H-REV107-1 Stimulates Growth in Non-Small Cell Lung Carcinomas via the Activation of Mitogenic Signaling

doi:10.2353/ajpath.2006.051341

Journal List > Am J Pathol > v.169(4); Oct 2006

Am J Pathol. 2006 October; 169(4): 1427–1439.

doi: 10.2353/ajpath.2006.051341.

PMCID: PMC1698850

H-REV107-1 Stimulates Growth in Non-Small Cell Lung Carcinomas via the Activation of Mitogenic Signaling

Irina Nazarenko,^* Glen Kristiansen,^* Sabine Fonfara,^* Raphaela Guenther,^* Cornelia Gieseler,^* Wolfgang Kemmner,^† Reinhold Schafer,^* Iver Petersen,^* and Christine Sers^*

From the Institute of Pathology,^* Charité University Medicine Berlin, Berlin; and Max-Delbrueck-Centrum,^† Berlin, Germany

Accepted July 17, 2006.

This article has been cited by other articles in PMC.

Abstract

H-REV107-1, a known member of the class II tumor suppressor gene family, is involved in the regulation of differentiation and survival. We analyzed H-REV107-1 in non-small cell lung carcinomas, in normal lung, and in immortalized and tumor-derived cell lines. Sixty-eight percent of lung tumors revealed positive H-REV107-1-specific staining. Furthermore, survival analysis demonstrated a significant association of cytoplasmic H-REV107-1 with decreased patient survival. This suggested that H-REV107-1, known as a tumor suppressor, plays a different role in non-small cell lung carcinomas. Knock-down of H-REV107-1 expression in lung carcinoma cells inhibited anchorage-dependent and anchorage-independent growth whereas overexpression of H-REV107-1 induced tumor cell proliferation. Consistent with results of the survival analysis, cytoplasmic localization of the protein was essential for this growth-inducing function. Analysis of signaling pathways potentially involved in this process demonstrated that overexpression of H-REV107-1 stimulated RAS-GTPase activity, ERK1,2 phosphorylation, and caveolin-1 expression in the cell lines analyzed. These results indicate that H-REV107-1 is deficient in its function as a tumor suppressor in non-small cell lung carcinomas and is required for proliferation and anchorage-independent growth in cells expressing high levels of the protein, thus contributing to tumor progression in a subset of non-small cell lung carcinomas.

Lung cancer is one of the most frequent causes of death from neoplastic malignancies worldwide. Although advances in therapy have provided some improvement in overall survival, outcomes remain poor, with less then 15% of patients with lung carcinomas surviving the first 5 years.¹ Numerous genetic alterations such as mutations of the p53, RB, and p16^INK4A tumor suppressor genes have already been identified in lung cancer,² yet the investigation of new molecular markers and candidate genes that might be helpful for therapy is a central focus of research in many laboratories. Genes belonging to the class II tumor suppressors are of particular interest because their down-regulation and inactivation in distinct tumors has a reversible character. Their re-expression can be achieved either via activation of positive regulators or by abrogation of negative regulation, for instance through treatment with pharmaceutical agents.^3–6 Once reactivated, the class II tumor suppressors affect main signal transduction pathways regulating cell differentiation, proliferation, and programmed cell death. For instance, caveolin-1 is down-regulated in breast, ovarian, and small cell lung carcinomas. Its re-expression in cell lines derived from these tumors can be achieved via treatment with 5-aza-2′deoxycytidine and leads to the inhibition of cell growth and survival.^7–10

The H-REV107-like genes comprise a novel class II tumor suppressor family.^11,12 Two members of this family, murine H-rev107-1 and human HRASLS, were demonstrated to be suppressed in tumors and tumor cell lines due to promoter methylation, and their re-expression was observed after treatment with 5′-aza-deoxycytidine.^13,14 In contrast, loss of the human H-REV107-1 gene in ovarian carcinoma cells is the result of a diminished expression of its positive regulator, the interferon-regulatory factor 1 (IRF-1). In these cells, H-REV107-1 expression can be reconstituted on interferon-γ induction and subsequently leads to apoptosis.⁵

In the current article, we describe our analysis of the H-REV107-1 expression in human non-small cell lung carcinomas (NSCLCs) and in a subset of immortalized and tumor-derived cell lines. We investigate a role of H-REV107-1 in the regulation of cell proliferation and identify signal transduction pathways that might be involved in the transmission of the H-REV107-1-dependent effects. Furthermore, we address the question whether expression and distribution of the H-REV107-1 protein correlates with clinicopathological parameters and patient survival.

Materials and Methods

Tumor Samples

Seventy-five tumor samples lsqb]35 adenocarcinomas (ADCs) and 40 squamous cell carcinomas (SCC)] were analyzed using tissue microarray. In addition, 15 ADCs and SCCs and six normal lung samples were immunostained on conventional slides. All patients underwent thoracotomy for tumor resection at the Charité Hospital between 1995 and 2002. No adjuvant radiotherapy or chemotherapy was applied before surgery. Clinicopathological characteristics according to TNM criteria (UICC) are summarized in Table 1. The histopathological diagnosis was established according to the World Health Organization guidelines.¹⁵

TABLE 1

Relationship between Cytoplasmic and Nuclear Expression of H-REV107-1 and Various Clinicopathological Parameters

