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Cancer Research 67, 10379, November 1, 2007. doi: 10.1158/0008-5472.CAN-07-0677
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Fragile Histidine Triad–Mediated Tumor Suppression of Lung Cancer by Targeting Multiple Components of the Ras/Rho GTPase Molecular Switch

Gitanjali Jayachandran1, Ji-ichiro Sazaki1, Masahito Nishizaki1, Kai Xu1, Luc Girard2, 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, The University of Texas M. D. Anderson Cancer Center, Unit 445, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-563-9143; Fax: 713-794-4901; E-mail: lji{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The fragile histidine triad (FHIT) gene has been shown to function as a tumor suppressor gene in vitro and in vivo. However, the mechanism of its action is still largely unknown. To elucidate the molecular mechanism and biological pathway in FHIT-mediated tumor suppression, we used a complementary gene and protein expression profiling with DNA microarray and ProteinChip technologies to quantitatively monitor cellular changes in gene and protein expression and discover the molecular targets of FHIT in non–small cell lung carcinoma (NSCLC) cells. The Ras/Rho signaling pathway was identified as one of the unique biological pathways associated with FHIT activity. A significantly down-regulated expression of genes and proteins of multiple key components in the Ras/Rho GTPases molecular switch, including Ran, Rab, Rac, Rap, and Ral, was observed on gene and protein expression profiles and further validated by Western blot analysis. Ectopic activation of FHIT in FHIT-deficient H1299 cells also significantly reduced the invasive potential of tumor cells by down-regulating expression of RhoC, a potential marker of tumor cell invasion and metastases. A simultaneous knockdown of the expression of several key Ras/Rho signaling molecules using gene-specific small interfering RNAs (RHO-siRNA) targeting selected Rab11, Rac1, and Rap1 genes significantly inhibited tumor cell growth and induced apoptosis in NSCLC cells in vitro, and a local injection of RHO-siRNAs complexed with N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate:cholesterol nanoparticles inhibited tumor growth in A549 tumor xenografts in mice, mimicking the AdFHIT-mediated tumor-suppressing effect. These results suggest a new role of FHIT in down-regulating the Ras/Rho GTPase-associated oncogenic signaling pathway. [Cancer Res 2007;67(21):10379–88]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer causes more deaths than the next three most common cancers (colon, breast, and prostate) combined, and an estimated 172,570 new cases of lung cancer were diagnosed in 2005 (1). When tumors are localized, the 5-year survival rate is 49%, but that rate diminishes to 2% if the cancer has spread to distant organs. Unfortunately, only 16% of lung cancer cases are detected in early stages (2, 3). Early detection remains a challenge because malignancy is the result of an intricate, multifaceted network of molecular events that can include silencing or modifying tumor suppressor genes (TSG) and activating oncogenes. The fragile histidine triad (FHIT) TSG located in human chromosome 3p14.2 region is one of the most active fragile loci of the human genome (4, 5). Two thirds of large cell lung carcinomas and 73% of non–small cell lung cancers (NSCLC) show abnormalities of FHIT gene and gene products (5). Several independent studies have shown that ~85% of precancerous lesions (bronchial dysplasia) lack FHIT expression, strongly suggesting that inactivation of FHIT is an early event in the development of lung cancer (5–9). Observations along these lines have shown not just the importance of FHIT in carcinogenesis but also its potential to serve as an early biomarker for lung cancer (10–12). Researchers have worked to decipher the inactivation mechanism, which has been attributed mainly to methylation of the FHIT promoter (13, 14). Although supporting evidence for the role of FHIT as a tumor suppressor and inactivation mechanisms in human cancers has accumulated over the years, the precise molecular mechanism of its tumor-suppressive function has yet to be identified (6, 15–17).

To facilitate our understanding of molecular events and mechanisms in FHIT-mediated tumor suppression activities, we did a complementary gene and protein expression profiling with DNA microarray and ProteinChip technologies to systematically and quantitatively monitor cellular changes in gene and protein expression in NSCLC cells in the presence and absence of ectopic FHIT gene expression to narrow down the numerous possible signaling molecules in FHIT-mediated tumor suppression and experimentally confirmed the expression status and biological function of prospective candidates in vitro and in vivo. In this report, we focused on presenting our findings on the unique Ras/Rho GTPase signaling pathway targeted by FHIT gene activities in lung cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell culture. Human NSCLC cell lines A549 and H1299 were maintained in Ham's F12 and RPMI 1640, respectively, supplemented with 10% deactivated FCS in an incubator containing a humidified atmosphere at 37°C with 5% CO2.

Gene and protein expression profiling and analysis. H1299 (p53–/– and FHIT defect with an exon 5 deletion and a 120-bp insertion) and A549 (p53+/+ and FHIT defect with an exon 4 deletion and exon 3/exon 5 fusion) cells were transduced with recombinant adenoviral vector AdFHIT at varied multiplicities of infection (MOI). Adp53-, AdLacZ-, and AdEV-transduced (containing an empty expression cassette without gene insert) and PBS-treated cells were used as a positive, a nonspecific, a negative, and a mock control, respectively. Construction of all recombinant adenovirus vectors has been described previously (17). Cells were harvested 24, 48, and 72 h after treatment, and harvested cells were divided to simultaneously isolate total RNAs for gene expression profiling and to prepare protein lysates for protein expression profiling, respectively.

