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Molecular Cancer Research 5, 509-520, May 1, 2007. doi: 10.1158/1541-7786.MCR-06-0273
© 2007 American Association for Cancer Research
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Signaling and Regulation

Down-Regulation of Sprouty2 in Non–Small Cell Lung Cancer Contributes to Tumor Malignancy via Extracellular Signal-Regulated Kinase Pathway-Dependent and -Independent Mechanisms

Hedwig Sutterlüty1, Christoph-Erik Mayer1, Ulrike Setinek2, Johannes Attems2, Slav Ovtcharov1, Mario Mikula1, Wolfgang Mikulits1, Michael Micksche1 and Walter Berger1

1 Institute of Cancer Research, Department of Medicine I, Medical University Vienna and 2 Institute for Pathology and Bacteriology, Otto Wagner Hospital Baumgartner Höhe, Vienna, Austria

Requests for reprints: Hedwig Sutterlüty, Institute of Cancer Research, Medical University Vienna, Borschkegasse 8a, 1090 Vienna, Austria. Phone: 43-1-427765244; Fax: 43-1-427765196. E-mail: hedwig.sutterluety{at}meduniwien.ac.at


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non–small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase–induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC. (Mol Cancer Res 2007;5(5):509–20)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Lung cancer is the leading cancer-related death in the industrial world. Lung tumors are classified in small cell and non–small cell lung cancer (SCLC and NSCLC), the latter representing over 75% of all lung tumors. NSCLC are further subdivided into squamous cell carcinoma (SCC), adenocarcinoma, and large cell carcinoma (LCC). All types of lung tumors are associated with poor prognosis, and further progress in treatment strategies is believed to be dependent on a more extensive knowledge on critical pathways involved in tumor development and progression (1).

One of the characteristics frequently linked to human cancer is the deregulation of signal transduction via receptor tyrosine kinases (RTK). RTKs play an important role in the control of fundamental processes, including cell proliferation, migration, metabolism, survival, and differentiation. Due to amplifications and/or mutations of genes involved in signal transmission, these cellular processes turn insensitive to external regulatory signals (2). Several reports describe high frequency of alterations causing hyperactivation of RTK-regulated pathways in NSCLC. For example, the proto-oncogene K-Ras is mutated in 20% to 25% of the lung tumors (3), and mutations in the epidermal growth factor receptor are found in 10% of all NSCLC (4). In addition, overexpression of fibroblast growth factor (FGF)/FGF receptor family members are frequently found in NSCLC cell lines (5). Besides mutations in signal-transmitting genes, enhanced RTK signaling in tumors can be based on deletions or mutations of genes coding for proteins counteracting RTK-mediated signal transduction, thus often representing tumor suppressors (e.g., phosphatase and tensin homologue deleted on chromosome 10; ref. 6).

Recently, a new antagonist of RTK signaling has been identified in Drosophila, which was termed Sprouty (dSpry), because disruption of this gene led to irregular and excessive branching events in tracheal development. This phenotype is almost identical to the one observed by overexpression of the branchless receptor (an FGF orthologue in Drosophila; ref. 7). Subsequent studies have exemplified that Spry proteins, which comprise a family of four members in mammals, have a conserved function as modulators of RTK signaling mainly by interfering with the Ras/mitogen-activated protein kinase (MAPK) cascade in divergent species (8, 9). Additionally, mammalian Sprys were found to play a conserved function during organogenesis by adjusting growth factor–induced remodeling processes particular of branching tissues (10, 11). Multiple reports have shown that impaired Spry functions during embryogenesis cause malformation of specific organs (12-16), which can be rescued by reducing the respective growth factor doses. In line with these data, overexpression of different Spry members was reported to suppress sprouting events during lung development (17, 18) and angiogenesis (11, 19). In vitro, Spry proteins inhibit cell proliferation (20) and migration (21-23) by negatively interfering with RTK-mediated MAPK activation induced by various growth factors (summarized in ref. 8). However, whereas in Drosophila dSpry protein generally interferes with RTK signaling (24), in mammals, expression of Spry rather enhances MAPK activation in response to epidermal growth factor (25, 26). Multiple proteins involved in RTK-mediated signal transduction like Grb2, Raf1, c-Cbl, Shp2, and GAP1 were shown to interact with Spry proteins (27). Based on these findings, several mechanisms suggesting a Spry function via interfering with Ras activation, receptor degradation, and/or Raf kinase induction have been anticipated, but the exact molecular determinants underlying growth factor–specific signal transduction inhibition by Spry proteins are still unclear.

