cancer Res 2005; 65(3): 749-57)

Introduction The p53 tumor suppressor protein is involved in a variety of cellular processes, including cell cycle regulation and apoptosis (1, 2). It functions as a transcription factor with a high affinity for specific DNA target sequences in response to DNA damage or hypoxia and it also up-regulates the expression of target genes. The p53 protein functions to selectively destroy stressed or abnormal cells to prevent the progression to cancer (3). As shown in some cancers, the interaction of p53 with viral and cellular proteins leads to inactivation of p53 functions; therefore, restoration of wild-type p53 activity in tumor cells could be of immense therapeutic potential (1, 4). p53 function is regulated by several mechanisms including posttranslational modification, stabilization, protein interactions, and subcellular localization. One of the most effective mechanisms is regulation of p53 protein stability. Protein stability is regulated, in normal as well as malignant cells, by ubiquitin-dependent proteolysis. Murine double minute 2 (Mdm2) oncoprotein is the most important regulator of p53 nuclear export and degradation (4–6). Recent evidence shows that several proteins involved in oncogenesis, such as ADP ribosylation factor, oncogenic Ras, RB, and TSG101, also affect the stability of p53 by modulating Mdm2mediated degradation (reviewed in ref. 7). The high-risk human papillomavirus (HPV), which are implicated in the pathogenesis of cervical cancer, produce the E6 protein (8). A complete switch from Mdm2 to E6-dependent degradation of p53 has been shown to

Materials and Methods Cell Culture, Antibodies, and Plasmids. Human cervical HeLa cells and human lung carcinoma H1299 cells were maintained in DMEM or RPMI (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum and penicillin/streptomycin (100 units/mL) at 37jC in a humidified 5% CO2 incubator. The following antibodies were used in this study: anti-Msx1 antibody (Babco, Berkeley, CA), anti-p53 mABs [Do-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and PAb421 (Oncogene, San Diego, CA)] anti-phospho-specific p53 (Cell Signaling, Beverly, MA), mouse anti-E6 protein mAB (Ab-1, Oncogene), anti-GFP (Santa Cruz), anti-caspase 3 (Santa Cruz), anti-Bax (Santa Cruz), and anti-h-actin (Sigma, St. Louis, MO). The human Msx1 gene in vector pCB6+ (pCB6+/Msx1), pGEX-2T-Msx1 (1-297), and pGEX2T-Msx1 (1-165) were gifts from Dr. Cory Abate-Shen (UMDNJ-Robert Wood Johnson Medical School, NJ) and were reported previously (17). Green fluorescence protein (EGFP)–tagged Msx1 expression vectors for full-length [pEGFP/ Msx1 (1-297)], homeodomain-deleted [pEGFP/Msx1 (1-165)], and homeodomain alone [pEGFP/Msx1 (166-225)] were constructed by subcloning BamHI-HindIII fragment of pGEX2T-Msx1 (1-297) and BamHI-EcoRI fragment of pGEX2T-Msx1 (1-165) into BglII and HindIII sites [pEGFP/Msx1 (1-297)] or BglII and EcoRI sites [pEGFP/Msx1 (1-165)] of pEGFPC1 (Clontech, Palo Alto, CA), respectively. pEGFP/ Msx1(166-225) was generated into SalI and BamHI sites of pEGFP vector by PCR amplification of the homeodomain (165-225) of Msx1 using pEGFP (1-297) as a template with the oligonucleotides (5V-attgtcgacaaccgcaagcc-

Requests for reprints: Je-Ho Lee, Molecular Therapy Research Center, Sungkyunkwan University, Samsung Medical Center Annex 8F, 50 ILwon-dong, Kangnam-ku, Seoul, 135-710, Korea. Phone: 82-2-3410-3510; Fax: 82-2-3410-0044; E-mail: [email protected]. I2005 American Association for Cancer Research.

