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Cancer Research 67, 709, January 15, 2007. doi: 10.1158/0008-5472.CAN-06-3463
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Synergistic Tumor Suppression by Coexpression of FUS1 and p53 Is Associated with Down-regulation of Murine Double Minute-2 and Activation of the Apoptotic Protease-Activating Factor 1–Dependent Apoptotic Pathway in Human Non–Small Cell Lung Cancer Cells

Wu-Guo Deng1, Hiroyaki Kawashima1, Guanglin Wu1, Gitanjali Jayachandran1, Kai Xu1, John D. Minna2, Jack A. Roth1 and Lin Ji1

1 Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas and 2 Department of Internal Medicine and Pharmacology, Hamon Center for Therapeutic Oncology Research, The University of Texas Southwestern Medical Center, Dallas, Texas

Requests for reprints: Lin Ji, Department of Thoracic and Cardiovascular Surgery, Unit 445, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-745-4530; Fax: 713-794-4901; E-mail: lji{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FUS1 is a novel tumor suppressor gene identified in human chromosome 3p21.3 region. Loss of expression and deficiency of posttranslational modification of FUS1 protein have been found in a majority of human lung cancers. Restoration of wild-type FUS1 in 3p21.3-deficient human lung cancer cells exhibited a potent tumor suppression function in vitro and in vivo. In this study, we evaluated the combined effects of FUS1 and tumor suppressor p53 on antitumor activity and explored the molecular mechanisms of their mutual actions in human non–small cell lung cancer (NSCLC) cells. We found that coexpression of FUS1 and p53 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle–mediated gene transfer significantly and synergistically inhibited NSCLC cell growth and induced apoptosis in vitro. We also found that a systemic treatment with a combination of FUS1 and p53 nanoparticles synergistically suppressed the development and growth of tumors in a human H322 lung cancer orthotopic mouse model. Furthermore, we showed that the observed synergistic tumor suppression by FUS1 and p53 concurred with the FUS1-mediated down-regulation of murine double minute-2 (MDM2) expression, the accumulation and stabilization of p53 protein, as well as the activation of the apoptotic protease-activating factor 1 (Apaf-1)–dependent apoptotic pathway in human NSCLC cells. Our results therefore provide new insights into the molecular mechanism of FUS1-mediated tumor suppression activity and imply that a molecular therapy combining two or more functionally synergistic tumor suppressors may constitute a novel and effective strategy for cancer treatment. [Cancer Res 2007;67(2):709–17]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The pathogenesis of lung cancer involves a multistep process of genetic and molecular changes in oncogenes and tumor suppressor genes (1). Chromosomal abnormalities at the 3p21.3 region and expressional deficiencies in 3p21.3 genes are frequently found in most human lung cancers (2–4). The tumor suppressor genes in chromosome 3p21.3 function as "gatekeepers" in lung cancer and play a critical role in lung tumorigenesis (5). FUS1 is one of novel candidate tumor suppressor genes identified in a 120-kb homozygous deletion region in chromosome 3p21.3 in human lung cancer cells. Genomic alterations of the FUS1 gene and resultant loss of expression or deficiency of posttranslational modification of FUS1 protein have been found in a majority of human non–small cell lung cancers (NSCLC) and in almost all small cell lung cancers (6, 7). Several lines of experimental evidence have shown the tumor suppressor function of FUS1. The exogenous overexpression of FUS1 protein in 3p21.3-deficient human lung cancer cells significantly inhibits tumor cell proliferation in vitro and efficiently suppresses tumor growth and tumorigenicity in vivo by altering the cell cycle kinetics and inducing apoptosis models (6, 7). FUS1 is a myristoylated protein and myristoylation in its NH2 terminus is required for FUS1-mediated tumor suppression activity. A myristoylation-deficient mutant of the FUS1 protein abrogates its ability to inhibit tumor cell–induced clonogenicity in lung cancer cells in vitro and to suppress the growth of tumor xenografts and lung metastases in vivo (8). The tumor suppression function of FUS1 and several other potential 3p21.3 tumor suppressor genes is also found to be directly or indirectly associated with p53 activity. However, the exact molecular mechanism by which FUS1 exhibits antitumor activity is still unclear, and no information is available about the combination treatment with FUS1 and other tumor suppressors such as p53 for cancer gene therapy.

