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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC Oct 31, 2012.
Published in final edited form as:
PMCID: PMC3484890
NIHMSID: NIHMS409217

Identification of Novel Gene Expression Targets for the Ras Association Domain Family 1 (RASSF1A) Tumor Suppressor Gene in Non-Small Cell Lung Cancer and Neuroblastoma1

Abstract

RASSF1A is a recently identified 3p21.3 tumor suppressor gene. The high frequency of epigenetic inactivation of this gene in a wide range of human sporadic cancers including non-small cell lung cancer (NSCLC) and neuroblastoma suggests that RASSF1A inactivation is important for tumor development. Although little is known about the function of RASSF1A, preliminary data suggests that it may have multiple functions. To gain insight into RASSF1A functions in an unbiased manner, we have characterized the expression profile of a lung cancer cell line (A549) transfected with RASSF1A. Initially we demonstrated that transient expression of RASSF1A into the NSCLC cell line A549 induced G1 cell cycle arrest, as measured by propidium iodide staining. Furthermore, an-nexin-V staining showed that RASSF1A-expressing cells had an increased sensitivity to staurosporine-induced apoptosis. We then screened a cDNA microarray containing more than 6000 probes to identify genes differentially regulated by RASSF1A. Sixty-six genes showed at least a 2-fold change in expression. Among these were many genes with relevance to tumorigenesis involved in transcription, cytoskeleton, signaling, cell cycle, cell adhesion, and apoptosis. For 22 genes we confirmed the microarray results by real-time RT-PCR and/or Northern blotting. In silico, we were able to confirm the majority of these genes in other NSCLC cell lines using published data on gene expression profiles. Furthermore, we confirmed 10 genes at the RNA level in two neuroblastoma cell lines, indicating that these RASSF1A target genes have relevance in non-lung cell backgrounds. Protein analysis of six genes (ETS2, Cyclin D3, CDH2, DAPK1, TXN, and CTSL) showed that the changes induced by RASSF1A at the RNA level correlated with changes in protein expression in both non-small cell lung cancer and neuroblastoma cell lines. Finally, we have used a transient assay to demonstrate the induction of CDH2 and TGM2 by RASSF1A in NSCLC cell lines. We have identified several novel targets for RASSF1A tumor suppressor gene both at the RNA and the protein levels in two different cellular backgrounds. The identified targets are involved in diverse cellular processes; this should help toward understanding mechanisms that contribute to RASSF1A biological activity.

INTRODUCTION

The high incidence of loss of heterozygosity at 3p21.3 in many sporadic human cancers suggests that this locus harbors one or more critical TSGs3 (16). The minimum critical interval was narrowed to ~120 kb by the discovery of overlapping homozygous deletions in lung and breast tumor cell lines (7, 8). Eight candidate TSGs were cloned from this gene-rich region including CACNA2D2, PL6, 101F6, NPRL2/G21, BLU, RASSF1, FUS1, and LUCA2 (9). However, conventional mutation analysis did not reveal frequent mutations in any of the above candidate genes (912). Nevertheless, the long isoform of RASSF1, RASSF1A, was found to be down-regulated in many lung tumor cell lines, although expression of the shorter isoform, RASSF1C, was unaffected (9, 13). The promoter region of RASSF1A is associated with a CpG island, and bisulphite DNA sequencing demonstrated that RASSF1A was inactivated by promoter region hypermethylation in the majority of lung tumor cell lines (1315). This is supported by the observed reexpression of RASSF1A in cell lines treated with demethylating agents. Further evidence for the candidacy of RASSF1A as a major 3p21.3 TSG comes from in vitro and in vivo growth studies in which RASSF1A drastically reduced colony formation, suppressed anchorage-independent growth, and inhibited tumor formation in nude mice (13, 15). Subsequently, frequent RASSF1A methylation has been detected in many other tumor types, including SCLC and NSCLC; breast, kidney, prostate, and testicular cancer; neuroblastoma; phaeochromocytoma; and gastric and nasopharyngeal cancer, indicating that the inactivation of RASSF1A is important in the pathogenesis of many human cancers (1322).

RASSF1A is a Mr 39,000 (340 aa) protein containing two major putative functional domains including a diacylglycerol (DAG)-binding domain (50–101 aa) at the NH2 terminus. A RAS association (RA) domain (194–288 aa) in the COOH terminus (also found in the C isoform) suggests RASSF1 proteins function as RAS-effectors (9, 23). However, recent studies indicated that it is unlikely that RASSF1A or RASSF1C bind directly to RAS (24). RASSF1A does, however, heterodimerize with the closely homologous mouse RAS-GTP-binding protein, Nore1 (2425). Human NORE1 interacts with the proapoptotic protein kinase MST1 to mediate a novel RAS-regulated apoptotic pathway (26). RASSF1A also interacts with MST1, suggesting that there might be a close interplay between RASSF1A and NORE1 proteins in RAS-mediated apoptosis. Support for a role for RASSF1A in RAS-signaling pathways was implied by a recent study that compared the frequency of RASSF1A methylation with the incidence of K-RAS mutation in colorectal cancers (27). An inverse relationship between these events was detected in a significant number of cases.

A recent study in the NSCLC cell line NCI-H1299 suggested that RASSF1A might inhibit cell cycle progression (28). Thus RASSF1A induced G1-S phase cell cycle arrest and blocked accumulation of Cyclin D1. The latter point was confirmed using siRNA to eliminate endogenous RASSF1A from HeLa cells with the concomitant increase in Cyclin D1 protein.

These studies suggest that RASSF1A may have multiple functions. To further define the possible range of functions, we have used cDNA microarray technology to investigate the global impact of RASSF1A on gene expression in NSCLC. In addition, we investigated the consistency of candidate RASSF1A target genes among NSCLC cell lines and compared the profile of RASSF1A target genes in NSCLC and neuroblastomas.