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Cancer Research caggacgcct-3V and 5V-attggatcctcactgcagtctcttggcctt-3V). Homeodomain mutant of Msx1 (pEGFP-HD mutant) that has three point mutations (K168A, R170A, and F173A) was generated with the oligonucleotides (5Vaagaccaaccgcgcgcccgcgacgcctgccaccacagctcag-3V and 5V-ctgagctgtggtggcaggcgtcgcgggcgcgcggttggtctt-3V) using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The sequence of each Msx1 plasmid was verified by DNA sequencing. Glutathione S-transferase (GST)p53 and pCMV-p53 expression vectors were kind gifts from Dr. J. Choe (Korea Advanced Institute of Science and Technology, Daejeon, Korea). Transient Transfection and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assays. For all experiments, subconfluent cells were transiently transfected with the indicated plasmids using Effectene according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Expression of the transfected gene was identified by immunoblotting and fluorescence microscopy with appropriate antibodies. The MTT colorimetric assay was done according to the method previously described (18). In brief, HeLa cells were seeded onto 96-well plates at a density of 3 103 cells per well. After 24 hours, the cells were transfected with the indicated expression vectors. Tetrazolium agent (50 Ag MTT in 10 AL PBS) was added into each well followed by a 4-hour incubation, and then culture medium supernatant was removed from wells and the formazan was dissolved with 50 AL DMSO. After formazan solubilization, the absorbance of each well was determined using a microculture plate reader (SLT-Lab Instrument, Salzburg, Austria) at 540 nm. The growth inhibition was measured by the mean absorbance. Each experiment was done in triplicate. Apoptosis Assays. HeLa cells were seeded onto chamber slides at a density of 5 104 cells per well and then transfected with pEGFP/fulllength Msx1 or pEGFP vector for 24 hours using Effectene as described above. Cells were incubated with FITC-labeled Annexin V and propidium iodide for 15 minutes according to the supplier’s protocol (Boehringer Mannheim, Mannheim, Germany) and then analyzed on a FACS Vantage (Becton Dickinson, San Jose, CA). For evaluation of nuclear morphology, cells were fixed in methanol and stained with 4V,6-diamidino-2-phenylindole (DAPI, 1 Ag/mL in methanol) for 15 minutes, washed with 1 PBS thrice, followed by treatment with VectaShield (Vector Laboratories, Burlingame, CA) and examined under a fluorescence microscope (Zeiss, Hallbergmoos, Germany). To detect apoptotic cells in murine tumors, frozen sections of 8 Am were processed using DeadEnd Fluorometric terminal deoxynucleotidyl transferase nick end label (TUNEL) system (Promega, Madison, WI) according to the protocol provided by the manufacturer. Colony Formation Assays. HeLa and H1299 cells plated in 60-mm dishes were transfected with the indicated plasmid construct using Effectene (Qiagen). After a 24 hours transfection, cells were selected by growth in 500 Ag/mL to 1 mg/mL of geneticin (G418, Life Technologies) for 2 weeks. The geneticin-resistant colonies were stained with crystal violet. Experiments were repeated at least thrice. Immunoprecipitation and Immunoblotting. Transfected cells were lysed in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP40, 0.1% SDS, and 10 mmol/L sodium deoxycholate]. Portions (100 and 20 Ag, respectively) of total cell lysates were used for immunoprecipitation and immunoblotting. To determine the half-life of p53 protein, HeLa cells were transfected with control pCB6+ vector and pCB6+/Msx1. At 24 hours post-transfection, cells were incubated with 80 Ag/mL cycloheximide to inhibit protein synthesis. At the indicated time points, cells were washed with PBS twice and lysed in SDS sample buffer. Cellular extracts from each time point were resolved by SDS-10% polyacrylamide gels and subjected to immunoblotting using indicated antibodies and visualized by chemiluminescence (Amersham, Buckinghamshire, United Kingdom) essentially as described (19). Equal protein loading was confirmed by Ponceau S staining and by sequential incubation of the membrane with anti-h-actin antibody. The intensity of the p53 bands was determined by densitometric analysis of immunoblots. Isolation of RNA and Northern Blot Analysis. Total cellular RNA was isolated using TriZol reagent (Life Technologies). For blotting, 15 Ag of total RNA were loaded per lane on 1.2% agarose-formaldehyde gels and blotted onto nylon membrane. The indicated probe was labeled with the Rapidhybrid kit (Amersham) as described (19) and hybridized to the blot.