The p53 tumor suppressor is a well-established cellular gatekeeper in human cancers (9). The p53 gene is frequently mutated somatically or deleted in various human cancers. More than 50% of NSCLCs possess a mutation in this gene (10, 11). Exogenous overexpression of wild-type p53 gene has proved effective in suppressing the growth of tumor cells bearing mutated p53 in vitro. Gene therapy by introduction of the wild-type human p53 gene into p53-deficient tumors has also shown a significant tumor-suppressing efficacy both in animal models and in human clinical trials (12–15). Furthermore, the tumor suppression activity of p53 can be enhanced by combination with chemotherapeutic drugs or ionizing radiation in various human tumors (16–18). The tumor suppression function of p53 is mainly through its ability to induce cell cycle arrest or apoptosis in response to a variety of stress signals (19, 20). However, because many tumor cells, including lung cancer cells, express wild-type p53 and exert resistance to p53 gene transfer and most tumors are also heterogeneous with respect to their p53 status, gene therapy by p53 alone may be insufficient to suppress tumor cell growth. Based on the status of p53 and FUS1 genes and gene products in human lung cancer cells and the apparent links of these two tumor suppressor genes in the development and malignant progression of lung cancer, in this study, we coexpressed wild-type FUS1 and p53 genes by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate (DOTAP):cholesterol nanoparticle–mediated gene transfer in human NSCLC cells and analyzed the combination effects of FUS1 and p53 coexpression on tumor suppression activity in vitro and in vivo. We present here the first evidence that the enforced coexpression of FUS1 and p53 dramatically and synergistically inhibited tumor cell growth and induced apoptosis in both p53-sensitive and p53-resistant NSCLC cell lines. We also found that a systemic treatment with a combination of wild-type FUS1 and p53 nanoparticles in a human H322 lung cancer orthotopic mouse model synergistically inhibited the development and growth of tumors. In addition, we showed that the synergism of the FUS1- and p53-mediated tumor suppression is associated with the FUS1-mediated down-regulation of murine double minute-2 (MDM2) expression and the resultant accumulation of p53 protein, as well as the enhanced activation of the apoptotic protease-activating factor-1 (Apaf-1)–associated apoptotic pathway in human NSCLC cells. These findings may have important implication to understand the molecular mechanism of FUS1-mediated tumor suppression and to develop better therapeutic strategies for lung cancers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell cultures. Human NSCLC cell lines H1299, H460, A549, and H322 and normal human bronchial epithelial cells (HBEC) were used in the study. The status of FUS1 and p53 genes and gene products in these cell lines are summarized in Table 1 . The A549 cell line was maintained in Ham's F12 medium supplemented with 10% FCS. The H1299, H322, and H460 lines were maintained in RPMI 1640 supplemented with 10% FCS and 5% glutamine. Normal HBECs were obtained from Clonetics, Inc. (Walkersville, MD) and cultured in the medium supplied by the manufacturer. All cells were incubated in a humidified incubator supplied with 5% CO2.


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  		Table 1. The status of FUS1 and p53 genes and gene products in lung cancer and normal cells

 
Transient transfection. Recombinant expression plasmid vectors containing a wt-p53, a wt-FUS1, and a mut-FUS1 (N-myristoylation site mutated-FUS1) gene were used in the experiments. The plasmid vector containing a ß-galactosidase gene (LacZ) was used as a nonspecific negative control. Construction of these recombinant plasmid vectors has previously been described (8, 16). Cells were transfected with DOTAP:cholesterol–encapsulated plasmid DNA nanoparticles as previously described (16). In brief, 2 µL of DOTAP:cholesterol reagent and 4 µg of plasmid DNA were mixed, and the mixture was added to each well in a six-well plate and incubated for indicated days. The transfection efficiency was assessed by a parallel transfection with an equal amount of enhanced green fluorescent protein (GFP)–expressing plasmid vector with a similar backbone in each cell lines. The transfection efficiency was in the range of 40% to 60% in those cell lines. For small interfering RNA (siRNA) experiments, cells were treated with Apaf-1 or control siRNA oligos by Oligofectamine reagent (Invitrogen Corporation, Carlsbad, CA). The target sequences of the siRNAs were as follows: Apaf-1 siRNA, 5'-AAUUGGUGCACUUUUACGUGA-3'; scrambled nonspecific control siRNA, 5'-AAUGGUCCGAAUCUAGUGUUU-3'.

Immunofluorescence imaging analysis. Cells were first cultured in chamber slides and transfected with plasmid DNA for 48 h. Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min on ice. After rinsing twice with PBS, cells were permeabilized with 0.2% Triton X-100 for 10 min. For immunostaining, cells were incubated with rabbit anti-FUS1 and mouse anti-p53 antibodies diluted in PBS containing 5% bovine serum albumin for 1 h at room temperature. FITC-labeled antirabbit immunoglobulin Gs (IgG) and rhodamine-labeled antimouse IgGs (Chemicon, Temecula, CA) were diluted 1:200 in PBS and the cells were incubated with the antibodies for 30 min. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and then examined under an Eclipse E400 fluorescence microscope (Nikon, Tokyo, Japan).