Gene expression profiling was done on Affymetrix Human Genome HG-U133A arrays using the total RNAs isolated from AdFHIT-transduced NSCLC H1299 and A549 cells compared with those from empty vector (AdEV)- or AdLacZ-transduced cells. RNA was isolated using Trizol reagent (Invitrogen) and further purified using the RNeasy columns (Qiagen), and cRNA synthesis and hybridization were done using Affymetrix gene array analysis reagent kits according to the manufacturers' instructions and as described in details elsewhere (18, 19). The data were analyzed using either Affymetrix GeneChip 5.0 or dChip software (20) in a perfect match model to extract statistically significant (P ≤ 0.05) expression indexes that are up-regulated or down-regulated compared with controls. The differentially expressed genes were clustered among various treatment groups and cell lines. Expression of selected genes of interests was validated by a real-time reverse transcription and subsequent PCR using reagent kits and an ABI PRISM 7000 instrument (Applied Biosystems).

Proteomic analysis was done using cell lysates prepared from above NSCLC H1299 and A549 cells, with the same treatments and controls as used for gene microarray analysis. Protein lysates (100–200 mg) were fractionated based on isoelectric point (pI) into organic (pH 9, 7, 4, and 3) fractions using a microfractionation kit (Ciphergen Biosystems), and 5 to 10 µg of protein mixtures from each fraction were processed on SAX2 (strong anionic) and WCX2 (weak cationic) ProteinChip arrays (Ciphergen Biosystems) according to the manufacturer's instruction. Mass spectra were obtained using a surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), and mass data were analyzed using Biomarker analysis software (Ciphergen Biosystems). To facilitate the identification of the protein species of interests detected on the SELDI-MS profiles, we also developed a novel two-dimensional liquid chromatography (LC) method for the fractionation and separation of crude protein lysates. Briefly, for the one-dimensional LC, cell lysates were fractionated based on the pI values of proteins by fast protein liquid chromatography system (Bio-Rad Laboratories, Inc.) with a chromofocusing Mono-P column (GE Healthcare Biosciences). The pI-fractionated samples were either directly applied for protein profiling on ProteinChip arrays by SELDI-MS or subjected to the two-dimensional LC using a nonporous reverse-phase column by high-performance liquid chromatography (Bio-Rad Laboratories) for further protein separation and identification. The fractionated proteins were then further separated using SDS-PAGE. The protein bands on the gel were visualized by a silver stain (Sigma) designed for MS analysis and then dissected them from the gel. The isolated protein bands were digested in gel with trypsin (Roche Molecular Biochemicals), and the peptide digests were analyzed by using either SELDI-TOF-MS or LC/MS/MS. Proteins of interests were identified by using peptide mapping with Internet-based proteomic tools.3 Expression of the identified proteins was further verified and validated by Western blot analysis using available antibodies. A small portion of proteins with small mass (<40 kDa) were selectively identified due to the technical difficulties and instrumental limitations.

Small interfering RNA construction. Gene-specific small interfering RNAs (siRNAs) to several key Ras/Rho GTPase genes, including Rab11, Rac1, and Rap1, were designed to knock down their expression in tumor cells. Three pairs of oligonucleotides per gene were designed. The siRNAs were synthesized using a Silencer siRNA Construction kit (Ambion). The relative effects of these siRNAs on expression inhibition were determined by reverse transcription-PCR (RT-PCR) and Western blot analysis, and their biological functions were evaluated by cell growth inhibition and apoptosis induction analysis, respectively, in A549 and H1299 cells transfected with these siRNAs with LipofectAMINE 2000 reagent (Invitrogen). The following optimized siRNA pairs were selected and used for the subsequent experiments [Rab11, 5'-AACATTGTTATCATGCTTGTGCCTGTCTC-3' (antisense) and 5'-AACACAAGCATGATAACAATGCCTGTCTC-3' (sense); Rac1, 5'-AATTTGCTTTTCCCTTGTGAGCCTGTCTC-3' (antisense) and 5'-AACTCACAAGGGAAAAGCAAACCTGTCTC-3' (sense); and Rap1, 5'-AACATCTTTGATATGGCTGGACCTGTCTC-3' (antisense) and 5'-AATCCAGCCATATCAAAGATGCCTGTCTC-3' (sense)], and one pair of scrambled siRNA corresponding to each above gene-specific siRNA was used as a nonspecific control.

Cell viability assay. Cytotoxicity was evaluated in 96-well microtiter plates by using a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Roche Molecular Biochemicals) according to the manufacturer's instructions 72 h after treatment with siRNA. Details of the procedure have been described previously (21). The relative absorbance of siRNA-treated cells compared with untreated cells was measured in a microplate reader at a wavelength of 450 nm. Cell viability was calculated relative to the untreated control cells as 100%.