MATERIALS AND METHODS

Cell Culture and Transfection

The NSCLC cell lines A549 and NCI-H1299 and neuroblastoma cell lines CHP212 and SK-N-AS were obtained from American Type Culture Collection and maintained in DMEM supplemented (Invitrogen) with 10% FCS. Cells (1 × 104) were seeded and transfected with 1 μg of pcDNA3.1 or pcDNA3.1/RASSF1A using Fugene 6 reagent (Roche). Twenty-four h after transfection, DMEM was supplemented with 500 μg/ml Geneticin (Invitrogen). Surviving colonies were harvested and expanded in separate flasks 14 days later.

Transient transfection was set-up similarly using pEGFP-C1, pEGFP-C1/RASSF1A, pcDNA3/HA-RASSF1A, and Effectene reagent (Invitrogen). Cells were harvested using trypsin or lysis buffer (see below) 48 h after transfection.

Apoptosis Assay

Annexin-V binding was used to measure apoptosis. One × 105 cells were seeded in 6-well dishes. Sixteen h later, DMEM was supplemented with 1 μg/ml staurosporine (Roche). Cells were incubated for 4 h and were harvested in ice-cold PBS. Cells were washed once with annexin-V binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2) and incubated at room temperature with 5 μl each of annexin-V-PE (Phycoethrin) and 7-AAD (7-Amino-actinomycin D) in 100 μl of binding buffer (Annexin-V-PE apoptosis detection kit; BD PharMingen) for 15 min in the dark. 400 μl of binding buffer was then added to the cells and a Coulter Epics XL-MCL flow cytometer was used to measure Annexin-V-PE binding. The assay was repeated three times.

Cell Cycle Profiling

Forty-eight h after seeding 1 × 105 cells, cells were harvested, washed with ice-cold PBS, resuspended in 70% ethanol (−20°C) and stored at −20°C for 24 h. Cells were pelleted by centrifugation, and the ethanol was decanted. Cells were stained with propidium iodide, and DNA content was analyzed by flow cytometry using a Coulter Epics XL-MCL flow cytometer running System II software. Three independent experiments were conducted.

Microarray

RNA was extracted from 50–70% confluent cells using Trizol Reagent (Invitrogen) in accordance with the manufacturer’s instructions. Aliquots (25 μg) of RNA were spiked with bacterial-RNA mixture for control and was ethanol-precipitated. The RNA mix was resuspended in H2O, was incubated for 5 min at 70°C with 5 μg of anchored oligo-dT17, and was snap-chilled on ice. Cy3- or Cy5-labeled cDNA was generated by incubating the RNA/oligo-dT mix with 1× first-strand buffer [0.03 M DTT, 5 mM dNTP mix, 0.1 mM dCTP-Cy3 or dCTP-Cy5 (Amersham), and 400 units of Superscript II (Invitrogen)] for 2 h at 42°C. RNA was removed by hydrolysis in 0.05 M NaOH at 70°C for 20 min. Unincorporated nucleotides were removed using AutoSeq G-50 columns (Amersham). Cy3- and Cy5-labeled ss (single-stranded) cDNA generated from separate samples were combined with 6 μl of human cot1 DNA (1 μg/μl) and 7 μl of 3 M sodium acetate (pH 5.2) and were ethanol-precipitated. The Cy3/Cy5 ss cDNA/cot1 DNA pellet was resuspended in 8 μl of H2O and 40μl of hybridization buffer [5× SSC, 6× Denhardt’s solution, 60 mM Tris-HCl (pH 7.6)], boiled for 5 min and cooled at room temperature for 10 min. The hybridization mix was then applied to precooled (4°C) Hver1.2.1 cDNA microarrays [Microarray consortium (MACS)], was overlaid with a coverslip and incubated at 47°C for 12–24 h in a humidified atmosphere. Microarrays were washed sequentially with 2× SSC, 0.1× SSC/0.1% SDS, and 0.1× SSC and were air-dried by briefly spinning in a centrifuge to remove excess liquid. The relative binding of Cy3- and Cy5-labeled ss cDNA was measured using a LSI-Lumonics SA4000 scanner and GeneSpring Expression Analysis Software (Silicon Genetics) was used to analyze the data.

Real-Time RT-PCR

Quantitative values are obtained from the cycle number (Ct value) at which the increase in fluorescent signal associated with an exponential growth of PCR products starts to be detected by the laser detector of the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA) using the Perkin-Elmer Biosystems analysis software according to the manufacturer’s manuals.

The precise amount of total RNA added to each reaction mix (based on absorbance) and its quality (i.e., lack of extensive degradation) are both difficult to assess. We, therefore, also quantified transcripts of the TBP gene coding for the TATA box-binding protein (a component of the DNA-binding protein complex TFIID) as the endogenous RNA control, and each sample was normalized on the basis of its TBP content.

Results, expressed as N-fold differences in target gene expression relative to the TBP gene, termed “ Ntarget,” were determined by the formula: Ntarget = 2ΔCtsample, where ΔCt value of the sample was determined by subtracting the average Ct value of the target gene from the average Ct value of the TBP gene.

The Ntarget values of the samples were subsequently normalized so that the mean of the Ntarget values of the RASSF1A-null-transfected samples of each cell line would equal a value of 1.

Primers for the TBP and RASSF1A and the 15 target genes were chosen with the assistance of the computer programs Oligo 5.0 (National Biosciences, Plymouth, MN). To avoid amplification of contaminating genomic DNA, one of the two primers was placed, if possible, in a different exon. In general, amplicons were between 70 and 120 nucleotides. Agarose gel electrophoresis allowed us to verify the specificity of PCR amplicons.