Cell viability assay. Cell viability was analyzed by trypan blue staining–based cell count or by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Roche Molecular Biochemicals, Basel, Switzerland) as previously described (21). Briefly, the cells were plated in 96-well microtiter plates at 1,000 per well in 100 µL of medium and transfected with p53 and FUS1 plasmid-based nanoparticles. At indicated time after transfection, cell viability was quantified in a microplate reader by XTT assay according to the manufacturer's instructions. The percentage of viable cells was calculated in terms of the absorbency in FUS1- and p53-treated cells relative to the absorbency in LacZ-treated control cells.

Apoptosis assay. Apoptosis was measured by flow cytometry using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)–based fluorescence-activated cell sorting (FACS) analysis as previously described (21). In brief, cells were transfected with plasmids. At indicated time after transfection, cells were fixed in 1% paraformaldehyde, permeabilized with 70% ethanol, washed with PBS, and stained with propidium iodide solution containing 40 µg/mL propidium iodide and 10 µg/mL DNase-free RNase A. DNA fragmentation was analyzed by flow cytometry. The relative apoptotic cells were calculated in terms of the FITC-positive values in cells.

Animal studies. All mice were maintained and animal experiments done at the Animal Core Facility at The University of Texas M.D. Anderson Cancer Center. An orthotopic mouse model of human NSCLC H322 was used to evaluate the combined effect of systemic administration of the FUS1 and p53 nanoparticles in vivo. The animals used in this study were female nu/nu mice (4–6 weeks old) purchased from Charles River Laboratories (Wilmington, MA). Before tumor cell inoculation, mice were subjected to 3.5-Gy total body irradiation with an external 137Cs source. To establish orthotopic pleural tumors, the mice were inoculated with 2 x 106 H322 cells in 0.1 mL of PBS by intrathoracic injection with a 27-gauge needle. These mice were randomly divided into four groups (seven mice per group): LacZ, p53, FUS1, and p53 plus FUS1. Orthotopic tumors are usually established on the lung or on the inner thoracic membrane 10 to 14 days after tumor cell inoculation. On days 10, 13, and 16 after tumor inoculation, the mice were given systemic DOTAP:cholesterol–based nanoparticles by tail-vein injection at a dose of 25 µg of plasmid DNA and 10 nmol DOTAP:cholesterol in 100 µL of 5% dextrose in water per mouse, alone or in combination at a dose of 2.5 mg/kg. All mice were then killed and the total number and total weight of intrathoracic and pleural tumors of each mouse were examined in each animal. To determine FUS1 or p53 expression in tumors, two mice in each group were treated with the designated agents and killed 48 h later, and tumors >5 mm were harvested and freshly frozen. The protein expression in frozen tumor sections was detected with specific FUS1 or p53 antibody (1:100) and examined under a light microscope. The images were examined under a Nikon TC200 fluorescence microscope equipped with a digital camera.

Statistical analysis. Three separate experiments were done in duplicate and means and SEs were calculated. The Wilcoxon signed-rank test was used for statistical analysis and P < 0.05 was taken to indicate a significant difference. StatView 5.0 (Abacus Concepts, Inc., Berkeley, CA) and SAS (Cary, NC) software were used for all of the statistical analyses. To analyze the interaction between FUS1 and p53, we assume that treatment by FUS1 multiplies the number of cells by a certain amount, say p1, and that treatment by p53 multiplies the cell count by p2. Then, if there is no interaction, the combination of FUS1 and p53 should multiply the cell count by p1*p2. Because we are assuming a multiplicative effect, we take the log of cell count as the dependent variable, so that we can use a linear model. To account for the fact that the data are paired (i.e., if there is a different baseline for each experiment), we enter the experiment in the model as a random effect. Thus, we fit a linear model with log count as the dependent variable. The experiment is regarded as a random effect and the therapeutic agents FUS1 and p53 and their interaction are regarded as fixed effects. The estimation was done with Restricted Maximum Likelihood using the JMP software from SAS. The significance of interaction is evaluated by paired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Synergistic inhibition of tumor cell growth by coexpression of FUS1 and p53. To evaluate whether the combination treatment of FUS1 and p53 could exert synergistic tumor suppression activity, we coexpressed FUS1 and p53 genes by DOTAP:cholesterol nanoparticle–mediated gene transfer and analyzed the combined effects of these two tumor suppressors on cell growth in human NSCLC cell lines H1299, H460, A549, and H322 and in the normal HBECs. LacZ plasmid was used as a negative control. The transfection efficiency was 40% to 60% by examining the GFP-expressing cells in the GFP gene–transfected cell population. The expression of FUS1 and p53 proteins was detected by immunofluorescence staining in FUS1 and p53–cotransfected NSCLC cells lines. The fluorescent images showed nuclear staining (DAPI, blue) and cytoplasmic and nuclear staining for FUS1 (FITC, green) and p53 (rhodamine, red) in H1299 and A549 cells at 48 h after cotransfection. The overlay of images represented the coexpression of FUS1 and p53. As shown in Fig. 1A , the coexpression of FUS1 and p53 was seen in >50% of NSCLC cells. {chi}2 statistical analysis showed that these coincidences of the expression of both proteins in both types of cells were significant (P < 0.001), indicating that equivalent levels of expression of both the FUS1 and p53 proteins could be achieved in FUS1 and p53–cotransfected NSCLC cells. Consistent with previously reported results, endogenous FUS1 could not be detected in both H1299 and A549 cells, and endogenous p53 could be detected in A549 but not in H1299 cells (results not shown). The overexpression of FUS1 and p53 proteins could also be detected in H460 and H322 cells cotransfected with FUS1 and p53 genes (data not shown).