Apoptosis analysis by flow cytometry. Apoptosis after various siRNA treatments was quantified by flow cytometry as described previously (21). Cells were processed for flow cytometry by using an APO-BrdU kit (Phoenix Flow Systems, Inc.) according to the manufacturer's directions with slight modifications. Briefly, cells were collected 72 h after treatment with siRNA and fixed in 1% paraformaldehyde in PBS for 1 h. The cells were washed twice in PBS and resuspended in 70% ethanol at –20°C overnight. The fixed cells were incubated overnight in labeling solution containing terminal deoxynucleotidyl transferase and bromodeoxyuridine triphosphate at room temperature and processed for flow cytometry according to the manufacturer's instructions. The threshold for apoptotic cell populations is established using the positive and negative cell controls as provided and described by the manufacturer.

In vitro cell invasion assay. Effects of FHIT expression on the tumor cell–induced invasive potential were quantitatively determined by a fluorescence-based in vitro tumor cell invasion assay using a BD Falcon HTS FluoroBlok 24-Multiwell Insert System (BD Biosciences Discovery Labware) according to the manufacturer's instruction. The system consists of a cell culture insert containing a fluorescence blocking and microporous positron emission tomography membrane that blocks the passage of light at wavelengths 490 to 700 nm at >99% efficiency. The light emitted by fluorescent-labeled cells on the upper surface of the membrane is separated from those on the lower. This allows for real-time monitoring and quantification of invading or migrating cells without further manipulation. Stable clones of H1299 cells were established by transfection with a RhoC or a RhoC-green fluorescent protein (GFP) fusion protein expressing plasmid with a neomycin resistance selective marker. Briefly, the parental H1299, RhoC–expressing, and RhoC-GFP–expressing stable H1299 cells (1 x 105 in 0.75 mL cell culture medium) were respectively seeded into each insert well, the cell-containing inserts were placed into 24-well plate containing cell culture medium, and cells were incubated at 37°C with 5% CO2 overnight. Cells were treated with AdFHIT at MOI of 100 and 1,000 and untreated cells were used as a control. Seventy-two hours after transduction, cells that remained in the upper surface of the membrane of the inserts were stained with propidium iodide (red) and cells that invaded to the lower surface were stained with 4',6-diamidino-2-phenylindole (DAPI; blue). Fluorescence images were examined under a fluorescence microscope equipped with a digital camera. Fluorescence intensity was determined using a fluorescence microplate reader with an excitation wavelength of 538 and 485 nm and an emission wavelength of 620 and 530 nm for propidium iodide and DAPI, respectively. The relative cell invasive potential was represented by the ratio of DAPI to propidium iodide fluorescence intensity. Experiments were done at least thrice and with duplicate samples.

In vivo experiments. Animal experiments were conducted in compliance with the guidelines of both the NIH and The University of Texas M. D. Anderson Cancer Center. Double-blinded in vivo experiments were done with 6- to 8-week-old female nu/nu nude mice (Charles River Laboratories, Inc.) that had been injected with A549 cells s.c. Before tumor cell injection, all mice were subjected to 3.5 Gy total body irradiation from a 137C source. Thirty-six mice were randomly assigned to one of six treatment groups (six mice each). The treatment groups were (a) N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate (DOTAP):cholesterol (DC) alone; (b) Rab11-siRNA nanoparticle; (c) Rac1-siRNA; (d) Rap1-siRNA; (e) a combination of Rab11, Rac1, and Rap1 siRNAs; and (f) a combination of scrambled nonspecific control siRNAs of Rab11, Rac1, and Rap1. Animals were given three treatments of DC-siRNA nanoparticles (10 nmol DC:250 pmol siRNA in 100 µL PBS per treatment) by intratumoral injection within 1 week. Tumor volume was measured at regular intervals as described previously (22). Suppression of tumor growth in response to siRNAs of Ras/Rho genes was assessed by measuring tumor volume V (mm3) = a x b2 / 2, wherein a is the largest diameter and b is the smallest dimension. Two weeks after the last treatment, the animals were sacrificed and the tumors were dissected and weighed. Tumor volume increase was calculated relative to the starting tumor volume in each animal and averaged with each treatment group. Expression of siRNA-targeted Rab11, Rac1, and Rap1 genes and proteins was also analyzed by a quantitative real-time RT-PCR and by Western blotting, respectively, in flash frozen tumor samples collected from mice in each treatment group 48 h after siRNA injection.

Statistical analysis. For gene expression profiling, arrays were normalized and changes in gene expression were analyzed as described by Wright et al. (23), Li and Wong (20), and Dobbin et al. (19, 23). All experiments were done at least twice with duplicates or triplicates of samples. ANOVA and Fisher's test were used to compare the values of the test and control samples. P < 0.05 was considered statistically significant. StatView 5.0 software (Abacus Concepts, Inc.) was used for all statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FHIT-mediated differential gene and protein expression in NSCLC cells. We used a complementary gene and protein expression profiling with gene microarray and ProteinChip technologies to quantitatively analyze cellular changes in gene and protein expression and discover the molecular targets of the novel FHIT TSG in NSCLC cells. We did gene expression profiling analysis using the Affymetrix HG-U133A GeneChips in AdFHIT-transduced NSCLC H1299 and A549 cells compared with those of PBS-treated mock, empty vector AdEV-transduced negative, and Adp53-transduced positive (or training) controls. All of the gene expression data were analyzed by a perfect match model-based analysis (20) to extract statistically significant (P ≤ 0.05) expression indexes that are up-regulated or down-regulated compared with controls. The differentially expressed genes between AdFHIT (or Adp53) and empty vector control were clustered among various treatment groups and cell lines (Fig. 1A ). The AdEV-transduced and the PBS-treated cells in both H1299 and A549 cells exhibited similar gene expression files as shown by the hierarchal clustering analysis. The gene expression profiles in both H1299 and A549 cells transduced by AdFHIT showed the same hierarchal class, whereas profiles in cells transduced by Adp53 clustered distantly and were significantly different from those transduced by AdFHIT. The difference in the gene expression profiles between Adp53-transduced H1299 and A549 cells may partially be the reflection of the different status of endogenous p53 gene, the former is p53 null and the latter is p53 wild-type. These results suggest the specificity of gene profiles under the influence of ectopic expression of a given gene and gene product.