Yeast Two-Hybrid Analysis. Interactions of the Msx1 deletion mutants with human HPV-E6 and p53 proteins were analyzed by the yeast twohybrid system developed by Gyuris et al. (20). DNA fragments encoding fulllength human HPV-E6 and p53 proteins were isolated by PCR from the cDNA library of HeLa cells (Clontech) and cloned into pJG4-5 at unique EcoR1 and XhoI sites for in-frame fusion of each cDNA. The three-truncated constructs of Msx1 (Met1-Thr171, Met1-Thr231, and Asn172-Thr231) were also obtained by PCR by using specific primers and pCB6+/Msx1 as a template. Each Msx1 construct was inserted into pGilda vector (Clontech) using BamHI and XhoI sites to express it as a fusion protein with LexA. Each pGilda and pJG 4-5 fusion construct was introduced into the testing yeast strain, EGY48, and interaction between the fused proteins was determined by ability of the cells to grow on leucine-depleted medium containing 2% galactose, and by the formation of blue colonies on synthetic medium containing X-gal, 2% galactose, and 2% raffinose. The binding activity between HPV-E6 and Msx1 was also determined quantitatively by the relative expression level of h-galactosidase as described (21, 22). Indirect Immunofluorescence Analysis. Cells were seeded onto 4chamber slides, washed with 1 PBS, and fixed in 4% paraformaldehyde, permeabilized with 0.2% triton X-100, and blocked with 1% bovine serum albumin. Cells were incubated with anti-p53 antibody and subsequently incubated with rhodamine-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratory, Inc., West Grove, PA). Expression and localization of the proteins were observed under a confocal microscope (Bio-Rad, Hertfordshire, United Kingdom). Construction and Injection of Ad-Msx1 in Mouse Tumor Model. The 0.9-kb PCR fragment of Msx1 was subcloned into the BamHI site in the multiple cloning region of pDACMVp(A) to generate pDACMVMsx1 (AdMsx1). Ad-p53 construct was described previously (23). Each adenovirus and an adenovirus genomic DNA plasmid pJM17 (kindly provided by F. Graham, McMaster University, Ontario, Canada) were used to cotransfect a 293 packaging cell line. Viruses were purified through two rounds of CsCldensity gradient centrifugation, followed by dialysis against PBS containing 10% glycerol and 1 mmol/L MgCl2. Titers of the virus stocks were determined by plaque assay on lawns of 293 cells. To determine the effect of Ad-Msx1 in a murine tumor model, 5 106 HeLa cells were injected s.c. into 5-week-old female BALB/c mice (Charles River Laboratoratories, Atsugi, Japan). One hundred percent of mice developed s.c. tumors within 10 days. Ten animals from each group were injected s.c. with 5 108 plaque-forming units (pfu) of Ad-Msx1, Ad-gal, or Ad-p53. Tumor growth was determined by caliper measurements. Tumor volume was calculated by the following formula: tumor volume (mm3) = (a b 2)/2, where a = length in mm and b = width in mm. Statistical significance was determined by one-way ANOVA. This animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute. Samsung Biomedical Research Institute is an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility and abide by the Institute of Laboratory Animal Resources guide. Tumor volume and body weight was measured every other day. Crosssectioned tumors were stained with H&E for pathologic confirmation and TUNEL assay as described above.

Results Msx1 Induces Apoptosis in Human Cancer Cells. To explore the role of the homeobox protein Msx1 in human cancer cells, Msx1 overexpression was induced in HeLa cells. Transient overexpression of Msx1 caused a change in cell morphology and a dramatic reduction in cell growth (Fig. 1A and B). Previously, we also observed an inhibition of tumor cell growth by Msx1 in human ovarian carcinoma OVCAR3 cells (18). To confirm that the reduction in cell number represents apoptosis, we examined the morphology of DAPI-stained nuclei in HeLa cells overexpressing control vector and full-length Msx1. Full-length Msx1-overexpressing cells showed fragmented nuclei characteristic of apoptosis, whereas control cells had normal oval nuclei (Fig. 1C). Apoptosis

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Interaction of p53 with Msx1 Induces Apoptosis

120 minutes after cycloheximide treatment (Fig. 2B). This stabilization of p53 coincides with the decrease in the cellular levels of E6 protein. This strongly suggests that stabilization of p53 protein in Msx1 overexpressing cells can result from a delay of E6mediated p53 degradation. Because p53 protein phosphorylation is another key regulatory mechanism of p53, we compared the phosphorylation status of p53 protein between control vector- and Msx1-expressing vectortransfected cells. Using 5V NH2-terminal phospho-specific p53 antibodies, we observed a dramatic induction of phosphorylation of p53 at Ser15 in Msx1-overexpressing cells. In contrast, other phosphorylation sites showed no significant changes (Fig. 2C). Next, indirect immunofluorescence was carried out to determine the subcellular localization of p53 in the presence of full-length Msx1. Msx1-transfected HeLa cells had a clear nuclear accumulation of p53, in contrast with the absence of p53 in the nucleus of cells expressing control vector (Fig. 2D). These results lead to the possibility that Msx1 might have a novel function in p53 stabilization and subcellular localization. The Homeodomain of Msx1 can Interact with p53 Protein. To determine whether protein-protein interactions are involved in the stabilization of p53 protein by Msx1, we used yeast two-hybrid assays. To this end, we made four Msx1 constructs (Fig. 3A, top). We analyzed the interaction between Msx1 and p53. Full-length Msx1 (1-297) and a truncated form of Msx1 containing the homeodomain (1-225) interacted with p53 protein. However, a construct lacking the homeodomain (1-165) lost its interaction with p53, suggesting the importance of the homeodomain in the interaction with p53