Figure 1
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  		Figure 1. Synergistic suppression of tumor cell growth by FUS1 and p53 coexpression. A, human NSCLC cell lines (H1299, H322, A549, and H460) or normal lung cell lines (HBEC) were cotransfected with DOTAP:cholesterol–based nanoparticles including p53, wt-FUS1, or mut-FUS1 plasmid. LacZ was used as a plasmid control for transfection experiments. Coexpression of FUS1 and p53 was detected by immunofluorescence analysis (A). The immunofluorescence staining was done on H1299 and A549 cells at 48 h after cotransfection. Nuclei were stained by DAPI (blue). Green and red, FUS1 and p53 protein expression, respectively. Immunofluorescent images were overlaid to confirm the coexpression of FUS1 and p53. Cell viability was measured by counting living cells at 24, 48, and 72 h after transfection (B) or by XTT assay at 72 h after transfection (C). Relative cell viability was calculated versus that in cells transfected with control plasmid LacZ. Columns, mean of three separate experiments; bars, SE.

 
We next analyzed the effect of FUS1 and p53 coexpression on tumor cell growth by counting living cells and determined the viability of cells at 24, 48, and 72 h after transfection, respectively. Tumor cell growth was suppressed in a time course manner and inhibition reached its peak at 72 h after transfection in NSCLC cells (Fig. 1B). Cotransfection with wt-FUS1 and p53 in H1299 and A549 cells exhibited a significant and synergistic inhibition on tumor cell growth by comparison with those transfected with wt-FUS1 or p53 alone (plus control plasmid LacZ; Fig. 1B). By using an improved XTT assay, we also quantitatively determined the combined effect of FUS1 and p53 on tumor suppression activity. As shown in Fig. 1C, the coexpression of wt-FUS1 and p53 dramatically inhibited tumor cell growth in all four NSCLC cell lines cotransfected with p53 and wt-FUS1 at 72 h after cotransfection, and the combined effects of wt-FUS1 and p53 on cell growth inhibition were significantly synergistic as indicated by a statistical analysis using the JMP software (P < 0.001 in all lung cancer cell lines tested). Transfection with wt-FUS1 or p53 alone (plus control plasmid LacZ) was less effective than the combination of wt-FUS1 and p53. No significant inhibition on cell viability was observed in the LacZ-transfected control cells. There is no obvious association of the coexpression of wt-FUS1 and p53 with cell growth inhibition in human normal cell lines HBEC (Fig. 1C).

Myristoylation is required for FUS1-mediated tumor suppression activity in human lung cancer cells (8). To determine whether myristoylation site in FUS1 protein is also essential for the synergistic effect of FUS1 and p53, we constructed a plasmid vector expressing a myristoylation site–deficient mutant (mut-FUS1) in which the predicted myristoylation site of glycine (G2) was replaced with an alanine (A2) by site-directed mutagenesis, and analyzed the combination effect of the dysfunctional myristoylation-deficient mut-FUS1 and p53 on tumor suppression activity. Cotransfection in all four NSCLC cell lines with p53 and mut-FUS1 did not alter the immunoreactive FUS1 level (Fig. 1A) but resulted in an attenuated inhibition of cell growth by comparison with those cotransfected with p53 and wt-FUS1 genes (Fig. 1B and C), confirming the importance of myristoylation of FUS1 in the tumor suppression activity of FUS1.