Figure 1
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  		Figure 1. Differential gene and protein expression profiling in AdFHIT-transduced versus AdEV-transduced NSCLC cells by gene microarray and protein array-based SELDI-TOF-MS. A, hierarchal cluster analysis of gene expression profiles in AdFHIT- and Adp53-transduced H1299 and A549 cells. The gene microarray data were analyzed by a perfect match model-based analysis using a dChip program (20). Expression data were normalized against untreated cells (PBS) and integrated to extract statistically significant (P ≤ 0.05) expression indexes, with a change in gene expression larger than 2-fold in AdFHIT- or Adp53-transduced cells compared with the AdEV-treated controls 48 h after transduction. B, scatter plots of unique human genes (UGs) overexpressed and underexpressed in H1299 and A549 NSCLC cells transduced with AdFHIT or an empty vector. C, changes in the expression of genes in the Ras/Rho signaling pathway by ectopic expression of FHIT. Data were extracted and summarized from gene expression profiles from AdFHIT-transduced H1299 and A549 cells. Gene names and their access numbers are indicated. D, protein profiles in AdFHIT-transduced H1299 cells compared with AdEV-transduced cells. Protein lysates were fractionated by a pH gradient microanion exchange column and then loaded onto various ProteinChip arrays and profiled by SELDI-TOF-MS. Representative protein expression profile on a strong anionic exchange ProteinChip. Top two panels, portion of the mass spectra in AdEV- and AdFHIT-treated cells; middle panels, changes in protein expression based on the intensity of mass peaks; bottom panel, cluster and statistical analysis of changes in protein expression of individual proteins among treatment groups with repeated experiments. The position of the boxes indicates the abundance of the protein, the width indicates the range of differential expression, and the error bars indicate the SD of the expression among the samples. The differentially expressed proteins were further identified by methods described in Materials and Methods.

 
By comparing the gene expression profiles in cells transduced by AdFHIT to those transduced by AdEV, we were able to identify 165 and 227 genes differentially and specifically expressed (>2-fold changes) in H1299 and A549 cells, respectively, as shown in scattered plots (Fig. 1B). The effect of adenoviral vector backbone on global gene expression was minimal because <30 genes seemed to be significantly affected by the transduction with empty vector compared with PBS mock control (data not shown). A functional classification analysis of these differentially expressed genes revealed unique cellular targets and several signaling pathways specifically affected by ectopic expression of FHIT in NSCLC cells, including the significantly down-regulated expression of genes in the Ras/Rho GTPase superfamily, the cytoskeleton- and tubulin-forming components, and the growth factors and the up-regulated protein mediators in cell death and apoptosis pathways. Particularly, the ectopic expression of FHIT showed an extensive effect on activities of multiple genes and key components in Ras/Rho oncogenic signaling pathways in NSCLC cells as presented in Fig. 1C. The differentially expressed genes were listed in Supplementary Table S1. It seems that there are more of these Ras/Rho GTPase genes down-regulated than would be expected by chance. We therefore did a gene set enrichment analysis using a web-based DAVID/EASE tool program.4 More than 70% of genes in a set of 206 genes that showed significant expression changes in AdFHIT-transfected lung H1299 and A549 cells compared with the AdEV-treated cells are significantly involved in cellular and physiologic processes (P = 0.0000–0.0142); a group of eight genes (some of which is listed in Fig. 1C) that are enriched from the list of Gene Ontology terms and pathways exhibited a significant association with small GTPase-mediated signal transduction pathway (P = 0.0424).

We also simultaneously did a protein expression profiling analysis by a ProteinChip array-based SELDI-TOF-MS spectrometry in above AdFHIT-transduced and control cells to analyze changes in protein expression affected by FHIT activation (Fig. 1D). Some of these differentially expressed protein species on the mass spectra were further identified by methods briefly described in Materials and Methods. Due to the technical limitations, only a small fraction of cellular proteins (~40) that are either differentially up-regulated or down-regulated by FHIT tumor-suppressing activities in these AdFHIT-transduced NSCLC cells were identified. Surprisingly, >60% of these differentially expressed proteins identified in this very limited group of proteins also found their corresponding gene components identified by gene microarray. These proteins were either the direct products or translational isoforms of the same genes or proteins closely related to a particular gene family. Interestingly, consistent with discoveries on gene microarrays, expression of several unique cellular proteins associated with Ras/Rho GTPase family proteins was also found to be differentially regulated in NSCLC cells transduced by AdFHIT compared with those transduced by AdEV (Fig. 1D and E). For instance, expression of the RABIF protein in AdFHIT-transduced H1299 cells was significantly down-regulated to less than one third of its baseline level in the AdEV-treated cells (Fig. 1D and E).