The complexity of this regulatory network is further increased by the fact that Spry proteins themselves are subjected to regulation by RTK-induced signals at multiple levels (28, 29). Levels and activity of Spry proteins are induced in response to diverse growth factors. Expression patterns during embryonic development coincide with known sites of growth factor activity (especially of the FGF family), suggesting involvement in negative feedback loops (30, 31). In tissue culture, Spry protein expression levels are responsive to growth factor–induced signaling. In addition to the transcriptional up-regulation, it has been shown that a growth factor–dependent phosphorylation on Tyr55 in a conserved NH2-terminal region of Spry2 contributes to the inhibitory activity on the MAPK pathway (28, 32). This phosphorylation event also influences localization of the protein and its interaction with c-Cbl and Grb2 (33-35).

Because activation of diverse RTK-mediated signals is known to play major roles in development and progression of NSCLC, and because Spry2 is found to be expressed in normal rodent lung epithelium (36, 37), we hypothesized that Spry2 function might be deregulated in lung cancer. In this study, we show that Spry2 expression is frequently decreased in NSCLC compared with normal bronchial epithelium. Furthermore, we compared the consequences of ectopic Spry2 expression in NSCLC cell lines harboring wild-type (K-Raswt) or mutated (K-RasG12mut) K-Ras. Thus, we show that Spry2 down-regulation in NSCLC can contribute to tumor malignancy by interfering with K-Ras–mediated extracellular signal-regulated kinase (ERK) phosphorylation and another Ras-independent pathway yet to be identified.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Spry2 Expression Is Reduced in NSCLC Tissue
As an initial experiment, we compared Spry1 and Spry2 mRNA expression of normal and malignant tissues by performing reverse transcription-PCR analysis of matched cDNA pairs from human lung tumors and the corresponding bronchial epithelia. As shown in Fig. 1A , Spry2 mRNA was expressed in all tumor samples but was distinctly down-regulated compared with the respective normal lung tissues in four of five cases analyzed. The only sample showing no down-regulation of Spry2 in the tumor was derived from SCLC. In contrast, Spry1 mRNA was generally expressed at higher levels in the tumor compared with the normal lung tissue. Therefore, we conclude that Spry2 but not Spry1 mRNA is consistently down-regulated in NSCLC.


Figure 1
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  		FIGURE 1. Analysis of Spry expression in NSCLC tissues. A. Spry1 and Spry2 mRNA expressions were analyzed by reverse transcription-PCR using total RNA from tumor (T) and corresponding normal lung (N) tissues. ß-Actin was used as internal standard for normalization. The indicated expression ratios (T/N) were calculated after densitometric analysis of the respective pairs using Image Quant 5.0. B. Cells (VL-1) were infected with decreasing amounts of adenoviruses expressing either hSpry1 (left) or hSpry2 (right) and analyzed by Western blot using antibodies against Spry (Upstate Biotechnology) and Spry2 and Spry1 (both raised in our laboratory), as indicated. C and D. Paraffin-embedded sections of normal lung tissue were stained for Spry1 (C) and Spry2 (D; red) counterstained with hemalaun (nuclei in blue). NSCLC were stained using either Spry1 (F), Spry (G-I), or Spry2 (J-L) antibodies and evaluated as described in Materials and Methods. Adenocarcinoma with intermediate Spry2 expression (scored as 2) was stained using a primary antibody recognizing Spry1 (F), Spry (I), or Spry2 (J). As a control, the primary antibody was omitted (E). A representative adenocarcinoma (G and H) and SCC (K and L) showing weak Spry2 expression (scored as 1) and adjacent normal bronchial epithelium with strong Spry2 expression. Selected tumor regions (arrowheads) and normal bronchial epithelium (arrows) are emphasized. Bar, 500 µm.

 
To confirm our observations on protein levels, immunohistochemistry was done. Antibodies recognizing Spry1 and Spry2 were generated and purified. Using immunoblotting, the quality of the antibodies was tested and compared with a commercially available antibody (Fig. 1B). Before analyzing tumor tissue, normal adult lung tissues of control patients with nonmalignant diseases (n = 4) were stained using Spry1 and Spry2 antibodies. As representatively shown in Fig. 1C (Spry1) and Fig. 1D (Spry2), both Spry proteins were strongly expressed in the epithelium of the bronchus, whereas staining was weak in fibroblasts.