Tissue Microarray and Statistical Analysis

Tissue microarrays were done as described elsewhere.¹⁶ A complete array consisted of 127 samples derived from either tumor or peritumoral lung tissues. Antigen retrieval was achieved by pressure cooking in 0.01 mol/L citrate buffer, and then an H-REV107-1-specific antibody⁵ diluted in a background reducing buffer (DAKO, Hamburg, Germany) was added. Detection was accomplished using the LSAB-kit (DAKO). Two tissue microarrays were processed in two staining sessions, and the concordance of the staining patterns was controlled. Seventy-five tumor samples could be evaluated on both arrays and were included into statistical analysis. A clinical pathologist (G.K.) examined intensity of the staining and semiquantitatively scored it as absent, moderate, or strong for each case for cytoplasmic and nuclear staining alike. Moderate and strong positive signals were assembled into one group, indicated as positive in further statistical analysis. Fisher’s exact test and Spearman’s rho were used to determine the association of H-REV107-1 expression with clinicopathological parameters. Cumulative survival curves were calculated with data obtained from 51 patients with a 5-year follow-up documentation using the Kaplan-Meier method. Discrepancy in survival time was assessed with the log-rank test, P values <0.05 were considered significant. Multivariate survival analysis was done based on the Cox proportional hazard model. All calculations were performed with the software package SPSS, Version 11.5 (SPSS, Chicago, IL).

Immunohistochemistry and Immunocytochemistry

For immunohistochemistry paraffin sections were cut, mounted on slides, dewaxed, and gradually hydrated. For immunocytochemistry cells were grown on glass slides and fixed in acetone for 20 minutes. All slides were washed and incubated with either H-REV107-1 antibody or with CC16 antibody diluted in the background reducing buffer (DAKO). Detection was accomplished using the LSAB/AP kit (DAKO). The signal was then developed with the fuchsin substrate chromogen system (DAKO), followed by hematoxylin counterstaining. All slides were analyzed by conventional light microscopy using a Leitz DM RBE microscope, objective ×40/070 (Leica, Bensheim, Germany).

Immunofluorescence

For immunofluorescence, cells were grown on glass coverslips. Forty-eight hours after transfection, cells were fixed with 3% paraformaldehyde for 15 minutes, permeabilized using 0.2% Triton X-100 for 1 minute, washed, and incubated with an H-REV107-1 antibody (1:2000) in 1% bovine serum albumin/phosphate-buffered saline for 1 hour and with a secondary anti-rabbit AlexaFluor 594 antibody (Invitrogen, Karsruhe, Germany). Digital images were acquired using a Leica confocal microscope TCS SL (oil objective ×63, 2 ×zoom).

Northern Blot and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Northern Blots and Cancer Profiling Array I (Clontech, Palo Alto, CA) were performed as described.⁵ In brief, total RNA was separated by electrophoresis, blotted onto Hybond N nylon membrane and hybridized with an H-REV107-1-specific ³²P-labeled cDNA fragment. Hybridization was performed at 65°C using the Express Hyb Mix (Clontech). Quantitative evaluation of the Cancer Profiling Array I was performed by phospho-image analysis using a GS250 molecular imager (Bio-Rad, Hercules, CA). RT-PCR reactions were performed using the Access RT-PCR System (Promega, Madison, WI) and optimized by the use of 10 pmol of primers per reaction, 10 ng of template RNA, and 27 cycles for the amplification of PCR products. The sense oligonucleotide 5′-CTACGCAGCGAAATCGAGCC-3′ and the anti-sense oligonucleotide 5′-GTCGCAGGAGCTGGTGTGCAGC-3′ were used to amplify 190 bp of the H-REV107-1 gene. As a control a G3PDH-specific fragment was amplified using primers 5′-GAACGGGAAGCTTGTCATTCA-3′ and 5′-GTAGCCAAATTCGTTGTCATAC-3′. The amount of H-REV107-1 mRNA was semiquantitatively evaluated using the ImageJ freeware (http://rsb.info.nih.gov/ij/).

Plasmids and Short Interfering RNAs (siRNAs)

The HREVFL expression plasmid containing full-length H-REV107-1 has been described earlier.⁵ The HREVdN construct, encoding an HREV107-1 protein lacking 10 N-terminal amino acids, was generated by PCR using HREVFL plasmid as a template, the sense oligonucleotide 5′-GGAGACCTGATTGAGATTTTTCGC-3′ and the anti-sense oligonucleotide 5′-GTCATCGCTGACAGACAGTC-3′. This fragment was subcloned into the pcDNA3.1 expression vector (Invitrogen) and verified by sequencing. The HREV107-1-specific short interfering RNAs (siRNAs) were designed according to the Khvorova algorithm¹⁷ and synthesized using the Silencer siRNA construction kit (Ambion, Austin, TX) The HREVsiRNA1 target sequence is 5′-AACUGCGAGCACUUUGUGA-3′, and the HREVsiRNA2 target sequence is 5′-AAGGCCAUCGUGAAGAAGGAA-3′. As a control, a scrambled siRNA 5′-AAGGCCAUCGUGAAGAAGGAA-3′ was used.