was also confirmed by fluorescence-activated cell sorting analysis after double staining with Annexin V-FITC and propidium iodide. Full-length Msx1-overexpressing cells showed characteristics of apoptotic cells, namely a low forward scatter (FCS) and high side scatter (SSC) profile (Fig. 1D). These results indicate that full-length Msx1 overexpression induced apoptosis in cervical carcinoma cells harboring the HPV-E6 protein. Overexpression of Msx1 Stabilizes p53 Tumor Suppressor Protein and Increases its Nuclear Localization. To explore the underlying mechanism of apoptosis induction by Msx1, we examined the levels of p53 mRNA and protein in HeLa cells. Overexpression of Msx1 did not change the level of endogenous p53 mRNA, but it did increase the steady state level of endogenous p53 protein (Fig. 2A). Because the intracellular concentration of p53 is primarily regulated by modulation of its stability, we compared the kinetics of p53 degradation in cells transfected with control vector or full-length Msx1. Densitometric scanning showed that the halflife of p53 protein in control vector-transfected cells was 2-fold higher (60 minutes). There is an f2-fold increase in the half-life of p53 protein by Msx1. The increase in half-life of p53 protein in Msx1-overexpressing HeLa cells could be a consequence of its decreased polyubiquitination mediated by HPV-encoded E6 protein that recruits the cellular ubiquitin-protein ligase E6-AP. Thus, we examined the effect of Msx1 overexpression on E6 expression. Whereas the cellular level of E6 protein was detectable after 30 minutes in control vector-transfected HeLa cells, E6 level remained undetectable in Msx1-overexpressing cells until

Figure 1. Msx1 induces apoptosis in HeLa cells. A , Cells were transfected with control vector or full-length Msx1 expression vector and cell morphology was observed by light microscopy. B, cell growth was determined by MTT colorimetric assay at the indicated times after transfection with the indicated expression vectors. The bar graph shows normalized values relative to those of control. Each experiment was done in triplicate. C, HeLa cells transfected with the indicated expression vectors were stained with DAPI to examine the nuclei and then examined by fluorescence microscopy. D, apoptotic cell death by Msx1. Transfected HeLa cells with the indicated expression vectors were incubated with FITC-labeled Annexin V and propidium iodide for 15 minutes and then analyzed by fluorescence-activated cell sorting.


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Figure 2. Msx1 interacts with p53 protein. A, Msx1 induces p53 proteibut not RNA. Total cellular RNA and whole cell lysates were prepared from HeLa cells transfected with the indicated expression vectors. RNAs (20 Ag) were subjected to Northern blot analysis with the indicated 32P-labeled Msx1 or p53 probe. Protein levels were determined by Western blot analysis with the indicated antibody. B, Msx1 increases p53 protein half-life. At 24 hours post-transfection with control and full-length Msx1 expression vector, transfected HeLa cells were treated with cycloheximide for the indicated time. Whole cell lysates (30 Ag) were subjected to SDS-PAGE and subjected to Western blot analysis with the indicated antibody and then detected by enhanced chemiluminescence. Equal protein loading was confirmed by sequential incubation of the membrane with anti-h-actin antibody. The intensity of the p53 bands was determined by densitometric analysis. C, Msx1 induces p53 phosphorylation at Ser15. HeLa cells were transfected with vector alone or Msx1 expression vector. Whole cell lysates were subjected to Western blot analysis with antibodies to p53 (DO-1) or phospho-specific p53. D, Nuclear localization of p53 by Msx1 in the presence of E6. HeLa cells seeded onto chamber slides were transfected with pEGFP/Msx1 and then analyzed by indirect immunofluorescence using confocal microscopy. Msx1 was detected by the intrinsic fluorescence (green ), whereas p53 protein (red ) was detected by indirect immunofluorescence. Nuclei were stained with DAPI.

sufficient for interaction with p53. Our results suggest a novel function for the Msx1 homeodomain as a protein-protein interacting motif rather than as a DNA-binding domain. To identify the region of p53 needed for p53-Msx1 interaction, three major domains of p53 protein were tested in yeast two-hybrid assay. The NH2 terminus of p53 encompassing both the transactivation domain and