A comparative analysis of the gene and protein expression profiling revealed that multiple genes and key components, such as RAN, Rab, Rac, Rap, and Ral, in the Ras/Rho GTPase molecular switch were significantly down-regulated by ectopic FHIT expression in FHIT-deficient NSCLC cells. This intriguing observation led us to explore more closely the involvement of FHIT activity in Ras/Rho oncogenic signaling pathway in this study.

Validation of FHIT-mediated changes in protein expression in NSCLC cells by Western blot analysis. We did Western blot analysis with commercially available antibodies to further validate our findings on FHIT-mediated effects on Ras/Rho GTPase signaling pathway from gene and protein expression profiling (Fig. 2A ), and the intensity of immunoblot bands was quantified by density scan and levels of expression were calculated as a percentage in AdFHIT-transduced cells relative to those in untreated controls (Fig. 2B). The expression of Rab11 protein was nearly depleted in both AdFHIT-transduced H1299 and A549 cells compared with those of AdLacZ-transduced or PBS-treated cells. A significant reduction of expression of Rac1 (>60%) and Rap1 (>75%) and a lower-degree reduction of Ral (~30%) and CDC4 (~40%) proteins, respectively, were observed in both H1299 and A549 cells transduced by AdFHIT compared with AdLacZ- and PBS-treated controls. Expression of RAN seemed to be unaffected by AdFHIT transduction.


Figure 2
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  		Figure 2. Validation of the FHIT-mediated changes in protein expression by Western blotting. Key components in the Ras/Rho gene and protein families identified by gene and protein expression profiling in AdFHIT-transduced NSCLC cells were validated by using commercially available antibodies. The individual protein loadings and blots were normalized to those of ß-actin blots. A, bottom two panels, endogenous p53 expression and exogenous FHIT expression. B, the relative expression of each protein was represented as a percentage of the corresponding protein species in untreated cells (PBS), as determined by densitometry. Columns, band intensity readings in experiment repeats; bars, SE.

 
Targeting Ras/Rho genes associated with FHIT activity in NSCLC cells by siRNA-mediated gene silencing. To validate these Ras/Rho GTPase family proteins as candidate cellular targets by FHIT-mediated biological activity, we did reverse functional analysis by using gene-specific siRNAs to knock down expression of Ras/Rho genes and gene products and then evaluating their biological effects in NSCLC cells. Based on the results of gene and protein expression profiling and Western blot validation as well as computer-aided pathway analysis, we selectively designed gene-specific siRNAs (RHO-siRNAs) to three key components, Rab11, Rac1, and Rap1, in the Ras/Rho GTPase molecular switch, which have also been shown to be significantly down-regulated in NSCLC cells transduced by AdFHIT. These RHO-siRNAs were individually or together transfected into H1299 and A549 cells using LipofectAMINE transfection reagents, and their effects on gene expression were quantitatively analyzed by a real-time RT-PCR assay (Fig. 3A ). The AdFHIT-transduced cells were used for comparison. The expression of Rab11, Rac1, and Rap1 genes was markedly reduced in A549 and H1299 NSCLC cells after AdFHIT treatment compared with control cells treated with the transfection reagent (LipofectAMINE) alone or with AdLacZ. The expression of individual Rab11, Rac1, and Rap1 gene was significantly reduced (up to 70–90% reduction) in both H1299 and A549 cells by their corresponding gene-specific siRNA compared with the untreated control. A dramatic inhibition (up to 90%) on expression of each RHO genes was observed in cells transfected with combined RHO-siRNAs (Fig. 3A, siComb) and a down-regulated expression of one gene also seemed to have a negative regulation on the others, suggesting a cooperative interaction among these genes in the Ras/Rho GTPase signaling pathway.


Figure 3
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  		Figure 3. Evaluation of transcription knockdown by RHO gene-specific siRNAs in A549 and H1299 NSCLC cells by quantitative real-time RT-PCR. A, relative expression of Rab11, Rac1, and Rap1 in A549 (left) and H1299 (right) cells transfected with individual siRNAs (siRab11, siRac1, and siRap1) or with a combination of three RHO-siRNAs (siComb) in equal molarities. Cells transduced with AdFHIT and AdLacZ and treated with LipofectAMINE (LIPO) transfection reagent alone were used as a positive, a nonspecific, and a negative control, respectively. Amount of mRNA transcripts of RHO genes was determined by comparing with that of GAPDH gene in each cell line and in known amount of total human RNA standards. B, Western blot analysis of relative expression of Rab11, Rac1, and Rap1 in RHO-siRNA–treated H1299 cells, and heat shock protein 90 (Hsp90) was used as a loading control. The relative intensity of each Ras/Rho GTPase immunoblot band to that of Hsp90 loading control in each treatment is listed under the blot. C, effects of AdFHIT and RHO-siRNAs on viability of A549 cells by a XTT assay. The percent viability of AdFHIT-treated and RHO-siRNA–treated cells was calculated relative to that of LipofectAMINE-treated negative control. AdLacZ-transduced cells was used as nonspecific control for adenoviral vector–mediated gene transfer and scrambled siRNAs as nonspecific siRNA control, respectively. D, effects of AdFHIT and RHO-siRNA treatments on cell morphology and growth in A549 cell growth as shown by light microscopy. Magnification, x200. NSR-Comb, combined siRNAs.