Total RNA extracted from cell line samples was reverse-transcribed before real-time PCR amplification. PCR was performed using the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). The thermal cycling conditions included an initial denaturation step at 95°C for 10 min and 50 cycles at 95°C for 15 s and 65°C for 1 min. Experiments were performed with duplicates for each data point.

Northern Blotting

RNA (20 μg) was separated on standard agarose–formaldehyde gel at 100 V for 3 h. RNA was transferred by capillary blotting overnight onto Hybond N+ membrane (Amersham-Pharmacia Biotech) in Northern transfer buffer (Sigma). RNA was fixed onto the membrane by baking at 80°C for 2 h. Gene fragments (typically 500–700 bp) for use in probe synthesis were excised by restriction enzyme digestion from cDNA clones (Sanger Centre). cDNA probes were labeled with α-32P (Amersham) using a random priming kit (Roche) in accordance with the manufacturer’s instructions. The probes were purified from unincorporated nucleotides on Sephadex 50 columns (Roche) and denatured. Hybridization was performed overnight at 68°C in PerfectHyb Plus hybridization buffer (Sigma). Membranes were washed according to manufacturer’s instructions, exposed with phosphorimager cassettes, and analyzed on phosphorimager program ImageQuant (Molecular Dynamics).

Western Blotting and Immunofluorescence

Cells were grown to ~70% confluence and harvested in NP40-lysis buffer. Lysates were incubated on ice for 10 min, and sonicated for 60 s, and insoluble cell debris was removed by centrifugation for 5 min at 14,000 rpm at 4°C. Protein samples (20 μg each) were separated by SDS-PAGE (6–15%) and were electroblotted to Hybond-P membranes (Amersham Biosciences). Immobilized proteins were detected using appropriate primary and horeseradish-peroxidase secondary antibodies by ECL (Amersham Bioscience).

For immunofluorescence detection of CDH2, cells were seeded onto Superfrost glass slides (VWR International) 24 h before fixing in acetone for 15 min. All of the antibody incubations and washes were done with PBS. Immobilized anti-CDH2 was detected using Cy3-labeled rabbit antimouse (Sigma). Cells were counterstained and mounted using 4′,6-diamidino-2-phenylindole (DAPI)/Vector mount.

Antibodies

Anti-RASSF1A monoclonal antibody (eB114) was purchased from eBiosciences; anti-ETS2 (C-20) was purchased from Santa Cruz Biotechnology; anti-Procathepsin L (Ab-2) and anti-TGM2 (rabbit) were purchased from Calbiochem; anti-Thioredoxin was purchased from Serotec; anti-HA (HA-7), anti-DAPK1, and anti-CDH2/A-CAM (GC-4) were purchased from Sigma, anti-CCND3 (DCS22) was purchased from the monoclonal antibody service, Cancer Research United Kingdom; and anti-Cyclin D1 was kindly provided by G. Peters, Cancer Research UK (formerly, ICRF), London, United Kingdom.

RESULTS

Characterization of A549 Clones Stably Expressing RASSF1A

Transfection of RASSF1A into A549 Cells Induces G1 Cell Cycle Arrest and Down-Regulates Cyclin D1 Expression

We used propidium iodide incorporation to investigate the effect of RASSF1A on cell cycle progression in the NSCLC cell line A549 (Fig. 1A). RASSF1A expression resulted in an increase of cells in the G1 phase of the cell cycle 48 h after transfection compared with GFP-expressing cells [Fig. 1A(i)].

Fig. 1
A, cell cycle profiles of A549 cells transiently and stably expressing RASSF1A. (i), A549 cells transfected with GFP or RASSF1A-GFP were harvested and analyzed after 48 h incubation. (ii), representative clones of A549 (V.18 and Cl.5) were harvested for ...

To determine whether the G1-arrested phenotype was maintained after drug selection, stably transfected clones of A549 were then analyzed. Expression of RASSF1A was confirmed by RT-PCR (data not shown) and by Western blotting (Fig. 1B). A representative clone expressing RASSF1A (Cl.5) was found to have an increased cell population in the G1 phase of the cell cycle compared with the vector control clone (V18), indicating that the cell cycle effect observed in transient transfection is perpetuated after drug selection [Fig. 1A(ii)].

The effect of RASSF1A on cell cycle may be associated with a decrease in Cyclin D1 expression. Therefore we screened whole cell lysates from RASSF1A expressing clones by Western blotting to determine whether Cyclin D1 is down-regulated in A549. Cyclin D1 protein expression was drastically reduced in RASSF1A-expressing clones compared with vector controls; similar results were obtained in the neuroblastoma cell line CHP212 (Fig. 1B). These results indicate that the RASSF1A affects cell cycle progression and reduces Cyclin D1 expression in A549 cells.

RASSF1A Increases Sensitivity to Staurosporine-induced Apoptosis

RASSF1A may function as a mediator of apoptosis. Hence, RASSF1A-expressing cells may be more sensitive to apoptotic stimuli. We used annexin-V binding to measure the apoptosis of RASSF1A-expressing A549 cell derivatives incubated with staurosporine. RASSF1A-expressing cells showed a 29.1% increase in apoptosis relative to control cells after treatment with staurosporine (Fig. 1C).