Cell Culture, Transfection, and Proliferation Assay

Human primary bronchial epithelial (HBE) cells and small airway epithelial (SAE) cells were purchased from Clonetics. The bronchial epithelial cell line Y-BE (YP440) was kindly provided by Prof. S. Cheng (Cancer Institute, Beijing, China). The NSCLC cell lines D51, D54, D97, and D117 were derived from primary tumors of patients who underwent surgery at the Charité Hospital in 1997. The COLO699, COLO668, COLO677, and CPC-N cell lines were obtained from the German Collection of Microorganisms and Cell Cultures. All other cell lines were purchased from American Type Culture Collection (Rockville, MD). Cells were cultured either in L15 (Cambrex, Verviers, Belgium), RPMI 1640 (Biocompare, South San Francisco, CA), or Ham’s F12 (Invitrogen) medium supplemented with 10% fetal calf serum, 0.2 mmol/L glutamine, 100 U/ml streptomycin, and 100 U/ml penicillin. For SAE and HBE cell lines, the BEG medium (Cambrex) was used. For transfection, 2 × 10⁵ A549 and D51 cells per well were seeded into six-well plates and transfected with 2 and 4 μg of plasmid DNA, or with 0.5 and 2 nmol/L siRNAs, respectively, using Lipofectamine 2000 transfection reagent (Invitrogen). For proliferation assays 2 × 10³ A549 and D51 cells per well were seeded into 96-well plates. To measure anchorage-independent growth, 96-well plates were precoated with polyHEMA (poly-2-hydroxyethyl methacrylate; Sigma, St. Louis, MO). Cell growth was monitored after 24, 48, 72, and 96 hours using an MTT assay (Roche Diagnostics, Mannheim, Germany), according to the recommendations of the supplier. All assays were performed as hexaplicates and repeated at least once.

Subcellular Fractionation, Immunoblotting, RAS- and Ral-GTPase Activity Assay

For fractionation, cells were harvested in hypotonic lysis buffer: 10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L phenyl-methyl sulfonyl fluoride, and Complete protease inhibitor cocktail tabs (Roche). Crude nuclei were recovered by centrifugation at 3000 × g for 5 minutes and discarded. The membrane fraction was separated from the supernatant by centrifugation at 60,000 × g for 30 minutes and diluted in 1× sodium dodecyl sulfate buffer. To collect cytoplasmic proteins, a methanol/chloroform precipitation was used.¹⁸ Purity of the fractions was controlled by immunoblotting with an antibody against α-tubulin (cytoplasmic marker).

To detect endogenous H-REV107-1 protein, cells were lysed in TNE buffer: 10 mmol/L Tris-HCl, pH7.8, 150 mmol/L NaCl, 1% Nonidet P-40, 1 mmol/L ethylenediaminetetraacetic acid, and Complete protease inhibitor cocktail tabs (Roche). To detect RAS and p-ERK, cells were lysed directly in 2× sodium dodecyl sulfate buffer. The antibodies used included H-REV107-1,⁵ pan-RAS, and caveolin-1 antibody (BD Biosciences, San Jose, CA), phospho-p44/42 MAP kinase, phospho-Akt/protein kinase B (PKB), phospho- focal adhesion kinase (FAK), phospho-epidermal growth factor receptor (EGFR), GST (Cell Signaling Technology, Danvers, MA), FAK, and EGFR (BD Biosciences). A pan-actin antibody (Chemicon, Temecula, CA) and an α-tubulin antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) were used as loading controls. To estimate the amount of active RAS and RalA proteins, we used a RAS-GTPase, and Ral-GTPase activation assays (Chemicon), accordingly. The procedure was performed according to the manual of the supplier. The amount of recovered RAS-GTP was semiquantitatively evaluated using ImageJ freeware.

Results

H-REV107-1 Expression in Normal Lung Epithelium and in NSCLC

Several years ago we described H-REV107-1 as a class II tumor suppressor widely expressed in normal tissues and down-regulated or lost in human ovarian and breast tumors and in tumor-derived cell lines.^5,19 To determine whether H-REV107-1 might play a role in the development of lung cancer, we investigated its expression using the Cancer Profiling Array I (BD Clontech), with the lung sample portion representing 21 cDNA pairs derived from eight ADCs, 11 SCCs, two malignant carcinoid tumors, and their corresponding normal counterparts of individual patients. The histopathological classification of the tumor probes used for cDNA preparation can be viewed at http://www.clontech.com/clontech/techinfo/manuals/PDF/7841–1.pdf. The complete cDNA Array containing 241 cDNA pairs derived from 10 different tumor types and normal tissues is included in Supplemental Figure 1 at http://ajp.amjpathol.org. Semi-quantitative analysis showed increased H-REV107-1 expression in four of eight ADC and 3 of 11 SCC. A re-duction of H-REV107-1 mRNA was observed in four of eight ADC, in 5 of 11 SCC, and in one malignant carcinoid tumor (Figure 1A)

. Because of this variability in H-REV107-1 mRNA expression, we examined H-REV107-1 protein expression in normal bronchial epithelium and in a subset of NSCLCs using conventional immunohistochemistry and a lung tissue microarray.

FIGURE 1

H-REV107-1 expression in human NSCLCs and in normal tissue. A: The Cancer Profiling Array I, representing cDNA probes from matched tumor and normal tissue, was hybridized with H-REV107-1 probe. cDNA represented normal lung tissue is spotted in the upper (more ...)

Immunohistochemical analysis of normal lung bronchial epithelium revealed a predominantly nuclear H-REV107-1-specific staining in the mature luminal layer (Figure 1B

; left, red arrow), whereas the proliferating basal cell layer showed no immunoreactivity (Figure 1B

; left, black arrow). Absorption of the H-REV107-1 antiserum against the specific H-REV107-1 peptide⁵ before immunohistochemistry was used to control for the specificity of the antibody (Figure 1B

, right).