(Fig. 3B, top). To further confirm that the homeodomain is essential for interaction between Msx1 and p53, the Msx1 homeodomain alone (166-225) was fused to another protein, pLexA. The efficiency of the interactions between the pLexA-Msx1 homeodomain fusion and p53 was comparable to those obtained with full-length Msx1 (Fig. 3B, top). The Msx1 homeodomain alone was essential and


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homeodomain of Msx1 is important for the stabilization of p53 and induction of its apoptotic function. Mammalian Msx1 expression constructs tagged with enhanced EGFP coding sequence were transiently expressed in HeLa cells. Transfected HeLa cells were photographed to confirm the expression of the Msx1-expressing construct and to examine the induction of apoptosis. The full-length Msx1 construct induced apoptosis in HeLa cells, as shown by the presence of fragmented nuclei stained with DAPI (Fig. 4A, top). In contrast, HeLa cells transfected with either Msx1 lacking the homeodomain or control GFP vector did not induce apoptosis, as shown by the presence of intact oval nuclei (Fig. 4A, bottom). To confirm the effect of the Msx1 homeodomain on apoptosis, we first selected transfected cells with medium containing G418, and then assayed colony formation of each Msx1 construct. HeLa cells transfected with EGFP vector or homeodomain-deleted Msx1 expression vector showed significant colony formation (Fig. 4B).

PXXP domain was found to be involved in the interaction with the homeodomain of Msx1 (Fig. 3B , bottom). To examine the interaction between p53 and Msx1 in human cells, HeLa cells were cotransfected with GST-p53 and pCB6/Msx1-expressing vectors or each expression vector alone. A GST pull-down assay on cotransfected cells revealed precipitation of both Msx1 and p53, indicating interaction between Msx1 and p53 in HeLa cells (Fig. 3C). Next, we were interested in determining the subcellular localization of E6 in Msx1-overexpressing HeLa cells. Indirect immunofluorescence analysis showed strong accumulation of E6 and Msx1 in the nucleus of Msx1-expressing cells, whereas E6 was present in the cytoplasm of control HeLa cells (Fig. 3D). This cytoplasmic localization of E6 is consistent with previous results by Liang et al. (24). The Homeodomain of Msx1 Is Necessary for Induction of Apoptosis and p53 Stabilization. Next, we explored whether the

Figure 3. Msx1 interacts with p53. A, schematic representation of Msx1 and p53 protein, showing the deletions and homeodomain of Msx1 and transactivation and PXXP domain of p53. B, Msx1 interacts with p53 and the homeodomain and transactivation and PXXP domain are necessary for their interactions. To test interactions between Msx1 and p53, both Msx1 and p53 were expressed as pGilda and pJG 4-5 fusion proteins in yeast. h-Galactosidase lift assays were done in the presence of X-gal to determine the binding activity of these constructs. Positive interactions were revealed by cell growth for 3 days at 30jC on leucine-depleted medium, as well as by the formation of blue colonies on medium containing X-gal. Quantitation of h-galactosidase activity by ONPG assays. h-Galactosidase activity was normalized to the value obtained with full-length Msx1 or full-length p53. C, interaction of Msx1 with p53 in HeLa cells. HeLa cells were cotransfected with GST, GST-p53, and pCB6/Msx1 expression vectors as indicated and then whole cell lysates were prepared for GST pull-down assays. After pull-down with glutathione-agarose beads, immunoblot analysis was done using anti-p53 (Do-1) and Msx1 antibodies. Immunoblot analysis revealed that the GST-p53 fusion protein efficiently pulled down Msx1, demonstrating an interaction between Msx1 and p53 in HeLa cells. D, E6 colocalizes with Msx1 in the nucleus of Msx1-transfected HeLa cells. HeLa cells were transfected with pEGFP/Msx1 and then the localizations of E6 and Msx1 were analyzed as in Fig. 2D . Merged, merged view of nuclear colocalization (yellow ) of E6 and Msx1.


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Figure 4. The homeodomain of Msx1 is necessary for induction of apoptosis and p53 stabilization. A, The homeodomain of Msx1 is necessary for inducing apoptosis in HeLa cells. HeLa cells were transfected with the indicated GFP expression vectors. After 24 hours, expression of Msx1 and subsequent nuclear morphology were observed by fluorescence microscopy after staining with DAPI. B, Msx1 homeodomain is necessary for growth suppression of HeLa cells. HeLa cells transfected with the indicated vectors as described in part were selected with 800 Ag/mL of G418 for 2 weeks, and surviving colonies were stained with crystal violet. C, homeodomain is necessary for inducing stabilization of p53. HeLa cells were transfected with the indicated expression vectors and then whole cell lysates were prepared for immunoblot analysis to examine the steady state level of p53. Whereas full-length Msx1 increased the level of endogenous wild-type p53 protein, the Msx1 mutant lacking the homeodomain had no effect in stabilizing p53 protein. D, stabilization of exogenous wild-type p53 by Msx1 in p53-negative H1299 cells. H1299 cells were cotransfected with the indicated expression vectors and then the level of p53 was examined by immunoblot analysis. E, stabilization of p53 is dependent on the intracellular concentration of Msx1. H1299 cells were cotransfected with a fixed amount of p53 expressing vector and the indicated amount of Msx1 full-length expression vector and then subjected to immunoblot analysis with the indicated antibody. The steady state level of p53 protein increases as the level of Msx1 expressed increases.