Synergistic activation of apoptosis by coexpression of FUS1 and p53. One of the molecular events induced by tumor suppressors is apoptosis. To examine the molecular mechanism by which coexpression of FUS1 and p53 synergistically inhibited tumor cell growth, we did FACS analysis with TUNEL reaction and propidium iodide staining to examine apoptosis-mediated DNA fragmentation and cell cycle kinetics in the FUS1 and p53–cotransfected NSCLC cells. Significant and synergistic induction of apoptosis (TUNEL positive cell population) was observed in all four NSCLC cell lines cotransfected with wt-FUS1 and p53 nanoparticles (Fig. 2A ). In contrast, cotransfection with p53 and the dysfunctional myristoylation-deficient mutant of FUS1 (mut-FUS1) did not show a synergistic effect on apoptosis induction. Transfection with wt-FUS1 or p53 alone (plus control plasmid LacZ) had less effects than the combination expression of wt-FUS1 and p53 on the number of apoptotic cells (Fig. 2A), indicating that the wt-FUS1 and p53–cotransfected NSCLC cells might have become more apoptotic. The magnitude of and the trend toward apoptosis induction in these FUS1 and p53–cotransfected cells paralleled the degree and the trend toward tumor cell growth inhibition (Fig. 1C). The correlation coefficients between the relative cell viability and the relative apoptotic cell populations in the wt-FUS1 and p53–cotransfected cells versus plasmid control LacZ were significant (P < 0.05) in all four NSCLC cell lines, suggesting that the synergistic tumor suppression by the coexpression of wt-FUS1 and p53 might be mediated by the synergistic induction of apoptosis. No significant increase of apoptosis was observed in normal cell line HBEC cotransfected with wt-FUS1 and p53 nanoparticles (Fig. 2A). We also examined the presence of sub-G1 population in the FUS1- and p53-transfected cells and found a synergistic increase of sub-G1 cell population at 72 h after cotransfection in NSCLC cells cotransfected with p53 and wt-FUS1 but not mut-FUS1 gene (results no shown).


Figure 2
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  		Figure 2. Synergistic induction of apoptosis by FUS1 and p53 coexpression. Human NSCLC cell lines (H1299, H322, H460, or A549) and normal cell line HBEC were cotransfected with DOTAP:cholesterol–based nanoparticles containing p53, wt-FUS1, or mut-FUS1 genes. The LacZ-transfected cells were used as negative control. At 72 h after transfection, the apoptosis was determined by TUNEL-based FACS analysis in conjunction with propidium iodide staining. A, the apoptoses are represented by relative percentages of TUNEL-positive cells versus that in untreated cells. B, the activation of caspases was analyzed by detecting the cleavage products of caspase-9 and caspase-3 by Western blot analysis. Top, representative blot; bottom, densitometry of the blot. Columns, mean of three separate experiments done in duplicate; bars, SE.

 
Activation of caspases is believed to be vital in the apoptosis pathway driven by tumor suppressors. To evaluate the synergistic role of apoptotic response in the wt-FUS and p53–cotransfected cells, we measured the combined effect of the FUS1 and p53 coexpression on caspase activation in NSCLC cells. As shown by the cleavage products detected by Western blot in Fig. 2B, both caspase-9 (an upstream caspase) and caspase-3 (an downstream caspase) were strongly cleaved from their inactive precursor forms (procaspase-9, 47 kDa; procaspase-3, 32 kDa) into active fragments (cleaved caspase-9, 10 kDa; cleaved caspase-3, 17 kDa) in the wt-FUS1 and p53–cotransfected NSCLC cells. Caspase-9 and caspase-3 were also activated in the cells cotransfected with mut-FUS1 and p53, or transfected with p53 or wt-FUS1 alone (plus control plasmid LacZ), but their activation was attenuated. These results suggest that the observed synergism in tumor suppression by wt-FUS1 and p53 coexpression may be due to a synergistic induction of apoptosis mediated by the enhanced activation of caspases.