We examined H-REV107-1 expression in 35 ADCs and 40 SCCs assembled on a tissue microarray. Twenty-one tumors (28%) displayed a predominantly nuclear staining (Figure 1C

, left) similar to the staining observed in the mature luminal layer of the normal bronchial epithelium (Figure 1B)

, 27 tumors (36%) displayed cytoplasmic staining (Figure 1C

, right), three tumors exhibited strong staining in both the cytoplasm and the nucleus (4%), and 24 tumors (32%) were H-REV107-1-negative. This was the first evidence that H-REV107-1, characterized until now as a class II tumor suppressor, is expressed in a considerable portion of human lung carcinomas.

The observation that H-REV107-1 is expressed in the differentiated cell layers of normal bronchial epithelium and at the same time is highly expressed in a subset of tumors prompted us to analyze whether other proliferating cells in normal lung tissue harbor H-REV107-1 protein. To address this issue, we examined H-REV107-1 expression in local repopulating cells of bronchiole, in Clara cells (Figure 1D)

. As a marker for Clara cells, we used the CC16 protein (Figure 1D

, left). Homogenous H-REV107-1-specific staining was observed in the cells of bronchiolar epithelium including Clara cells (Figure 1D

, right), but neither direct, nor reverse correlation can be seen between H-REV107-1 and CC16 staining (Figure 1D)

. This suggests that the H-REV107-1 protein can be expressed both in the nonproliferating and in the proliferating cells in normal lung tissue.

Potential Prognostic Relevance of the H-REV107-1 Protein Localization

The pronounced differences observed in the intracellular localization of the H-REV107-1 protein prompted us to test whether the nuclear or cytoplasmic staining might correlate with standard clinical parameters. First, we tested the correlation between cytoplasmic and nuclear H-REV107-1 using Fisher’s exact test and Spearman’s rho linear relationship. Both tests revealed a strong inverse correlation of these two parameters with a P value below 0.001, and Spearman’s rho −0.509. Afterward, we calculated an individual correlation between cytoplasmic or nuclear H-REV107-1 with tumor grading, size, nodal status, and disease stage according to UICC, but no significant correlation was detected (Table 1).

We addressed whether a distinct localization of the H-REV107-1 protein might correlate with the survival time of the patients and found a significant correlation of cytoplasmic H-REV107-1 with shortened survival (24 months versus 41 months, P = 0.0165; Figure 1E

). Other significant prognostic parameters were tumor grading, nodal status, and tumor stage (Table 2). We performed a multivariate progression analysis based on the Cox proportional hazard model to test the individual value of each parameter. Cytoplasmic expression of H-REV107-1 (P = 0.033), nodal status (P = 0.047), and tumor grading (P = 0.025) appeared to be independent prognostic factors for shortened overall survival (Table 3).

TABLE 2

Univariate Survival Analysis (Kaplan-Meier): Survival Times of 51 Patients with Adenocarcinoma or Squamous Cell Carcinoma According to Clinicopathological Parameters or H-REV107-1 Localization

TABLE 3

Multivariate Survival Analysis (Cox Regression Model)

Impact of siRNA-Mediated H-REV107-1 Knock-Down on Anchorage-Dependent and Anchorage-Independent Growth in Lung ADC Cells

To test the hypothesis drawn from the statistical analysis that H-REV107-1 plays a growth stimulatory role, we addressed the consequences of H-REV107-1 knock-down for the proliferation in lung ADC cell lines. First, we analyzed expression of this gene in 34 human lung epithelial and lung cancer cell lines (Supplemental Table 1 at http://ajp.amjpathol.org). H-REV107-1 mRNA was not expressed in the normal SAE and HBE cells, and, among four immortalized bronchial epithelial cell lines, only 9609 cells expressed a low level of H-REV107-1. Fifteen of 16 NSCLC cell lines expressed H-REV107-1 mRNA, but only three of twelve SCLC cell lines revealed an H-REV107-1-specific signal (Figure 2A

; Supplemental Table 1 at http://ajp.amjpathol.org). We have described earlier that expression of the H-REV107-1 protein might be controlled at the posttranscriptional levels.^5,19 Therefore, we examined the presence of H-REV107-1 protein in several cell lines showing a strong H-REV107-1 mRNA signal. We detected low levels of the H-REV107-1 protein in 9 of 12 cell lines analyzed, and four were considered to be negative, confirming that expression of this protein is controlled at both transcriptional and posttranscriptional levels (Figure 2B)

FIGURE 2

H-REV107-1 expression in cell lines derived either from normal bronchial epithelium or from lung tumors. A: Northern blot analysis of H-REV107-1 mRNA in immortalized lung epithelial cell lines and lung carcinoma cell lines. HBE and SAE are primary bronchial (more ...)