apoptosis (Fig. S1B and C). Taken together, overexpression of Msx1 homeodomain alone can stabilize p53 tumor suppressor and induce apoptosis of HeLa cells. To examine whether stabilization of wildtype p53 protein by Msx1 is dependent on the intracellular concentration of Msx1, p53-null human lung carcinoma H1299 cells were transfected with wild-type p53 expression vector alone or cotransfected with p53 and full-length Msx1. Coexpression of fulllength Msx1 stabilized exogenously expressed wild-type p53 protein compared with wild-type p53 alone (Fig. 4D). This p53 stabilization by Msx1 led to decreased colony formation in wild-type p53 and Msx1-coexpressing H1299 cells (data not shown). Cotransfection of H1299 cells with a fixed amount of wild-type p53 expression vector and increasing amounts of full-length Msx1-expressing vector revealed that p53 protein levels gradually increased in parallel to increasing amounts of Msx1 (Fig. 4E). Our results indicate that overexpression of Msx1 was capable of stabilizing exogenous p53 in a concentration dependent manner in p53 null human cancer cells. Msx1 Suppresses Tumor Growth in Nude Mice. To confirm the role of Msx1 in tumor growth, exponentially growing HeLa cervical cancer carcinoma cells were injected s.c. into immunodeficient BALB/c nude mice in groups of 12 to 15. Tumor growth

In contrast, expression of full-length Msx1 dramatically inhibited colony formation. Furthermore, the steady state level of p53 protein was increased in HeLa cells transfected with full-length Msx1, whereas no stabilization of p53 protein was observed in cells transfected with pEGFP vector or homeodomain-deleted Msx1 (Fig. 4C). To further address whether homeodomain alone is sufficient for the stabilization and induction of apoptosis, pEGFPHD alone [pEGFP-Msx1 (166-225)], and pEGFP-Msx1 HD mutant harboring three point mutations (amino acid positions at 168, 170, and 173) in the homeodomain of a full-length Msx1 was prepared. This Msx1 mutation has alanine substitutions in the homeodomain. As shown in Fig. S1A, expression of the homeodomain alone can stabilize p53 protein compared with control pEGFP vector alone (Fig. S1A, lane 3). In contrast, the homeodomain point mutant of Msx1 abrogates its ability to stabilize p53 protein (Fig. S1A, lane 4). Expression of each pEGFP-Msx1 construct in HeLa cells was determined by immunoblotting the same blot with antiGFP antibody as shown in Fig. S1A (bottom). Next, we examined whether the homeodomain of Msx1 alone is capable of inducing apoptosis. The homeodomain alone is capable of inducing apoptosis in HeLa cells, whereas homeodomain mutants did not induce


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and morphology were analyzed over 30 days. Palpable tumors were detected within 2 weeks. When tumor volume reached 100 mm3 in size, 5 108 pfu of adenovirus expressing full-length Msx1, h-gal, or p53 was injected into tumors. No significant difference in mouse body weight was observed among the groups in the 30 days after adenovirus injection (data not shown). There was no significant difference in tumor growth among the different groups during the initial 4 days after adenovirus treatment. After 7 days, the Ad-Msx1–treated group began to show slowed tumor growth, whereas the Ad-p53- or Ad-h-gal–treated group showed continued increase in tumor mass. At the time of mouse sacrifice, an f2-fold reduction in tumor size was observed in the AdMsx1–treated group compared with control Ad-h-gal–treated group (Fig. 5A). Ad-h-gal–treated tumors were large, round, and highly vascularized whereas Ad-Msx1–treated tumors were small, collapsed, and pale in appearance (Fig. 5A). Consistent with our previous results, Ad-p53 treatment effectively suppressed the growth of tumors (25). To explore whether suppression of tumor growth in the Ad-Msx1–treated group involved apoptosis, the immunoblot of tumor tissue lysates were examined with antibodies to proteins involved in apoptosis. Expression of cell deathrelated proteins such as caspase 3 and Bax was dramatically increased in Ad-Msx1–treated tumors (Fig. 5B). To further confirm the induction of apoptosis by treatment with Ad-Msx1, TUNEL assays were done with frozen sections of each tumor. Significantly more apoptotic cells were detected in Ad-Msx1–treated tumors as well as in Ad-p53–treated tumors compared with Ad-h-gal–treated tumors. These results indicate that Msx1 overexpression is capable of suppressing tumor growth by inducing apoptosis in vivo.