 
Inhibition of tumor cell growth by RHO-siRNAs mimics the AdFHIT-induced tumor suppression activities in NSCLC cells. To further establish the biological relevance of these Ras/Rho GTPases to FHIT-mediated tumor suppression activities, we analyzed effects of the down-regulated Ras/Rho GTPases by RHO-siRNAs on tumor cell growth and apoptosis in comparison with those AdFHIT-mediated effects in NSCLC cells. A marked inhibition on expression of the endogenous Rab11, Rac1, and Rap1 proteins was detected in A549 cells transfected by these RHO-siRNAs (Fig. 3B), confirming the effectiveness of these siRNAs in knockdown expression of gene products. A significant decrease in the cell viability was detected in A549 cells treated with individual siRNAs of Rab11 (P = 0.005), Rac1 (P = 0.015), or Rap1 (P = 0.001) or a combination of all three (P = 0.0012) compared with those treated with their corresponding scrambled nonspecific siRNA controls or to the untreated cells (Fig. 3C and D). The combined treatment was highly cytotoxic to the NSCLC cells and was comparable with the effect of AdFHIT treatment, suggesting a similar biological consequence between AdFHIT and siRNA-mediated down-regulation of these Ras/Rho GTPase activities in lung cancer cells.

Induction of apoptosis by siRNA-mediated down-regulation of Ras/Rho GTPase activities in NSCLC cells. One of the mechanisms in FHIT-mediated tumor suppression is induction of apoptosis in cancer cells (12, 17). Induction of apoptosis was also detected in NSCLC H1299 (Fig. 4A ) and A549 (Fig. 4B) cells treated with individual siRNAs against Rab11, Rac1, and Rap1 or combined siRNAs, as shown by flow cytometry analysis. A moderate and varied level of apoptosis induction was detected in H1299 and A549 cells treated by individual RHO-siRNAs compared with those untreated control cells. However, a dramatically increased level of apoptosis induction was obtained in H1299 (>65%) and A549 (>55%) cells treated by the combination of all three siRNAs, a level slightly higher than that induced by AdFHIT (~45%). These results suggested that a high level of apoptosis induction could be achieved by simultaneously targeting multiple key elements in Ras/Rho oncogenic signaling pathway.


Figure 4
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  		Figure 4. Induction of apoptosis in AdFHIT-transduced and RHO-siRNA–treated NSCLC cells by flow cytometry. H1299 (A) and A549 (B) cells were transduced with AdFHIT or with the siRNA specific to Rab11, Rac1, and Rap1 genes or a combination of all three RHO-siRNAs (siComb) and a mixture of scrambled siRNAs was used as a nonspecific control (siNSRs). Representative flow cytometry profiles were shown on the top of charts. The percentage of apoptotic cells was presented in H1299 and A549 cells after various treatments for 72 h. Columns, average of three separated experiments with duplicated samples; bars, SE.

 
Inhibition of the RhoC-mediated tumor cell invasion by ectopic FHIT expression. In addition to those FHIT-targeted Ras/Rho genes and proteins identified by gene and expression profiling, we also found that activation of FHIT showed a negative regulation on the biological activity mediated by RhoC, a novel Rho family protein that has been shown to play an important role in tumor cell–induced invasion and metastasis (24). To verify the potential of RhoC in lung tumor cell–induced invasion and metastases, we established stable clones of H1299 cells that constitutively express a high level of RhoC or RhoC-GFP fusion protein. A fluorescence-based in vitro tumor cell invasion assay was used to quantitatively evaluate effects of ectopic expression of RhoC and FHIT on tumor cell–induced invasion (Fig. 5A and B ). The parent H1299 cell has very low level of endogenous RhoC expression (Fig. 5C) and exhibits a low potential of migration and invasion, as indicated by low percentage of invading or migrating cells (Fig. 5A). However, activation of RhoC dramatically increased (>25-fold) the H1299 cell-induced invasion in both H1299-RhoC and H1299-RhoC-GFP sublines (Fig. 5A and B). In contrast, ectopic activation of FHIT significantly reduced the invasive potential in these RhoC-activated H1299 cells when transduced by AdFHIT in a dose-dependent manner (Fig. 5A and B). A dramatic inhibition of RhoC protein expression was also detected in AdFHIT-transduced stable RhoC-expressing H1299 cells as shown by Western blot (Fig. 5C). These results clearly showed the specific and negative regulation of FHIT on RhoC-mediated tumor cell invasion or migration.