Regarding the potential relevance of the H-REV107-1 intracellular localization in human tumors, we addressed the distribution of the endogenous H-REV107-1 protein in cultured cells. Two ADC cell lines, A549 and D51, displaying the highest levels of the H-REV107-1 protein (Figure 2B)

were chosen for this analysis. A549 cells revealed a weak homogenous staining of the H-REV107-1 antigen in the cytoplasm, whereas the nuclei were negative (Figure 3A

, left, enlarged icon). In contrast, immunocytochemical analysis of D51 cells demonstrated a heterogeneous expression of H-REV107-1. It seems that the protein is only detectable in near confluent areas of the culture, whereas subconfluent D51 cells were H-REV107-1-negative (Figure 3A

, middle). It was also remarkable that a fraction of the H-REV107-1-positive D51 cells contained the protein only in the cytoplasm similar to A549, whereas in some cells we observed an additional strong staining of the nuclei (Figure 3A

, middle, enlarged icon). To control for the specificity of the H-REV107-1 antibody, we stained COS-7 cells, which are known to be H-REV107-1-negative, and did not obtain any cross-reactivity (Figure 3A

, right). These data demonstrate that in lung ADC cell lines, H-REV107-1 protein is either localized in the cytoplasm or in both the nuclei and the cytoplasm, exhibiting a strong H-REV107-1-specific signal.

FIGURE 3

H-REV107-1 intracellular localization in A549 and D51 cells and effect of H-REV107-1 suppression on cell proliferation. A: Immunocytochemistry was done using the H-REV107-1-specific antibody. A part of the image was digitally magnified (a small icon in (more ...)

To demonstrate a direct effect of H-REV107-1 on cell proliferation, we suppressed H-REV107-1 expression using RNA interference (RNAi). Two different short interfering RNAs (siRNAs) specific for H-REV107-1 and one unspecific scrambled siRNA were used in this experiment. We monitored growth of the A549 and D51 cells transfected with different combinations of the siRNAs for up to 4 days (Figure 3B

, left). The level of H-REV107-1 mRNA was controlled using RT-PCR (Figure 3B

, right). As shown in Figure 3B

, an individual application of either HREVsiRNA1 or HREVsiRNA2 resulted in a suppression of H-REV107-1 mRNA up to 35%, leading to a moderate inhibition of cell proliferation compared to the transfection with scrambled siRNA in both cell lines (Figure 3B)

. A more effective knock-down effect was achieved by a combination of two H-REV107-1-specific siRNAs. We obtained a re-duction of H-REV107-1 mRNA up to 50% in A549 cells and up to 60% in D51 cells (Figure 3B

, right). This stronger suppression of H-REV107-1 completely abolished growth when compared to the cells transfected with control siRNAs (Figure 3B

, left). Although A549 cells grew significantly more slowly on poly-HEMA compared to D51 cells, in both cell lines the inhibitory effect of the H-REV107-1 knock-down on cell proliferation was clearly observed and suggested a direct correlation between the amount of H-REV107-1 and the capacity of the cells for anchorage-dependent and anchorage-independent growth. This confirmed a role of H-REV107-1 in the regulation of proliferation in lung ADC cells retaining expression of this protein.

Role of the H-REV107-1 Intracellular Localization in the Proliferation-Inducing Effect

We also sought to investigate the effect of an increased level of the H-REV107-1 protein on cellular growth and to determine whether cytoplasmic localization is essential for H-REV107-1 action. Earlier studies had demonstrated that overexpressed H-REV107-1 protein is bound to intracellular membranes via its C-terminal membrane-binding domain. Deletion of this domain led to a loss of the protein from the membrane fraction and its redistribution through the cytoplasm.²⁰ In a recent study, we identified a second functional domain of H-REV107-1 on the N terminus of the protein. Deletion of both the C and the N termini resulted in a diffuse distribution of H-REV107-1 through the nucleus and the cytoplasm (unpublished data). To assess if the N terminus determines the intracellular localization of the protein in lung tumors, we generated a truncated mutant HREVdN lacking 10 N-terminal amino acids. Then we overexpressed the wild-type protein and the HREVdN mutant in A549 and D51 lung ADC cells and performed immunofluorescence analysis to define how the proteins are distributed within the cell. As depicted in Figure 4A

, wild-type H-REV107-1 protein (HREVFL) has an exclusively cytoplasmic localization in A549 cells. In D51 cells, it was concentrated preferably in peripheral area of the cytoplasm and at the cell membrane; in addition, a weak staining of the perinuclear region and of the nucleus was observed (Figure 4A)

. In contrast to the full-length construct, the HREVdN deletion mutant is strictly localized to the perinuclear region in both A549 and D51 cells, confirming a role for the N terminus in intracellular distribution (Figure 4A)

FIGURE 4

Role of the cytoplasmic localization for H-REV107-1 function. Two expression constructs, HREVFL and HREVdN, were transfected into A549 and D51 cells. A: Immunofluorescence was used to analyze the distribution of the ectopically expressed H-REV107-1 wild-type (more ...)

We used the HREVFL and HREVdN constructs to determine whether increased levels of the H-REV107-1 protein will lead to a higher proliferation rate of lung tumor cells and if cytoplasmic localization might play a role this effect. We transiently transfected A549 and D51 cells with HREVFL and with the HREVdN expression constructs and monitored cellular growth during 4 days. As a control, we used the pcDNA3.1 plasmid. Again, A549 cells had a lower proliferation rate on the poly-HEMA-coated plates compared with D51 cells (Figure 4B

, bottom). Overexpression of the H-REV107-1 wild-type protein led to a significant increase of anchorage-dependent and anchorage-independent growth, whereas the HREVdN mutant had no effect on cell proliferation compared to pcDNA3.1 (Figure 4B)

. These observations confirm that high expression of H-REV107-1 leads to an increased proliferation of lung tumor cells and that the localization of the protein within the cells plays a role.