To evaluate staining intensities of the malignant tissues with respect to the ones observed in corresponding normal epithelial cells, preferentially, samples from the tumor boundary that included also unaffected healthy tissue were used. In all cases analyzed, normal bronchial epithelium and tumor tissue stained positively for Spry1 and Spry2, whereas in the control slide, in which the primary antibody had been omitted, staining was completely negative (Fig. 1E). Consistent with the expression data obtained by reverse transcription-PCR (Fig. 1A), Spry1 expression was up-regulated in 6 of 10 tumor samples analyzed. Two of the other four samples showed a comparable intensity in staining of the tumor and the adjacent normal epithelium (Fig. 1F). Only 2 of 10 samples revealed down-regulated Spry1 protein levels in the tumor sections. Therefore, we conclude that Spry1 is not commonly down-regulated in NSCLC.

Hence, immunohistochemical staining of Spry2 was done in 25 tissue sections from surgical NSCLC specimens (11 adenocarcinoma, 11 SCC, and 2 LCC). Nineteen of 25 tumor sections stained with a commercially available antibody (Spry) contained also normal bronchial epithelium and thus could be evaluated. In no case was Spry2 expression in the epithelium weaker than in the tumor. In 7 of 19 (37%) cases, Spry2 expression was comparable or only weakly reduced in the tumor sections (score 3), whereas 12 of 19 (63%) tumors displayed a distinctly weaker Spry2 staining compared with the adjacent normal epithelium. Twenty-one percent of the tumors were scored as 1 (Fig. 1G and H), and 42% were scored as 2 (Fig. 1I). To validate the expression analysis, the sections were re-stained with a second Spry2 antibody raised against the NH2-terminal region of hSpry2 (Fig. 1J-L). Staining of the tumor samples with the Spry2 antibody from a second source confirmed that Spry2 is down-regulated in tumor tissues of NSCLC patients.

With regard to histology, 4 adenocarcinoma and 3 SCC scored as 3; 3 adenocarcinoma, 4 SCC, and 2 LCC scored as 2; and 2 adenocarcinoma and 2 SCC had almost completely lost Spry2 expression (score 1). On average, less Spry2 was detected in SCC compared with adenocarcinoma, although the difference was not significant. Regarding tumor stage, no significant differences were found in Spry2 expression. However, it has to be mentioned that only material from patient stage I to IIIb were available for analysis. Additionally, the few (n = 3) tumor samples staged as T4 had clearly reduced Spry2 expression (score 1 and 2). With regard to differentiation, no effect on Spry2 expression level was detectable (mean: G1, 2.2; G2, 2.3; G3, 2.0).

NSCLC Cell Lines with High Levels of Spry2 Expression Harbor Mutations in K-Ras
Spry2 expression levels of 15 NSCLC cell lines were determined by Northern and Western blot analyses. mRNA and protein levels of Spry2 correlated significantly in the investigated cell lines (linear regression, P < 0.001; compare Fig. 2A and B ). Furthermore, we observed that all cell lines derived from metastases (VL-5 to VL-8 and Calu-3) showed particularly low Spry2 expression. In parallel to Spry2, MAPK activity in NSCLC cells was measured using an antibody recognizing phosphorylation of ERK (pERK). No correlation was found between the basic levels of pERK and Spry2 expression in NSCLC cell lines (Fig. 2A).


Figure 2
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  		FIGURE 2. Characterization of NSCLC cell lines. A. Using Western blot, endogenous expression of Spry2 was analyzed in 15 NSCLC cell lines and in human embryonic lung fibroblasts WI38. Equal amounts of protein (150 µg as determined by protein concentration measurement using bovine serum albumin as a standard) were loaded in each lane. Note that none of the common loading controls reflected the protein amounts, but, with the exception in the SK-LU-1 cells, ribosomal protein L4 did. Therefore, protein concentration determination was verified by staining a second SDS-PAGE gel with Coomassie Blue. The resulting Western blots were sequentially probed with the indicated antibodies. Levels of Spry2 protein were quantified by densitometry analysis and normalized to WI38 as indicated. AC, adenocarcinoma. B. Northern blots were used to determine Spry2 mRNA levels in NSCLC-derived cell lines. After densitometric analysis, the Spry2 expression levels were indicated as ratio to 18S rRNA. C. Mutation analysis of K-Ras in NSCLC cell lines was done by PCR, inserting an artificial RFLP (BstXI) discriminating wild-type and mutated codon 12 (G12mut). Note that Calu-6 is mutated at codon 61 (+) as described in ref. 39. D. Western blots of selected cell lines were done to detect Spry2 expression with (+) or without (–) 5-azacytidine (AZA) treatment (10 µM for 3 d).