Figure 5
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  		Figure 5. Effects of forced expression of FHIT on RhoC activity and tumor cell–induced invasion and RhoC activity in H1299 cells. A, representative fluorescence images of the invasive potential of stable H1299 clones expressing RhoC. The parental cells (H1299) and stable clones ectopically expressing RhoC (H1299+RhoC) or RhoC-GFP fusion proteins (H1299+RhoC/GFP) were transduced by AdFHIT at MOI of 100 and 1,000, and untransduced cells were used as controls. The invasive potential was analyzed by a fluorescence-based in vitro tumor cell invasion assay using a BD Falcon HTS FluoroBlok 24-Multiwell Insert System. Noninvasive cells [stained by propidium iodide (PI), red] remained on the upper layer of the membrane and the invasive cells (stained by DAPI, blue) penetrated the membrane and were shown on the opposite layer. Images were taken 48 h after transduction. B, quantification of fluorescence intensity was determined by a fluorescence microplate reader. The relative invasive potential was determined by the ratio of fluorescence intensity of DAPI-stained cells to propidium iodide–stained cells. Columns, average of three individual experiments with duplicate samples among experiments; bars, SD. C, Western blot analysis of RhoC expression in AdFHIT-transduced cells.

 
Inhibition of tumor growth by intratumoral injection of DC nanoparticle-complexed RHO-siRNAs in human lung cancer xenograft mouse model. To validate our observations from in vitro experiments and to explore the antitumor therapeutic potential of using gene-specific siRNAs targeting Ras/Rho GTPase oncogenic signaling pathway, we carried out in vivo experiments using a s.c. A549 tumor xenograft mouse model. Nude mice were inoculated with A549 cells by s.c. injection of 1 x 106 cells per mouse in 100 µL PBS at the right flank site. When the average tumor size reaches ~0.5 mm in diameter, animals were randomly divided into six treatment groups. DC nanoparticles were used to deliver RHO-siRNAs in vivo. Animals were treated with DC-siRNA nanoparticles of individual siRab11, siRac1, and siRap1 or a combination of all three RHO-siRNAs, and animals treated with DC reagent alone and with a combination of the scrambled siRNAs of three RHO genes were used as a negative and nonspecific siRNA control, respectively. Animals were given three treatments of DC-siRNA nanoparticles at 10 nmol DC:250 pmol siRNA in 100 µL PBS per treatment by intratumoral injection within 1 week. A moderate inhibition on tumor growth was observed in animals treated with siRab11 compared with those treated with DC alone or nonspecific siRNA (Fig. 6A ). Animals treated with siRac1 nanoparticles showed a similar growth profile as that of nonspecific siRNA, although an apparent reduction in tumor volumes could be seen in this treatment group compared with the DC treatment control group (Fig. 6A). A significant reduction in tumor volumes was seen in animals treated with siRap1 (P < 0.0002) and with a combination of all three siRNAs (P < 0.0001) compared with both controls (Fig. 6A). The effect of the knockdown of RHO GTPases in vivo by a combined RHO-siRNAs on NSCLC growth is consistent with that observed in vitro, suggesting that a simultaneous inhibition of multiple key molecules in Ras/Rho GTPase molecular switch may be an effective therapeutic strategy for lung cancer. The knockdown of the targeted Rab11, Rac1, and Rap1 gene and protein expression by siRNAs were also confirmed by a quantitative real-time RT-PCR (Fig. 6B) and by Western blot (Fig. 6C) analysis, respectively, in tumors collected from mice treated by RHO-siRNAs for 48 h.


Figure 6
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  		Figure 6. Effects of RHO-siRNAs on tumor growth and RHO gene and protein expression in nude mice bearing s.c. A549 tumors. A, effect on tumor growth in nude mice. Synthetic siRNAs were complexed with DC nanoparticles for in vivo delivery. Tumor-bearing animals were treated with individual siRNA, siRac1, siRap1, or siRab11, alone or together (siComb), by s.c. injection as described in detail in Materials and Methods. Cells treated with DC nanoparticle alone were used as a control. Relative tumor growth was calculated as changes in the total tumor volumes among mice in each treatment group at different time points after treatment compared with those at start points. Points, two repeated experiments were done; bars, SE. Effects of RHO-siRNAs on expression of the targeted Rab11, Rac1, and Rap1 gene (B) and protein (C) in tumors. Total RNAs and crude protein lysates were prepared from tumor samples in mice treated with RHO-siRNAs for 48 h and quantified by real-time RT-PCR and Western blot analysis, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Genomic abnormalities, genetic mutations and deletions, transcriptional alterations, and translational defects of FHIT gene and gene products have been frequently found in lung cancer and other human cancers (5, 6, 15, 25). We previously showed that restoration of wild-type FHIT function by a recombinant adenoviral vector–mediated gene transfer significantly inhibited tumor cell growth in FHIT-deficient NSCLC cells by induction of apoptosis and alteration of cell cycle kinetics and inhibited tumor growth, tumorigenesis, and metastases in human NSCLC mouse models (17). Accumulating evidence is supporting the role of FHIT as a tumor suppressor in vitro and in vivo (6, 11, 12). FHIT is a multifunctional protein and involved in many important cellular processes, although the precise molecular mechanisms and signaling pathways mediated by FHIT activities remain largely unknown (6, 12, 26). For example, the apoptotic pathway triggered by FHIT in lung cancer cells was shown to be mediated by FADD and caspase-8–associated cell death pathway but independent of the Bcl-2 or Bcl-x(L)–associated mitochondrial pathway (27). The path of FHIT-mediated apoptosis (28) and the patterns of FHIT-regulated cell cycle processes (17, 29, 30) seemed to be different from those induced by p53 tumor suppression activity in lung cancers. In this study, we used a novel approach of complementary gene and protein expression profiling to simultaneously monitor global changes in gene and protein expression modulated by FHIT activity in NSCLC cells. This approach allows us to identify several specific cellular targets, including RAN, Rab, Rac, Rap, and Ral, and unique signaling pathways mediated by FHIT action, which could otherwise never been revealed by conventional and isolated methods. Particularly, we discovered, for the first time, that multiple key molecules in the Ras/Rho GTPase molecular switch were negatively regulated by FHIT activity. The differential expression of these Ras/Rho genes and gene products modulated by ectopic activation of FHIT was validated by quantitative real-time RT-PCR and Western blot analysis and biologically justified by reverse functional analysis by gene-specific knockdown with RHO-siRNAs in vitro and in vivo.