Synergistic inhibition of tumor growth by the coadministration of FUS1 and p53 nanoparticles in vivo. To determine whether the synergistic tumor suppression function mediated by the coexpression of FUS1 and p53 observed in vitro could be reproduced in vivo, we evaluated the combined effects of FUS1 and p53 overexpression on tumor growth by systemic coadministration of wt-FUS and p53 nanoparticles via tail-vein injection in human H322 orthotopic lung cancer xenografts mouse models. Mice were divided into four treatment groups that received treatment thrice with DOTAP:cholesterol–based nanoparticles including expressing plasmids LacZ, wt-FUS1, p53, and wt-FUS1 plus p53. Each treatment group contained five mice. The overall effects of treatments on tumor growth were analyzed by measuring tumor number and tumor weight. The expression of FUS1 or p53 protein in these nanoparticle-injected tumor tissues was confirmed by immunohistochemical staining using anti-FUS1 or anti–wt-p53 antibodies, respectively (Fig. 3A ), showing the capability and efficiency of DOTAP:cholesterol nanoparticle–mediated gene delivery for systemic cancer treatment. Consistent with the results in vitro, the combined treatment with wt-FUS1 and p53 nanoparticles significantly inhibited tumor formation and growth in the H322 orthotopic lung tumor model (Fig. 3A). A synergistic inhibition in both the total tumor numbers (Fig. 3B) and the total fresh tumor weights (Fig. 3C) was seen in mice cotreated with wt-FUS1 and p53 nanoparticles in comparison with the results seen in the other control treatment groups. This result showed that coadministration of DOTAP:cholesterol–based wt-FUS1 and p53 nanoparticles produced a synergistic and more effective therapeutic efficacy for suppressing tumor growth in mice with human lung cancer xenografts.


Figure 3
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  		Figure 3. Synergistic inhibition of tumor growth in lung cancer mouse models by FUS1 and p53 coexpression. An orthotopic mouse model of human NSCLC H322 was used to evaluate the combined effect of systemic administration of the DOTAP:cholesterol–based FUS1 and p53 nanoparticles on tumor growth inhibition in vivo, as indicated in Materials and Methods. A, representative images of the thoracic necropsy (arrows, tumors) and immunohistochemical staining of FUS1 and p53 proteins in tumor samples from nanoparticle-injected mice. Tumor samples were prepared 48 h after injection. Columns, total tumor numbers (n = 5; B) and total fresh tumor weights (n = 5; C) in each mouse for each treatment group; bars, SE.

 
Increased accumulation of p53 and down-regulation of MDM2 expression by coexpression of FUS1 and p53. To elucidate the molecular mechanisms responsible for the synergistic tumor growth inhibition and apoptosis induction by FUS1 and p53 coexpression, we studied their mutual effects on the accumulation of the FUS1 and p53 proteins themselves in the FUS1 and p53–cotransfected NSCLC cells by Western blot analysis. The coexpression of wt-FUS1 and p53 resulted in a larger increase of p53 protein levels in all four NSCLC cell lines cotransfected with wt-FUS1 and p53 nanoparticles, independent of the endogenous p53 status in these cells (Fig. 4 ). By contrast, p53 protein levels were almost unaltered by coexpression of mut-FUS1 and p53. However, the levels of FUS1 protein were not affected by coexpression of wt-FUS1 and p53 (Fig. 4). These results indicate that the synergistic effect of these two tumor suppressor genes may be due to the FUS1-mediated accumulation of p53 protein, but not the reverse.


Figure 4
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  		Figure 4. Effects of FUS1 and p53 coexpression on p53 accumulation and MDM2 expression. Four NSCLC cell lines were cotransfected with DOTAP:cholesterol–based FUS1 and p53 nanoparticles. LacZ was used as a negative plasmid control for transfection experiments. At 72 h after transfection, cells were harvested and the protein levels of FUS1, p53, and MDM2 were analyzed by Western blot assay using specific anti-FUS1, anti-p53, and anti-MDM2 antibodies, respectively. The experiments were repeated thrice.

 
MDM2-mediated p53 degradation is a key pathway to regulate p53 accumulation or stabilization in response to different forms of stress (20). To determine whether the increased accumulation of p53 protein in the FUS1 and p53–cotransfected NSCLC cells is associated with the down-regulation of MDM2 expression, we analyzed the levels of MDM2 proteins by Western blot analysis. We detected a significantly decreased level of the 90-kDa MDM2 protein in the cells cotransfected with wt-FUS1 and p53 by comparison with the cells cotransfected with mut-FUS1 and p53, or transfected with wt-FUS1 or p53 alone (plus control plasmid LacZ; Fig. 4). Thus, these results suggest that the synergistic effect of wt-FUS1 and p53 on tumor suppression is mediated by the down-regulation of MDM2 expression and the resultant accumulation of p53 protein in these NSCLC cells.