Although 50% of all human tumors have been reported to have a point mutation in the p53 tumor suppressor gene, a nonmutational mechanism of p53 inactivation involving p53 stabilization and subcellular localization also plays an important part in p53 regulation in tumors with wild-type p53. In this study, we provide convincing evidence to show a novel function of homeobox Msx1 as a p53 regulator using in vitro and in vivo models. Our previous profiling of differentially expressed genes from human primary cervical tumor specimens, using cDNA expression array and Northern blot analysis, showed decreased expression of Msx1 in cervical cancer tissue compared with normal cervical tissue (26). Our findings show that overexpression of Msx1 stabilizes wild-type p53 and restores the apoptotic function of p53 even in the presence of the HPV-E6 oncoprotein which abrogates the apoptotic function of wild-type endogenous p53 expression. Our yeast two-hybrid assays, GST pull-down assays, and indirect immunofluorescence analysis showed that Msx1 protein binds to p53 through its homeodomain, and this interaction is important for wild-type p53 stabilization and accumulation in the nucleus, where it exerts its apoptotic function. Protein-protein interaction through the homeodomain of Msx1 can contribute to a defense mechanism against rapid degradation of wild-type p53 protein by E6, thus restoring the apoptotic function of wild-type p53 in cervical carcinoma cells harboring HPV-E6. Our in vivo experiments also show the overexpression of Msx1 can suppress tumor growth in nude mice. Thus, Msx1 might be a potential target for gene therapy in HPVpositive cervical carcinomas through inducing wild-type p53 accumulation in the nucleus, and subsequent apoptosis of the cancer cells. Interesting recent findings by Vassilev et al. (27) show that activation of p53 with Nutlins, small-molecule antagonists of MDM2, can be beneficial in cancers with wild-type p53. Likewise, overexpression of Msx1 can be used in treating HPV-positive cervical tumors, where wild-type p53 function is abrogated by overexpressed HPV E6 oncoprotein. In addition, Parc (Parkin-like protein) sequesters wild-type p53 in the cytoplasm and prevents the transport of p53 into the nucleus (28). Our results along with

Discussion The p53 gene is the most frequent target of genetic alteration thus far identified in human cancers. p53 plays a critical role in maintaining cellular homeostasis and tumor-free survival of the organism by modulating cell cycle progression or apoptosis (2).

Figure 5. Overexpression of Msx1 suppresses tumor growth in nude mice. A, intratumoral injection of Ad-Msx1 inhibits s.c. HeLa human cervical carcinoma growth as xenografts in nude mice (n = 10). Mean tumor volume was determined by caliper measurements. Bars, SE. Shown are gross tumors representative of groups immediately after resection. B, induction of apoptosis-related proteins in Ad-Msx1–treated tumors. Immunoblot of whole tumor lysates from each adenovirus were analyzed with indicated antibodies. Induction of caspase 3 cleavage and increase in Bax protein expression were detected in Ad-Msx1 treated tumors. C, induction of apoptosis in Ad-Msx1–treated tumors. Frozen sections of 8 Am of each tumor were processed for TUNEL assay and then stained with DAPI to visualize nuclei. Increase in the number of apoptotic cells (green fluorescence) was detected in Ad-Msx1–treated tumors as well as in Ad-p53–treated tumors.