Genes Differentially Regulated by RASSF1A

To determine changes in gene expression that result from the reintroduction of RASSF1A into A549, we have used a competitive hybridization-based approach to screen Hver1.2.1 cDNA microarrays containing ~6000 unique genes (does not include Cyclin D1). RNA was extracted from duplicate cultures of representative derivatives of A549 (RASSF1A-expressor Cl.5 and vector-control V.18). To reduce variations in gene expression caused by culture conditions, we harvested RNA from cells at 60–70% confluency, and 48 h after seeding, we generated cDNA labeled with Cy3 and Cy5 from each sample. Competitive hybridizations were done in duplicate using Cy3- and Cy5-labeled cDNA from each preparation. Furthermore, Cy3- and Cy5-labeled cDNA from each of the vector samples was used in duplicate control hybridizations to eliminate background noise caused by possible differences in labeling efficiency of the Cy dyes and variable genes. In total, eight Hver1.2.1 chips were screened: four RASSF1A versus vector and four control hybridizations. Data were analyzed using GeneSpring software. Essentially the GeneSpring software passed data sets that showed a significant increase or decrease. From these, data sets that showed significant changes in the control hybridizations were filtered. Stringency was set so that only the data sets with a minimum 2-fold change and agreement in four of four RASSF1A versus vector hybridizations were passed. Table 1 lists 66 genes arranged into functional groups derived from 74 data sets that have met the set criteria. Of the 66 genes differentially expressed in response to RASSF1A, 34 were induced, whereas 32 were down-regulated.

Table 1
Genes differentially expressed by RASSF1A in NSCLC cell line A549

Northern and Quantitative Real-Time RT-PCR Confirmation of Microarray Data

Six of the identified RASSF1A targets were selected for confirmation by Northern blotting in the same clones used for microarray analysis (Fig. 2). Quantification revealed a good correlation between the fold changes obtained in the microarray and Northern blotting for DUSP1, CA12, HPCAL1, ABCG2, SM22, and CTSB.

Fig. 2
Northern blot analysis showing the change in RNA expression of the indicated genes in RASSF1A expressing Cl.5 (+) and vector control V18 (−) A549 cells. Blots were stripped and reprobed for GAPDH as control for RNA loading.

Quantitative real-time RT-PCR was used to investigate the expression levels of 17 target genes in three RASSF1A-expressing clones of A549. The data obtained is presented in Table 2. There was complete correlation between the real-time RT-PCR results and the microarray data for all of the A549 clones analyzed. This included 6 induced genes (ZYX, CDH2, TPM1, ETS2, ANPEP, and SPARC) and 11 down-regulated genes (ITGB5, PIGPC1, ATP5H, DB1, DAPK1, CCND3, TXN, CTSL, EDG2, SPINT2, and CA12). This confirmed the microarray data and demonstrated that RASSF1A has a reproducible effect on the expression of these genes in A549. For control, two separate primer sets were used to quantify RASSF1A expression.

Table 2
Real-time RT-PCR showing fold change of target gene expression in A549 cellsa

In Silico Comparison of Target Gene Expression in RASSF1A-expressing A549 versus Other NSCLC Cell Lines of Known RASSF1A Status

We wished to determine to what extent the genetic background of individual cell lines might influence the expression profile of candidate RASSF1A target genes. Expression data from microarray experiments is made available on websites such as UCSC (University of California Santa Cruz) human genome project (http://genome-archive.cse.ucsc.edu/). Ross et al. (29) have investigated the variation in gene expression of a wide range of human tumor cell lines including NSCLC. The RASSF1A expression status of four of the NSCLC cell lines used (A549, NCI-H460, NCI-H332, and NCI-H23) is known. The RASSF1A promoter region in these cell lines is methylated and RASSF1A is not expressed. This information provided us with an opportunity to compare target gene expression in our A549 derivatives with the levels of target genes in this panel of NSCLC cell lines determined by Ross et al. (Fig. 3). The inclusion of A549 in the Ross study is a useful control. Expression data were available for 17 of 22 confirmed RASSF1A gene targets. Overall, the expression of the target genes in the NSCLC cell panel (not expressing RASSF1A), with the exception of DAPK1, was the opposite of that observed in RASSF1A-expressing A549 cells indicating that these genes are RASSF1A regulated. In the Ross data, TPM1 appears to be up-regulated in A549, whereas it seems to be down-regulated in the other NSCLC cell lines. Closer inspection of the Ross data shows that TPM1 is up-regulated 2-fold in A549, whereas we show a 5-fold increase in RASSF1A-expressing A549 cells. These differences are likely to be attributable to the differences in reference RNA used in each study.

Fig. 3
In silico comparison of target gene expression in RASSF1A-expressing A549 and other NSCLC cell lines of known RASSF1A expression status (shown on the right: −, not expressed; +, expressed). The expression levels of 17 RASSF1A gene targets determined ...

Analysis of RASSF1A Target Genes in Neuroblastoma Cell Lines

Having established that the profile of RASSF1A candidate target genes was similar between different NSCLC cell lines in silico, we wished to determine whether RASSF1A targets in NSCLC were also regulated in neuroblastoma cell lines. Real-time RT-PCR confirmation of the microarray data were extended to include RASSF1A expressing clones of two neuroblastoma cell lines SK-N-AS and CHP212. The expression data from the SK-N-AS and CHP212 cells corroborated the results obtained in the lung background for 10 of 17 genes (CDH2, TPM1, ETS2, ANPEP, PIGPC1, DB1, CCND3, TXN, CTSL, and CA12; Table 3; Fig. 4). This shows that the majority of the RASSF1A expression targets identified in A549 have relevance in the neuroblastoma background. The expression of seven genes, SPINT2, EDG2, ITGB5, SPARC, ZYX, DAPK1, and ATP5H, was either unaffected by RASSF1A in one of the two neuroblastoma cell lines (ZYX, SPARC, and ATP5H), or changed contrary to the effect observed in lung cancer (DAPK1 and ZYX), or agreed with the results seen in lung cancer but only in one of the neuroblastoma backgrounds (SPINT2, EDG2, ITGB5, and SPARC). This raises the possibility that RASSF1A may also have tissue-specific effects.