Regulation of Main Signal Transduction Pathways by Overexpressed H-REV107-1 in NSCLC Cells

Next, we intended to explore the underlying mechanisms, by which H-REV107-1 induces proliferation in NSCLCs. We investigated expression and phosphorylation of EGFR known to be frequently activated via amplification and mutation in NSCLCs.^21–24 As shown in Figure 5A

, EGFR is expressed in both cell lines. The low levels of phosphorylated EGFR (Tyr845) and a weak inhibitory effect of H-REV107-1 in A549 cells (Figure 5A

, left) suggest that H-REV107-1 induces cell proliferation independent of EGFR.

FIGURE 5

Effect of the H-REV107-1 overexpression on RAS, MAPK, and caveolin-1 expression and on RAS activity. A: Western blot analysis was used to examine the levels of total and phosphorylated EGFR after transfection of A549 and D51 cells with full-length HREV107-1 (more ...)

Because RAS oncogenes are activated by mutations in ~35% of NSCLCs²⁵ and a KRAS V12 mutation was identified in A549 cells,²⁶ we analyzed the status of KRAS in D51 cells and identified a V12 mutation. To define the proportion of activated RAS in A549 and D51 cells, we fractionated the protein extracts and analyzed the amounts of RAS in the cytoplasmic and in the membrane fractions, assuming that active RAS proteins are attached to the cell membranes.^27,28 As evident from Figure 5B

, most of the intracellular RAS is in the membrane fraction and migrates as a double band on a polyacrylamide gel (Figure 5B

, top). This observation indicates that A549 and D51 cells have a fraction of activated RAS proteins targeted to the membrane, and a smaller pool of inactive cytoplasmic RAS. Because secondary modifications such as farnesylation and palmytoylation are obligatory for a RAS membrane targeting,^29,30 the obtained double band in the membrane fraction might be reasoned by the presence of wild-type and mutated RAS isoforms, which are characterized by different electrophoretic mobility.³¹ To test this hypothesis, we treated A549 and D51 cells with farnesyl transferase inhibitors FTI-227 and FTP inhibitor II (Chemicon). The results, presented in Supplemental Figure 2 at http://ajp.amjpathol.org, demonstrate that the treated cells maintain two RAS-specific bands with lower electrophoretic mobility, corresponding to unmodified wild-type and mutated RAS proteins.³²

We then overexpressed full-length (HREVFL) and its deletion mutant (HREVdN) in both cell lines and analyzed the amount of total RAS (Figure 5C

, top). In HREVFL-transfected cells, the level of RAS proteins was slightly higher compared to the HREVdN- and pcDNA3.1-transfected cells (Figure 5C

, top). We also controlled the activation of RAS downstream pathways PI3-kinase, RalA, and RAF-MAPK.²⁵ No difference in RalA protein levels was seen after H-REV107-1 overexpression (Figure 5C)

, whereas a slightly higher amount of phosphorylated ERK1,2 was observed after HREVFL transfection in both cell lines (Figure 5C)

. In A549 cells, phosphorylated Akt/PKB was detected independent of H-REV107-1, whereas in D51 cells Akt/PKB seemed activated after H-REV107-1 overexpression (Figure 5C)

. Recently, a role of caveolin-1 (Cav1) in the regulation of survival and growth of NSCLC cells has been established.¹⁰ Similar to our observations of H-REV107-1, high expression of caveolin-1 is associated with a poor prognosis of patients with ADCs and SCCs.^33,34 Therefore, we tested if H-REV107-1 might regulate expression of caveolin-1. Indeed in D51 cells, but not in A549 cells, increased levels of Cav1 protein were observed after H-REV107-1 overexpression (Figure 5C)

. We next assessed the effect of forced H-REV107-1 expression on the activation of FAK because caveolin-1 was shown to play a role in integrin-mediated FAK activation.^10,35 In D51 cells exogenous full-length and also the N-terminally truncated H-REV107-1 increased the levels of total and phosphorylated FAK (Figure 5C)

. This indicates that although H-REV107-1 overexpression has an influence on FAK activation, it is rather unlikely that FAK is involved in H-REV107-1-mediated growth regulation because only full-length H-REV107-1 was able to induce cellular growth and Cav1 expression. These results suggest that MAPK signaling cascade, caveolin-1, and Akt/PKB might contribute to the H-REV107-1-dependent activation of proliferation in a subset of lung ADC cells.

To determine whether the moderate effect of H-REV107-1 on RAS expression in D51 and A549 cells might also influence RAS activity, we performed a RAS-GTPase activation assay (Figure 5D)

. In addition, we tested whether H-REV107-1 might influence RalA activity, without changing its expression level, because caveolin-1 was described to regulate RalA activity in prostate cancer cells.³⁶ For the assays, we used A549 and D51 cells transfected with HREVFL, HREVdN, and pcDNA3.1 plasmids. The active, GTP-bound form of RAS was recovered from the cells via co-immunoprecipitation using commercially available Raf1-RBD (Raf1-RAS binding domain)-coupled agarose slurry (Upstate). For the recovery of activated RalA, we used RalA-BP1 (RalA binding protein 1)-coupled agarose slurry (Upstate). A more than twofold RAS activation was observed 48 hours after transfection in the presence of the full-length H-REV107-1 protein in A549 cells (Figure 5D

, left). In contrast, only a moderate increase of RAS activity was monitored after H-REV107-1 transfection in D51 cells (Figure 5D

, right). No difference in RalA activity was seen in either cell lines (Figure 5D

, middle). These results confirm that H-REV107-1 may regulate expression and activity of RAS proteins in both ADC cell lines, albeit in A549 cells the effect of H-REV107-1 on RAS activity is obviously stronger.