Enhanced activation of the Apaf-1–dependent apoptotic pathway by coexpression of FUS1 and p53. Apaf-1 is a key factor in the mitochondrial apoptotic pathway, including the induction of apoptosis by the p53 tumor suppressor (22, 23). To determine whether the synergistic tumor suppression function by coexpression of FUS1 and p53 is also involved in the Apaf-1–mediated apoptotic pathway, we first measured the combined effect of FUS1 and p53 on the expression of Apaf-1 protein in NSCLC cells by Western blot analysis. We detected a larger increase of Apaf-1 protein levels in all four NSCLC cell lines cotransfected with wt-FUS1 and p53 genes, but a smaller increase in the cells cotransfected with mut-FUS1 and p53, or transfected with wt-FUS1 or p53 alone (plus control plasmid LacZ; Fig. 5A ). This result suggests a possible association of the synergistic tumor suppression by coexpression of FUS1 and p53 with the Apaf-1–dependent apoptotic pathway. To further confirm the involvement of the Apaf-1–mediated apoptotic pathway in the wt-FUS1– and p53-mediated synergistic tumor suppression function, we blocked the expression of Apaf-1 by siRNA technology in the wt-FUS1 and p53–cotransfected NSCLC cells and determined the effect of inhibition of Apaf-1 expression on the function of wt-FUS1 and p53. An inhibition of Apaf-1 expression in the Apaf-1 siRNA–treated H1299 and A549 cells was detected by Western blot analysis (Fig. 5B). We also found that the treatment with Apaf-1 siRNA largely abolished the synergistic effect of wt-FUS1 and p53 on tumor cell growth inhibition and apoptosis induction (Fig. 5B). By contrast, no changes in cell viability and apoptosis were seen on treatment with a nonspecific control siRNA (Fig. 5B). These results therefore indicate that the synergistic tumor suppression induced by coexpression of wt-FUS1 and p53 is implicated in an Apaf-1–dependent pathway in NSCLC cells.


Figure 5
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  		Figure 5. Effect of FUS1 and p53 coexpression on the Apaf-1–dependent apoptotic pathway. NSCLC cells were cotransfected with DOTAP:cholesterol–based FUS1 and p53 nanoparticles. LacZ was used as a negative control. At 72 h after transfection, cells were harvested and protein levels of Apaf-1 were analyzed by Western blot assay with a specific anti–Apaf-1 antibody (A). For the siRNA experiment, H1299 and A549 cells were cotransfected with FUS1 and p53 plasmids plus Apaf-1 or scrambled nonspecific control siRNA by Oligofectamine reagent for 72 h. The LacZ plasmid was used as a negative control. B, mean cell viability and apoptosis determined by XTT and TUNEL-based FACS assays, respectively. Bars, SD. The experiments were repeated thrice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent advances in understanding lung cancer biology and genetics have suggested novel treatment strategies. Optimization of combinations of standard and novel therapeutic agents may improve the outcome of treatment for lung cancer (24). In this study, we used DOTAP-cholesterol nanoparticle–mediated gene transfer to introduce and coexpress the functionally synergistic tumor suppressor genes (tumor suppressor genes) FUS1 and p53 in human NSCLC cells and in a human lung cancer mouse model and quantitatively analyzed the combined effects of these two tumor suppressor genes on tumor suppression activity. We found that the enforced overexpression of FUS1 and p53 genes resulted in a significant and synergistic inhibition of tumor growth in vitro and in vivo. To our knowledge, this is the first report on the combined introduction of FUS1 and another functionally synergistic tumor suppressor gene in human lung cancers. From a clinical perspective, many human cancers, including NSCLCs, are highly resistant to conventional treatments such as surgery, radiotherapy, and chemotherapy (25). However, inactivation of multiple tumor suppressor genes has shown a synergistic effect on tumor development and proliferation (26). Almost all of NSCLCs have deficient FUS1 expression (6–8) and >50% of NSCLCs carry p53 mutations (11). Thus, combined introduction and enforced coexpression of the synergistic tumor suppressor genes FUS1 and p53 may constitute a rational and effective therapeutic strategy for NSCLCs or other cancers.

A key role of tumor suppressors is conferred by their induction of apoptosis. Assessment of the combined effects of FUS1 and p53 tumor suppressors on apoptosis is vital in understanding the molecular mechanism by which coexpression of FUS1 and p53 synergistically inhibits tumor cell growth. The results from our study also showed that the level of apoptosis is synergistically induced when NSCLC cells were cotransfected with these two tumor suppressor genes. Moreover, the level of activation of caspase cascades was in accord with that of apoptosis in the FUS1 and p53–cotransfected NSCLC cells. These results are consistent with the interpretation that coexpression of FUS1 and p53 enhances the induction of apoptosis, which results in the synergistic inhibition of tumor growth.