Fig. 4
Venn diagram showing the cell line distribution of RASSF1A-regulated genes.
Table 3
Real-time RT-PCR data showing the fold change of target gene expression in neuroblastoma cell linesa

Protein Confirmation of Microarray Data

To establish whether RASSF1A-induced effects seen at the RNA level translated to changes in protein levels, we examined the expression of ETS2, CTSL, TXN, DAPK1, CDH2, and CCND3 in lung and neuroblastoma backgrounds using a combination of Western blotting and immunofluorescence. Protein levels of CTSL, TXN, and CCND3 were greatly reduced in RASSF1A-expressing cells of A549 and CHP212 in agreement with changes seen at the RNA level (Fig. 5). DAPK1, also, was drastically down-regulated in stably transfected A549 cells. Induction of ETS2 and CDH2 by RASSF1A was confirmed by immunoblotting in both lung and neuroblastoma lineages. The change of target gene expression seen by Western blotting correlated with the level of RASSF1A protein expression in these clones. The induction of CDH2 was further corroborated at the cellular level by immunofluorescence staining (Fig. 6).

Fig. 5
Western blot analysis showing protein level changes of RASSF1A-regulated genes. Protein lysates from control clones (A549 V18 and CHP212 V1) and independent RASSF1A-expressing clones (A549 Cl.1, Cl.5 and CHP212 Cl.1, Cl.3) were separated on polyacrylamide ...
Fig. 6
Immunofluorescence staining showing induction of CDH2 in RASSF1A-expressing A549 cells. (i) and (ii), negative control stains of A549-vector control (V.18) cells and the RASSF1A-expressing clone Cl.1, respectively. (iii) and (iv), CDH2 staining in A549 ...

Regulation of RASSF1A Target Genes in Transient Assay

To further confirm the candidacy of the identified targets as RASSF1A-regulated genes, we set up a transient transfection assay. Fig. 7 shows that both CDH2 and TGM2 are strongly induced 48 h after transfection with RASSF1A in both A549 and NCI-H1299 cell lines, in agreement with our findings in stable clones. This effect was reproducible (n = 2) and also suggests that the targets identified in A549 are relevant in other NSCLC cell lines.

Fig. 7
Immunoblot detection of RASSF1A-induced expression of CDH2 and TGM2. A549 and NCI-H1299 cells were transfected with pcDNA3HA (−) or pcDNA3HA-RASSF1A (+) and were harvested in lysis buffer 48 h after transfection. Proteins were resolved by PAGE ...

DISCUSSION

RASSF1A is a major 3p21.3 TSG with a high incidence of epigenetic inactivation in many common sporadic human cancers. Exogenous overexpression of RASSF1A has a profound effect on tumor cell growth in vitro and in vivo; however, the mechanisms of RASSF1A tumor suppression are not yet understood. We have demonstrated that RASSF1A-induced cell cycle arrest in NSCLC A549 cells is consistent with findings in NSCLC NCI-H1299 (28). Furthermore, we show that stable exogenous overexpression of RASSF1A sensitized A549 cells to staurosporine-induced apoptosis. Subsequently, we used cDNA microarrays to gain insight into possible functions of RASSF1A. Thus, we identified 66 genes differentially up- or down-regulated by RASSF1A by at least 2-fold in the NSCLC cell line A549. We confirmed the changes in RNA expression by Northern blotting and or quantitative real-time RT-PCR of 22 genes. Human tumorigenesis is a multistep process, and RASSF1A-induced changes in gene expression might be influenced by the genetic and epigenetic background of the cell line (and the tumor type). To determine whether RASSF1A target gene analysis in a range of NSCLC cell lines would be consistent with that obtained in A549, we correlated the changes in candidate target gene expression in four NSCLC cell lines analyzed by Ross et al. (29) using microarrays. Remarkably the expression pattern of 16 of 17 target genes were confirmed by in silico analysis using the data deposited by Ross et al. Having obtained consistent results within a single tumor type, we then compared target gene expression in NSCLC and neuroblastoma cell lines. Ten genes were confirmed in two neuroblastoma cell lines (CHP212 and SK-N-AS) including CDH2, TPM1, ETS2, ANPEP, PIGPC1, DB1, CCND3, TXN, CTSL, and CA12, which indicated that these RASSF1A targets are common in lung and neuroblastoma cell lineages. Western analysis of six of these genes, ETS2, CTSL, TXN, DAPK1, CDH2, and CCND3, demonstrated that changes in RNA levels were paralleled by changes in protein expression in both cell backgrounds. Furthermore, RASSF1A-induced expression of CDH2 was demonstrated by immunofluorescence. Interestingly, there seemed to be a correlation between the change in target gene expression and RASSF1A expression at the protein level in A549 cells. Transient transfection in A549 and NCI-H1299 was used to further confirm the role of RASSF1A in regulating the expression of target genes CDH2 and TGM2. This highlights the importance of RASSF1A in the regulation of these genes and indicates that the targets identified in A549 are also important in other NSCLC cell lines. Our analysis of RASSF1A target genes in NSCLC and neuroblastoma suggests that RASSF1A has pleiotropic effects on tumor cell biology affecting several pathways important in tumorigenesis, including cell cycle progression, cell adhesion, cell migration, angiogenesis, transcription, and apoptosis.