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human breast tumors (33). In addition, a recently identified nuclear protein kinase, homeodomain-interacting protein kinase-2 (HIPK2), acts as a transcriptional corepressor for homeoproteins (34). HIPK2 phosphorylates p53 at Ser46 and regulates p53-induced apoptosis as well as p53 activity (35). It would be interesting to explore the possible involvement of HIPK2 in Msx1-mediated apoptosis. The p53 protein can directly engage in two major apoptotic pathways involving stimulation of both death receptor signaling and mitochondrial perturbations (36). Our results show the induction of apoptotic proteins involved in mitochodrial perturbation (Fig. 5B). Further studies will be required to elucidate a possible involvement of Msx1 in defense mechanisms against genotoxic stress in normal cells. Based on our findings, we suggest the following working model to explain the role of Msx1 as a regulator of p53. Msx1 binds to p53 in the nucleus and inhibits its nuclear export thereby stabilizing it against HPV-E6–mediated degradation in the cytoplasm. It is equally possible that Msx1 binds to E6 and is translocated to the nucleus, where E6-mediated ubiquitination cannot occur. In either case, accumulated p53 in the nucleus can exert its apoptotic function. Because we have observed nuclear colocalization of Msx1, p53, and E6 proteins, the possibility of interaction among the three proteins cannot be excluded. Taken together, a novel function of Msx1 as a regulator of the p53 tumor suppressor in human tumors suggests that Msx1 can exert its tumor suppressive effects through inducing apoptosis. However, Msx1 can exert its tumor suppressive effect through inhibiting angiogenesis. It is worthwhile to identify Msx1 target genes and interacting proteins to explore these possibilities and to elucidate the apoptotic signaling pathways to develop therapeutic drugs for the treatment and prevention of tumors with wild-type p53 with impaired function. It is obvious that further study on this novel role of Msx1 will be required and would contribute to our understanding of the mechanism of Msx1-induced p53 stabilization and restoration of the apoptotic function of p53.

these results show the importance of nonmutational mechanisms in p53 inactivation. Homeobox genes are highly expressed in early embryogenesis but decline thereafter (reviewed in ref. 11). However, our previous observation of the high level of Msx1 expression in the normal cervix and decreased expression in cervical tumors has suggested that Msx1 has important functions in normal cervical tissue (26). Here, we propose a novel function of Msx1 as a regulator of the p53 tumor suppressor function in normal differentiated tissues. We presented two major findings. First, our observations show that overexpression of Msx1 can inhibit tumor growth by stabilizing and promoting nuclear translocation of p53, thereby restoring its apoptotic function. Second, our results showed clearly that the Msx1 homeodomain is essential both for physical interaction with p53 and for inducing apoptosis. Our findings suggest that Msx1 binds to wild type p53 in the nucleus through its homeodomain and protects p53 from E6-mediated degradation in the cytoplasm. This stabilization of p53 can subsequently restore the apoptotic function of p53 in human cancer cells with nonfunctional wild-type p53. To further answer whether Msx1dependent stabilization of p53 protein is a general phenomenon, we tested the effect of Msx1 on p53 stabilization in p53 null H1299 human lung cancer cells transfected with Msx1 (Fig. 4D) as well as in p53 null human osteosarcoma Saos2 cells (data not shown). In addition, our results showed that Msx1 induces phosphorylation of p53 at Ser15, a residue important for p53 stabilization and apoptotic activity (29, 30). Because phosphorylated p53 at Ser15 was increased by Msx1 overexpression, we have tested whether phosphorylation of p53 at Ser 15 (p53 S15A) was involved in its interaction with Msx1 in HeLa cells. Our preliminary data suggests that S15A mutation decreases their interaction significantly (Fig. S3A). Consistent with our yeast two-hybrid data showing the involvement of the NH2-terminal 100 amino acids of p53 in interaction with Msx1, mutation of all potential phosphorylation residues at the NH2-terminal of p53 abolishes p53 stabilization by Msx1 (Fig. S3B). However, our preliminary results do not contradict the notion that increased stability of p53 may be regulated by its interaction with Msx1. Our preliminary data suggest HPV E6 interacts with Msx1 (Fig. S2). Phosphorylation of Ser15 in p53 may be important for its interaction with E6. Further investigation is required to clarify the significance of Ser15 of p53 in the stabilization of p53 by Msx1. NH2terminal phosphorylation of p53 at Ser15 by ataxia-telangiectasia mutated protein kinase is important in the subsequent modification of the distant COOH-terminal lysine residues through enhanced recruitment of p300/CBP/PCAF (31, 32). Recent evidence suggests a critical connection between p53 and homeobox HoxA5 gene in

References

Acknowledgments Received 6/28/2004; revised 10/22/2004; accepted 11/18/2004. Grant support: SRC grant from Korea Science and Engineering Foundation. 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 Michael Atchison, Wei Zhang, and Ralf Krahe for their critical reading and invaluable comments on the article; Cory Abate-Shen, F. Graham, and Joonho Choe for their gifts of Msx1 constructs [pCB6+/Msx1, pGEX-2T-Msx1 (1-297), and pGEX-2T-Msx1 (1-165)], pJM17, and GST-p53 expression plasmids, respectively; Mihyun Kim for technical assistance with TUNEL assay and fluorescence microscopy; and Jennifer Macke and Seunghee Hong for editing the text.

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