The stability of p53 is the key to the maintenance of multiple cellular functions such as apoptosis and cell cycle arrest. The p53 tumor suppressor gene is activated in response to diverse cellular stresses, such as those caused by DNA-damaging agents, and to oncogenic signals through mechanisms that result in the accumulation or stabilization of wild-type p53 protein (27, 28). Although the sensitivity of NSCLC cells to FUS1-mediated tumor suppression was not significantly correlated with the p53 gene status in the cells tested, coexpression of wild-type FUS1 and p53 massively increased the accumulation of p53 in cells, the latter resulting in a synergistically enhanced inhibition of tumor growth. The activity of p53 has been shown to be tightly regulated by divergent extracellular and intracellular signals through the mechanisms that result in degradation, stabilization, or accumulation of p53 protein. MDM2 is one of the negative regulators of p53 protein. It can block p53 function when overexpressed (29). Multiple cellular pathways also exist in the regulation of MDM2 activity. One of the mechanisms may be imposed via a negative feedback pathway of p53 and MDM2. The introduction of exogenous p53 induces the overexpression of endogenous MDM2, which, in turn, results in rapid degradation of p53 protein in the ubiquitin-proteasome system (27, 30, 31). MDM2 interacts directly with p53 in the NH2-terminal domains (32). As a result of this interaction, MDM2 directly targets p53 for degradation by proteolysis and blocks the tumor suppression activity of p53 (33). In this study, we observed a significant reduction of MDM2 proteins in NSCLC cells cotransfected with wt-FUS1 and p53. Thus, we speculated that the synergistic effect of FUS1 and p53 on tumor suppression may be related to the FUS1-mediated down-regulation of MDM2 protein and the resultant accumulation of p53 protein in NSCLC cells. Our findings implicate an important molecular pathway in the regulation of FUS1-mediated tumor suppression faction.

Apaf-1 is a transcriptional target of p53 and a key factor in the mitochondrial apoptotic pathway. Transcriptional regulation of Apaf-1 has been implicated in many important biological processes such as the development of the mammalian central nervous system (34, 35). Several studies have also shown that overexpression of Apaf-1 sensitizes tumor cells to apoptosis mediated by chemotherapeutic agents (36). Although p53 induces a number of proapoptotic genes besides Apaf-1, expression of Apaf-1 has been shown to be essential for p53-dependent apoptosis. Apaf-1 activates caspases in a cytochrome c–dependent manner. It recruits procaspase-9 into a multimeric complex and triggers its autoactivation. This, in turn, results in the cleavage of procaspase-3 and the ensuing proteolytic cascade (37–39). In our study, we also explored whether the synergistic effect of FUS1 and p53 on tumor suppression is implicated in the Apaf-1–dependent apoptotic pathway. We found that coexpression of FUS1 and p53 up-regulated the expression of Apaf-1 and enhanced the Apaf-1–dependent activation of caspase cascade in NSCLC cells. This result coincides with the increased accumulation of p53. Inhibition of Apaf-1 expression by a specific Apaf-1 siRNA attenuated the synergistic effects of FUS1 and p53. Therefore, our finding suggests that the synergistic tumor suppression function of FUS1 and p53 may be involved in the facilitated Apaf-1–associated mitochondrial apoptotic pathway.

In summary, our study showed that the coexpression of FUS1 and p53 synergistically suppressed NSCLC cell growth and induced apoptosis in vitro and in vivo. We also identified a novel molecular mechanism for FUS1-mediated tumor suppression function as summarized in Fig. 6 . We conclude that the coexpression of FUS1 and p53 increases the accumulation of p53 protein by down-regulating expression of MDM2 and enhances the activation of caspases and apoptosis by up-regulating the Apaf-1–dependent apoptotic pathway through the transcription factor activity of p53. Thus, our findings suggest that a treatment targeting multiple pathways by combination delivery of functionally synergistic tumor suppressors such as FUS1 and p53 may be an effective therapeutic strategy for human lung cancer and other cancers.


Figure 6
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  		Figure 6. Schematic illustration of the FUS1- and p53-mediated pathway in tumor suppression. Coexpression of FUS1 and p53 down-regulates MDM2 expression, resulting in increased p53 accumulation, and up-regulates the Apaf-1–dependent apoptotic pathway, causing enhanced activation of caspases and apoptosis.

 

   Acknowledgments
 
Grant support: National Cancer Institute, NIH, Specialized Program of Research Excellence grants CA70907, CA71618, and MMHCC U01CA10535201; Department of Defense TARGET Lung Cancer Programs grant DAMD17-02-1-070; a W.M. Keck Gene Therapy Career Development grant; M.D. Anderson Cancer Center Support Core grant CA16672; and a grant from the Tobacco Settlement Funds as appropriated by the Texas State Legislature.

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 Dr. Atkinson (Department of Biostatistics and Applied Mathematics, M.D. Anderson Cancer Center, Houston, TX) for statistical analysis support and David Galloway for manuscript editing.

Received 9/18/06. Revised 11/ 7/06. Accepted 11/15/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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