Our data suggests that regulation of the cell cycle may be just one mechanism through which RASSF1A regulates cell proliferation. Exogenous overexpression of RASSF1A inhibited cell cycle progression, which was consistent with the observed down-regulation of Cyclin D1 and D3 protein levels. In addition, we found that RASSF1A affects the expression of genes involved in the regulation of cell growth such as Diazepam binding inhibitor (DBI) and Spermidine/spermine N1-acetyltransferase (SSAT). At this stage, it is not possible to determine whether the effect of RASSF1A on the cell cycle and the observed down-regulation of cyclins is a direct effect or a downstream effect of altered expression of genes that regulate cell growth.

Cell-cell adhesion and cell-substratum adhesion are thought to affect cell migration, proliferation, and apoptosis. Through proteins such as N-cadherin (CDH2), Zyxin (ZYX) and Tropomyosin1 (TPM1), RASSF1A may influence these cellular functions, which ultimately affect cell behavior. Loss of expression of CDH2, ZYX, and TPM1 has been linked with cell transformation, gain of contact-independent growth, and development of metastasis (3035). RASSF1A-expressing cells have reduced contact-independent growth and form fewer metastases in nude mouse assays. Hence, it is tempting to speculate that RASSF1A-induced expressing of CDH2, ZYX, and TMP1 may contribute to this phenotype.

A characteristic of many tumors is the ability to change and reshape the ECM especially during angiogenesis and migration. Our data suggests that RASSF1A plays a role in regulating these processes by down-regulating Cathepsin L (CTSL) and increasing the expression of SPARC (Secreted protein acidic and rich in cysteine/Osteonectin). CTSL is a cysteine proteinase that is active against substrates such as elastin, collagen, actin laminin, and fibronectin (36). In neoplasia, CTSL promotes tumor cell invasion and metastasis (37). SPARC is a multifunctional matricellular protein. Among its reported effects are the inhibition of breast tumor cell line proliferation and the inhibition of growth and angiogenesis in neuroblastoma (38, 39).

Modulation of target gene expression can be achieved in different ways, including at the level of transcription. RASSF1A affects the expression of some transcriptional regulators, including ETS-2, that belong to the ETS family of transcription factors, which are important downstream targets of the RAS/RAF/MEK/MAPK-signaling pathway (40). Phosphorylation of specific residues in ETS-2 by MAP-kinase is essential for RAS-mediated ETS-2 activation. However, whereas ETS-2 activation by RAS is important during transformation, increased expression of ETS-2 has been shown to reverse RAS-mediated transformation (41). At this stage, it is not clear which of the RASSF1A-induced changes in gene expression are attributable to the secondary effects of alterations in transcription factor levels.

Resistance to apoptosis is an important part of tumorigenesis. RASSFIA is suggested to be a mediator of RAS-induced apoptosis. Consistent with this, we showed that RASSF1A sensitized cells to staurosporine-induced apoptosis. However, our results suggest that RASSF1A down-regulates the expression of Death-associated protein kinase1 (DAPK1) in stable clones of A549 cells but not in neuroblastoma cell lines, which showed increased expression by real time RT-PCR. This variance still needs to be resolved. It has been shown that promoter methylation has a role in inactivating DAPK1 in some cancers. However, recent studies show that DAPK1 is unmethylated in A549 cells, suggesting that its unresponsiveness is unlikely to be caused by promoter methylation (42). Interestingly, we show that RASSF1A strongly induced the expression of Transglutaminase 2 (TGM2), which is also involved in apoptosis (reviewed in 43). TGM2 functions both as a calcium-dependent transglutaminase and as a G-protein (Gh) modulating phospholipase activity (44) and also has roles in bone ossification, wound healing (45), cell adhesion (46), and cell signaling (47). This suggests that through TGM2, RASSF1A may not only affect apoptosis but several other important cellular functions.

Some of the RASSF1A expression targets such as CTSL and TPM1 are also modulated by the oncogene RAS during transformation (35, 38, 41, 48). Regulation of such genes implies that one of the functions of RASSF1A may be to regulate the expression of RAS gene targets. How this is achieved needs further investigation. However, it is interesting to note that DUPS1, a regulator of MAP-kinase activity, and ETS-2 are among the list of RASSF1A gene targets.

Overall, our global approach to characterizing the role of RASSF1A raises the possibility that it functions as a regulator of a number of key processes important for tumor progression, which supports its status as a major 3p21.3 TSG.

Footnotes

1Supported in part by SPARKS (Sports Aiding Medical Research for Kids), The Wellcome Trust, Cancer Research United Kingdom, and National Cancer Institute, NIH, Grants P50 CA70907 and CA71618.

3The abbreviations used are: TSG, tumor suppressor gene; NSCLC, non-small cell lung cancer; aa, amino acid(s); siRNA, small interfering RNA; RT-PCR, reverse transcription-PCR; ECM, extracellular matrix.