Discussion

Members of the H-REV107 family have been described to be down-regulated in tumors and to exert a tumor-suppressing or differentiation-inducing potential.^5,37 In contrast to these observations, our current study suggests that H-REV107-1 might act to favor cellular growth in a subset of NSCLCs. Interestingly, analysis of the H-REV107-1 expression using the Cancer Profiling Array I shows increased H-REV107-1 cDNA levels not only in lung but also in colon, stomach, and rectum cancer, suggesting that also in these tumors H-REV107-1 does not exert a growth-suppressing function.

To assess a role of H-REV107-1 in NSCLCs, we suppressed H-REV107-1 expression in two positive cell lines using RNAi and found that the reduced levels of H-REV107-1 correlate with the inhibition of anchorage-dependent and anchorage-independent growth. On the contrary, overexpression of wild-type H-REV107-1 increases cell proliferation and anchorage-independent growth, supporting the idea that the protein plays a growth-promoting role in NSCLCs.

Opposite regulation and function in different tumors and tumor subtypes have already been described for other tumor suppressors. The serine protease inhibitor maspin suppresses breast cancer progression in vivo and in vitro,^38–40 yet in estrogen receptor-positive breast cancer, high levels of maspin correlate with shorter survival time.⁴¹ Caveolin-1 acts as a growth suppressor in breast and ovarian cancers,^7,42 but induces proliferation in NSCLCs¹⁰ and in prostate cancer cells.³⁶ It is remarkable that in lung tumors, the growth-promoting effect of caveolin-1 is restricted to NSCLCs, whereas in small cell lung carcinomas (SCLCs), the protein is down-regulated and has retained its tumor-suppressing properties.¹⁰ Such a differential expression and function of an individual gene in NSCLCs versus SCLCs is known for other tumor suppressors. For instance, the pRB tumor suppressor is absent in 90% of SCLCs but only 10 to 15% of NSCLCs⁴³; PTEN is mutated in ~30% of SCLCs but only 5% of NSCLCs.^44,45 Similar to these observations, H-REV107-1 was expressed in NSCLCs and NSCLC cell lines but lost in most SCLC cell lines analyzed. This indicates a potentially different role of H-REV107-1 in these two types of lung cancer. In view of the differences between SCLCs and NSCLCs, we restricted our current analysis of H-REV107-1 expression and function to NSCLs, whereas an impact of H-REV107-1 on cell proliferation in SCLCs remains to be elucidated.

The immunohistochemical analysis of normal and malignant lung tissues revealed a distinct distribution of the H-REV107-1 protein in normal bronchial epithelium, in Clara cells, and in lung carcinomas. In normal lung epithelium, H-REV107-1 was localized preferably to the nuclei of the nonproliferating mature luminal layer, whereas the proliferating basal cells remained negative. This suggested a role of the protein in the differentiation of lung bronchial epithelium. This assumption is further supported by the observation that in cell lines derived from the proliferating normal bronchial epithelium such as SAE and HAEB, H-REV107-1 was hardly detectable even at the mRNA level. Although a differentiation-inducing function for H-REV107-1 has not been reported, the H-REV107-1-related protein TIG3/RIG1 promotes keratinocyte terminal differentiation via the activation of type I transglutaminase.⁴⁶ Interestingly, in bronchiolar epithelium H-REV107-1 revealed a uniform staining that was detectable mainly in the nucleus. Additional staining with the Clara cell marker CC16 suggests that H-REV107-1 is expressed in Clara cells, characterized as a major progenitor cell population likely to represent airway stem cells.⁴⁷ Recently, a pulmonary stem cell population termed bronchioalveolar stem cells (BACS) was identified in mice. The authors suggested that these cells act as progenitor cells for Clara cells and alveolar epithelial cells type II (AT2) and demonstrated a direct association of BACS with lung tumorigenesis in vivo.⁴⁸ Although Clara cells show a characteristic staining pattern in bronchial epithelia, H-REV107-1 is evenly distributed and expressed in all cells, indicating that Clara cells are likely to express H-REV107-1, too. Furthermore, we investigated the presence of the Clara cell marker in A459 and D51 cells. A549 cells were described earlier to exert features of the type II alveolar epithelial cells⁴⁹ and did not express CC16. In contrast, D51 cells stained positive for the Clara cell marker, suggesting that D51 cells are of Clara cell origin (data not shown). These observations suggest to us that the H-REV107-1 protein might have a function in stem cells of the lung, yet this hypothesis requires a more detailed analysis of the H-REV107-1 expression in normal lung and during lung development.