Our finding of Ras/Rho GTPases as direct cellular targets of FHIT may provide a biological support for an earlier crystallographic study that suggested the FHIT-substrate complex to be an active signaling molecule for its tumor suppression activity and to be functionally parallel to the Ras-GTP complexes (15, 31, 32). FHIT has also been shown to bind to tubulin and associated with actin in vitro and promotes cytoskeleton assembly (33). On the other hand, the assembly of Rho GTPases has been shown to act as a molecular switch to control an actin cytoskeleton-associated oncogenic signaling transduction pathway in cancer cells through their interaction with multiple target or effector proteins (34–37). Our findings suggest that the FHIT-targeted down-regulation of the Ras/Rho GTPases may be mediated through a negative regulation of actin cytoskeleton-associated signaling transduction by a direct or indirect FHIT-Rho GTPase protein interaction. Our findings would warrant further studies in these aspects.

In addition to those FHIT-targeted Ras/Rho GTPases identified by gene and protein expression profiling, we also found that activity of RhoC was significantly and specifically down-regulated by ectopic expression of FHIT in NSCLC cells. RhoC GTPase is a member of the Ras/Rho superfamily of small GTP-binding proteins that are involved in a wide spectrum of cellular functions, including cytoskeletal organization, cell cycle progression, cell transformation, adhesion, migration, and invasion (35, 38–40). Activation of RhoC is significantly correlated with the potential of tumor invasion and metastasis by a functional genomics study and to be absolutely required for metastasis in a mouse melanoma model (24). RhoC but not RhoA has also been shown to play an essential role in tumor cell migration, invasion, and metastases in NSCLC cells (41) and in lung cancer mouse models (42). Although information has been gathered about the role of RhoC in cancer metastasis, the mechanism by which it exerts its effect in lung cancer is still not completely understood. Ikoma et al. (42) proposed that RhoC functions by stimulating matrix metalloproteinases. They also speculated that other signaling molecules in diverse pathways may function as effectors of RhoC contributing to the metastatic potential of tumor cells.

The finding of the Ras/Rho GTPase molecular switch as a direct target of FHIT activity encouraged us to explore the potential for therapeutic targeting of key components in Ras/Rho oncogenic signaling pathway for suppressing tumor cell growth in NSCLC. We used gene-specific siRNAs to knock down expression of multiple Rho GTPase Rab11, Rac1, and Rap1 gene products in NSCLC cells. We found that a simultaneous inhibition of three Ras/Rho GTPase genes by a combination of three RHO-siRNAs significantly suppressed tumor cell growth by induction of apoptosis in vitro and in lung cancer mouse models, although variations were noted in studies with regard to the effect of individual siRNA treatments. Because Ras/Rho GTPases act through a network interaction among member proteins and with target proteins or effectors to ensure coordinated control of their and other cellular processes (39, 43, 44), our finding emphasizes the importance of targeting multiple molecules rather than inhibiting a single gene on a signaling network for achieving an optimal therapeutic outcome.

In conclusion, our data showed the potential of using complementary gene and protein expression profiling for the identification of specific cellular targets and signaling pathways mediated by a specific gene product in a complex biological network. These findings clearly linked FHIT tumor-suppressing function to the regulation of apoptosis, tumor cell proliferation, progression, and metastasis and provided insight into the molecular mechanism of FHIT action. A clear understanding of the molecular pathway by which FHIT functions would allow us to identify novel cancer therapeutic targets and develop a pathway-targeted molecular cancer therapy, as shown by the promising antitumor therapeutic efficacy obtained in human lung cancer mouse models using gene-specific siRNA nanoparticles designed to simultaneously target multiple key molecules in the Ras/Rho GTPase oncogenic signaling pathway.


   Acknowledgments
 
Grant support: National Cancer Institute, NIH Specialized Program of Research Excellence grants CA70907, CA71618, and RO1CA116322 (L.J.) and Mouse Models of Human Cancers Consortium grant U01CA10535201; Department of Defense TARGET Lung Cancer Programs grant DAMD17-02-1-0706; M. D. Anderson Cancer Center Support Core Grant CA16672; and 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. Charlotte Clark (Ciphergen Biosystems) for her technical support on ProteinChip array analysis and protein identification and Christine Wogan for manuscript editing.


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

3 http://prowl.rockefeller.edu/prowl-cgi/profound.exe Back

4 http://david.abcc.ncifcrf.gov/home.jsp Back

Received 2/21/07. Revised 8/ 3/07. Accepted 8/30/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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