References

1. Wistuba II, Behrens C, Virmani AK, Mele G, Milchgrub S, Girard L, Fondon JW, III, Garner HR, McKay B, Latif F, Lerman MI, Lam S, Gazdar AF, Minna JD. High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res. 2000;60:1949–1960. [PubMed]
2. Martinez A, Fullwood P, Kondo K, Kishida T, Yao M, Maher ER, Latif F. Role of chromosome 3p12-p21 tumour suppressor genes in clear cell renal cell carcinoma: analysis of VHL-dependent and VHL-independent pathways of tumori-genesis. Mol Pathol. 2000;53:137–144. [PMC free article] [PubMed]
3. Martinez A, Walker RA, Shaw JA, Dearing SJ, Maher ER, Latif F. Chromosome 3p allele loss in early invasive breast cancer: detailed mapping and association with clinicopathological features. Mol Pathol. 2001;54:300–306. [PMC free article] [PubMed]
4. Maitra A, Wistuba II, Washington C, Virmani AK, Ashfaq R, Milchgrub S, Gazdar AF, Minna JD. High-resolution chromosome 3p allelotyping of breast carcinomas and precursor lesions demonstrates frequent loss of heterozygosity and a discontinuous pattern of allele loss. Am J Pathol. 2001;159:119–130. [PMC free article] [PubMed]
5. Fullwood P, Marchini S, Rader JS, Martinez A, Macartney D, Broggini M, Morelli C, Barbanti-Brodano G, Maher ER, Latif F. Detailed genetic and physical mapping of tumor suppressor loci on chromosome 3p in ovarian cancer. Cancer Res. 1999;59:4662–4667. [PubMed]
6. Ejeskar K, Aburatani H, Abrahamsson J, Kogner P, Martinsson T. Loss of heterozygosity of 3p markers in neuroblastoma tumours implicate a tumour-suppressor locus distal to the FHIT gene. Br J Cancer. 1998;77:1787–1791. [PMC free article] [PubMed]
7. Wei MH, Latif F, Bader S, Kashuba V, Chen JY, Duh FM, Sekido Y, Lee CC, Geil L, Kuzmin I, Zabarovsky E, Klein G, Zbar B, Minna JD, Lerman MI. Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG. Cancer Res. 1996;56:1487–1492. [PubMed]
8. Sekido Y, Ahmadian M, Wistuba II, Latif F, Bader S, Wei MH, Duh FM, Gazdar AF, Lerman MI, Minna JD. Cloning of a breast cancer homozygous deletion junction narrows the region of search for a 3p21.3 tumor suppressor gene. Oncogene. 1998;16:3151–3157. [PubMed]
9. Lerman MI, Minna JD. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res. 2000;60:6116–6133. [PubMed]
10. Angeloni D, Wei MH, Duh FM, Johnson BE, Lerman MI. A G-to-A single nucleotide polymorphism in the human α2δ2 calcium channel subunit gene that maps at chromosome 3p21.3. Mol Cell Probes. 2000;14:53–54. [PubMed]
11. Angeloni D, Duh FM, Wei MF, Johnson BE, Lerman MI. A G-to-A single nucleotide polymorphism in intron 2 of the human CACNA2D2 gene that maps at 3p21.3. Mol Cell Probes. 2001;15:125–127. [PubMed]
12. Honorio S, Gordon K, MacCartney D, Agathanggelou A, Latif F. Identification of a single nucleotide polymorphism in the human α2δ2 calcium channel subunit gene. Mol Cell Probes. 2001;15:391–393. [PubMed]
13. Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer GP. Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet. 2000;25:315–319. [PubMed]
14. Agathanggelou A, Honorio S, Macartney DP, Martinez A, Dallol A, Rader J, Fullwood P, Chauhan A, Walker R, Shaw JA, Hosoe S, Lerman MI, Minna JD, Maher ER, Latif F. Methylation-associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene. 2001;20:1509–1518. [PubMed]
15. Burbee DG, Forgacs E, Zochbauer-Muller S, Shivakumar L, Fong K, Gao B, Randle D, Kondo M, Virmani A, Bader S, Sekido Y, Latif F, Milchgrub S, Toyooka S, Gazdar AF, Lerman MI, Zabarovsky E, White M, Minna JD. Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst (Bethesda) 2001;93:691–699. [PubMed]
16. Astuti D, Agathanggelou A, Honorio S, Dallol A, Martinsson T, Kogner P, Cummins C, Neumann HP, Voutilainen R, Dahia P, Eng C, Maher ER, Latif F. RASSF1A promoter region CpG island hypermethylation in phaeochromo-cytomas and neuroblastoma tumours. Oncogene. 2001;20:7573–7577. [PubMed]
17. Dammann R, Yang G, Pfeifer GP. Hypermethylation of the cpG island of Ras association domain family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus, occurs in a large percentage of human breast cancers. Cancer Res. 2001;61:3105–3109. [PubMed]
18. Morrissey C, Martinez A, Zatyka M, Agathanggelou A, Honorio S, Astuti D, Morgan NV, Moch H, Richards FM, Kishida T, Yao M, Schraml P, Latif F, Maher ER. Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma. Cancer Res. 2001;61:7277–7281. [PubMed]
19. Byun DS, Lee MG, Chae KS, Ryu BG, Chi SG. Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma. Cancer Res. 2001;61:7034–7038. [PubMed]
20. Kwong J, Lo KW, To KF, Teo PM, Johnson PJ, Huang DP. Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res. 2002;8:131–137. [PubMed]
21. Kuzmin I, Gillespie JW, Protopopov A, Geil L, Dreijerink K, Yang Y, Vocke CD, Duh FM, Zabarovsky E, Minna JD, Rhim JS, Emmert-Buck MR, Linehan WM, Lerman MI. The RASSF1A tumor suppressor gene is inactivated in prostate tumors and suppresses growth of prostate carcinoma cells. Cancer Res. 2002;62:3498–3502. [PubMed]
22. Honorio S, Agathanggelou A, Wernert N, Rothe M, Maher ER, Latif F. Frequent epigenetic inactivation of the RASSF1A tumour suppressor gene in testicular tumours and distinct methylation profiles of seminoma and nonseminoma testicular germ cell tumours. Oncogene. 2003;22:461–466. [PubMed]