In addition, H-REV107-1 might act in a different way depending on the intracellular localization of the protein. A nuclear localization typical for the differentiated layers of bronchial epithelium is linked to a growth-suppressing function, whereas a cytoplasmic localization might be responsible for a growth-promoting effect. This hypothesis is supported by data obtained from the tumor samples and from the investigation of the H-REV107-1-induced modifications in signaling cascades. Within the NSCLCs, we observed either nuclear or cytoplasmic distribution of the H-REV107-1 protein. Only 3 of 75 cases had a strong H-REV107-1 signal in both the nucleus and the cytoplasm. The patients with cytoplasmic H-REV107-1 seemed to face an unfavorable prognosis compared with those harboring the nuclear protein. Statistical analysis revealed that the cytoplasmic distribution of the H-REV107-1 protein could be used as an independent prognostic marker. Given the fact that overexpressed H-REV107-1 stimulates RAS activation and caveolin-1 expression in the cell lines, one would assume that cytoplasmic H-REV107-1 acts as a positive regulator of signaling; however, nuclear H-REV107-1 does not have access to the same effectors. A similar connection between the biological function and the subcellular localization was recently reported for maspin, whose nuclear localization, as opposed to a combined nuclear and cytoplasmic distribution, has been associated with increased survival of NSCLC patients.^50,51 In addition, other groups demonstrated that nuclear localization of maspin contributes to a favorable prognosis in malignancies arising from breast and ovary, in contrast to cytoplasmic maspin that was correlated with a high tumor grade and shorter overall survival in ovarian carcinoma patients.^52,53

A possible mechanism underlying different intracellular localizations of a protein has been detected in the tumor suppressor gene encoding p53. An Arg283His mutation was identified as the basis of an aberrant cytoplasmic localization, leading to a modest transforming activity of the protein in primary rodent fibroblasts.⁵⁴ We examined whether H-REV107-1 might be mutated in lung cancer cell lines yet we detected only wild-type H-REV107-1 cDNA, suggesting that other unknown mechanisms are responsible for the regulation of H-REV107-1 intracellular distribution.

Previously we described that the endogenous H-REV107-1 protein is hardly detectable in proliferating cells.^5,19 In contrast, we now found H-REV107-1 protein in six of nine NSCLC cell lines expressing H-REV107-1 mRNA. This observation is consistent with the idea that H-REV107-1 is deficient in its role as a tumor suppressor in NSCLCs. Whether H-REV107-1 can function in a growth-promoting or growth-inhibiting manner dependent on the cell type and whether these different functions directly correlate with its intracellular distribution are currently unclear. Regarding the very low levels of the H-REV107-1 protein in some NSCLC cell lines, we cannot exclude that H-REV107-1 has simply lost its function here and is not required for growth.

When we analyzed expression and activity of growth regulators known to play a role in lung cancer development, we detected an influence of H-REV107-1 onto distinct components governing survival-stimulatory signaling. In NSCLCs, EGFR is frequently activated via mutations in the tyrosine kinase domain or via gene amplification.^21,22 Although both the A549 and D51 cell lines expressed EGFR, we detected only low levels of phosphorylated receptor, suggesting that either other members of the EGFR family such as HER2 and HER3 or its downstream effectors such as RAS are activated in these cells. Consistently, KRAS and ERK1,2 are frequently activated in lung cancer.^25,55 In both A549 and D51 cells, KRAS is mutated in the V12 position.²⁶ Most interestingly, these cell lines differ in their response to H-REV107-1 overexpression. Although H-REV107-1 strongly induced RAS activity in A549 cells, the effect on RAS activation was considerably weaker in D51 cells and also ERK activation was higher in A549 cells expressing H-REV107-1 compared with D51 cells. This suggests that alternative, RAS-independent signaling cascades might be activated in lung ADC cells in response to H-REV107-1 overexpression. Thus, in D51 cells, it is likely that caveolin-1, recently described as a positive regulator of cell proliferation in NSCLCs,¹⁰ might partially mediate the effects of H-REV107-1 overexpression. Consequently AKT/PKB, known to be activated via caveolin-1-dependent inhibition of PP1 and PP2A phosphatases,³⁶ was activated in D51 but not in A549 cells after forced H-REV107-1 expression. Another caveolin-1 effector, FAK,¹⁰ does not seem to be involved in H-REV107-1-dependent growth regulation.

These results demonstrate that H-REV107-1 activates different signal transduction pathways in lung ADC cells effecting their growth capacity. Whether such targeting of distinct pathways by H-REV107-1 in A549 and D51 cells is attributable to the cells’ origin as alveolar type II and Clara cells, respectively, cannot be determined at the moment. The observed RAS activation, phosphorylation of ERK1,2, Akt/PKB, and up-regulation of caveolin-1 were detectable only 48 to 72 hours after transfection, suggesting that H-REV107-1 is not a direct regulator of these proteins. Further experiments are now required to identify the H-REV107-1 effectors mediating the proliferation-promoting effect of H-REV107-1 in lung carcinomas and to understand the impact of intracellular localization on its function.

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Acknowledgments

We thank Britta Beyer, Yu Yongwei, Danny Bashin, Anne Siekhaus, Kristin Lucht, and Natalia Asselborn for technical support.

Footnotes

Address reprint requests to Christine Sers, Institute of Pathology, Charité Universitaetsmedizin Berlin Schumannstr. 20/21, D-10117 Berlin, Germany. E-mail: christine.sers/at/charite.de.

Supported by the Deutsche Krebshilfe (grant 10-1803 to C.S.) and by the Berliner Programm zur Frauenfoerderung im Bereich der Natur- und Technikwissenschaften (to I.N.).

Supplemental material for this article can be found on http://ajp.amjpathol.org.

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