23. Vos MD, Ellis CA, Bell A, Birrer MJ, Clark GJ. Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis. J Biol Chem. 2000;275:35669–35672. [PubMed]
24. Ortiz-Vega S, Khokhlatchev A, Nedwidek M, Zhang XF, Dammann R, Pfeifer GP, Avruch J. The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1. Oncogene. 2002;21:1381–1390. [PubMed]
25. Vavvas D, Li X, Avruch J, Zhang XF. Identification of Nore1 as a potential Ras effector. J Biol Chem. 1998;273:5439–5442. [PubMed]
26. Khokhlatchev A, Rabizadeh S, Xavier R, Nedwidek M, Chen T, Zhang XF, Seed B, Avruch J. Identification of a novel Ras-regulated proapoptotic pathway. Curr Biol. 2002;12:253–265. [PubMed]
27. van Engeland M, Roemen GM, Brink M, Pachen MM, Weijenberg MP, de Bruine AP, Arends JW, van den Brandt PA, de Goeij AF, Herman JG. K-ras mutations and RASSF1A promoter methylation in colorectal cancer. Oncogene. 2002;21:3792–3795. [PubMed]
28. Shivakumar L, Minna J, Sakamaki T, Pestell R, White MA. The RASSF1A tumor suppressor blocks cell cycle progression and inhibits cyclin D1 accumulation. Mol Cell Biol. 2002;22:4309–4318. [PMC free article] [PubMed]
29. Ross DT, Scherf U, Eisen MB, Perou CM, Rees C, Spellman P, Iyer V, Jeffrey SS, Van de Rijn M, Waltham M, Pergamenschikov A, Lee JC, Lashkari D, Shalon D, Myers TG, Weinstein JN, Botstein D, Brown PO. Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet. 2000;24:227–235. [PubMed]
30. Birchmeier W, Behrens J. Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness. Biochim Biophys Acta. 1994;1198:11–26. [PubMed]
31. Takeichi M. Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Biol. 1993;5:806–811. [PubMed]
32. Bremnes RM, Veve R, Hirsch FR, Franklin WA. The E-cadherin cell-cell adhesion complex and lung cancer invasion, metastasis, and prognosis. Lung Cancer. 2002;36:115–124. [PubMed]
33. Reyes-Mugica M, Meyerhardt JA, Rzasa J, Rimm DL, Johnson KR, Wheelock MJ, Reale MA. Truncated DCC reduces N-cadherin/catenin expression and calcium-dependent cell adhesion in neuroblastoma cells. Lab Investig. 2001;81:201–210. [PubMed]
34. Prasad GL, Fuldner RA, Cooper HL. Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene. Proc Natl Acad Sci USA. 1993;90:7039–7043. [PMC free article] [PubMed]
35. Hirota T, Morisaki T, Nishiyama Y, Marumoto T, Tada K, Hara T, Masuko N, Inagaki M, Hatakeyama K, Saya H. Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor. J Cell Biol. 2000;149:1073–1086. [PMC free article] [PubMed]
36. Dilakyan EA, Zhurbitskaya VA, Vinokurova SV, Gureeva TA, Lubkova ON, Topol LZ, Kisseljov FL, Solovyeva NI. Expression of cathepsin L and its endogenous inhibitors in immortal and transformed fibroblasts. Clin Chim Acta. 2001;309:37–43. [PubMed]
37. Premzl A, Puizdar V, Zavasnik-Bergant V, Kopitar-Jerala N, Lah TT, Katunuma N, Sloane BF, Turk V, Kos J. Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases. Biol Chem. 2001;382:853–857. [PubMed]
38. Dhanesuan N, Sharp JA, Blick T, Price JT, Thompson EW. Doxycycline-inducible expression of SPARC/Osteonectin/BM40 in MDA-MB-231 human breast cancer cells results in growth inhibition. Breast Cancer Res Treat. 2002;75:73–85. [PubMed]
39. Chlenski A, Liu S, Crawford SE, Volpert OV, DeVries GH, Evangelista A, Yang Q, Salwen HR, Farrer R, Bray J, Cohn SL. SPARC is a key Schwannian-derived inhibitor controlling neuroblastoma tumor angiogenesis. Cancer Res. 2002;62:7357–7363. [PubMed]
40. Wasylyk B, Hagman J, Gutierrez-Hartmann A. Ets transcription factors: nuclear effectors of the Ras-MAP-kinase signalling pathway. Trends Biochem Sci. 1998;23:213–216. [PubMed]
41. Foos G, Garcia-Ramirez JJ, Galang CK, Hauser CA. Elevated expression of Ets2 or distinct portions of Ets2 can reverse Ras-mediated cellular transformation. J Biol Chem. 1998;273:18871–18880. [PubMed]
42. Paz MF, Fraga MF, Avila S, Guo M, Pollan M, Herman JG, Esteller M. A systematic profile of DNA methylation in human cancer cell lines. Cancer Res. 2003;63:1114–1121. [PubMed]
43. Autuori F, Farrace MG, Oliverio S, Piredda L, Piacentini M. “Tissue” transglutaminase and apoptosis. Adv Biochem Eng Biotechnol. 1998;62:129–136. [PubMed]
44. Chen S, Lin F, Iismaa S, Lee KN, Birckbichler PJ, Graham RM. α1-Adrenergic receptor signaling via Gh is subtype specific and independent of its transglutaminase activity. J Biol Chem. 1996;271:32385–32391. [PubMed]
45. Upchurch HF, Conway E, Patterson MK, Jr, Maxwell MD. Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix bound enzyme. J Cell Physiol. 1991;149:375–382. [PubMed]
46. Gentile V, Thomazy V, Piacentini M, Fesus L, Davies PJ. Expression of tissue transglutaminase in Balb-C 3T3 fibroblasts: effects on cellular morphology and adhesion. J Cell Biol. 1992;119:463–474. [PMC free article] [PubMed]
47. Nakaoka H, Perez DM, Baek KJ, Das T, Husain A, Misono K, Im MJ, Graham RM. Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function. Science (Wash DC) 1994;264:1593–1596. [PubMed]
48. Gal S, Gottesman MM. The major excreted protein of transformed fibroblasts is an activable acid-protease. J Biol Chem. 1986;261:1760–1765. [PubMed]
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