 
Because Spry expression is reported to be activated by growth factor–induced signals as part of a negative feedback loop, we analyzed the NSCLC-derived cell lines with respect to K-Ras mutations by artificial RFLP (38). Our mutation analysis focused on alterations in codon 12 of the K-Ras oncogene because in NSCLC, more than 80% of the Ras mutations affect this codon (3). As shown in Fig. 2C, 5 of 15 investigated cell lines were mutated at K-Ras codon 12. In addition, Calu-6 is known to harbor a K-Ras mutation at codon 61 (39). K-Ras mutations were found only in cell lines established from adenocarcinoma and LCC, whereas all investigated SCC-derived cell lines were K-Raswt. High levels of Spry2 expression were exclusively detected in cell lines identified as K-Ras mutated (Calu-6, A-427, and VL-4; Fig. 2A and C). However, not all of the K-Ras–mutated cell lines showed high Spry2 expression levels.

To address whether epigenetic inactivation of the promoter is responsible for the down-regulation of Spry2 in NSCLC, all tumor cell lines with reduced Spry2 expression were treated with the demethylating agent 5-azacytidine. As seen in Fig. 2D, only 3 of 12 cell lines (SKLU-1, VL-2, and VL-9) contained slightly elevated Spry2 expression levels after treatment with 5-azacytidine. These data indicate that only in few cases is hypermethylation of the Spry2 promoter involved in reducing Spry2 expression in lung cancer.

Inhibition of MAPK Activation by Spry2 Expression Is Only Observed in Cell Lines with Homozygous Wild-type K-Ras alleles
The adenoviral system was used to analyze the function of ectopic Spry2 expression on MAPK activation in selected NSCLC cell lines. For these experiments, we selected normal embryonic lung fibroblasts (WI38), two NSCLC cell lines homozygous for K-Raswt and three cell lines harboring K-RasG12mut, susceptible for adenoviral infection and distinguishable in expression levels of Spry2 protein (see Fig. 2). First, logarithmically growing cells were infected with control or Spry2-expressing adenoviruses, and 48 h after infection, cells were harvested to investigate the influence of Spry2 on Ras/MAPK activity. Immunodetection of pERK proteins revealed that ectopic Spry2 expression reduced MAPK activity in all cell lines with K-Raswt (Fig. 3A, left ). In contrast, Spry2 failed to inhibit or even enhanced ERK phosphorylation in cell lines expressing K-RasG12mut (Fig. 3A, right). To ensure that inhibition of ERK phosphorylation by Spry2 is restricted to cell lines homozygous for K-Raswt, we repeated the experiments including FGF2. Therefore, cells were infected with the respective adenoviruses and cultured in reduced serum levels (2%) supplemented with FGF2 (20 ng/mL) for 2 days. Resembling logarithmically growing cells, no inhibition of ERK phosphorylation by Spry2 was observed in cell lines harboring K-RasG12mut (Fig. 3A). Next, we studied the influence of Spry2 on ERK phosphorylation following serum stimulation in those cell lines harboring a constitutively active K-Ras (A-549, VL-4, and VL-2). As a control, we included WI38 cells. Cells were serum starved and infected with control or Spry2-expressing adenoviruses. After 2 days, cells were stimulated with 20% serum for 5, 10, 15, or 20 min. Cells were lysed in SDS sample buffer and analyzed by Western blot using the respective antibodies. In all the cell lines expressing K-RasG12mut, ectopic Spry2 expression failed to inhibit ERK phosphorylation, whereas in the WI38 control, MAPK activation was reduced by ectopic Spry2 expression (Fig. 3B).


Figure 3
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  		FIGURE 3. Effect of ectopic Spry2 expression on MAPK activity in selected cell lines. A. Logarithmically growing cells (FCS) as well as cells cultured in reduced serum (2%) supplemented with 20 ng/mL FGF2 (FGF2) were infected with control and Spry2 adenoviruses and analyzed by Western blot 48 h after infection using the indicated antibodies. The indicated ratios of pERK/ERK were calculated after densitometric analysis of the respective pairs using Image Quant 5.0. Ratios of hSpry2-infected cells are given relatively to those controls. B. Cells were serum starved and infected with control and Spry2 adenoviruses, respectively. Two days after serum withdrawal, the cells were stimulated with 20% FCS and analyzed. Representative Western blots showing the influence of Spry2 expression on ERK phosphorylation in WI38 in comparison with the selected cell lines harboring K-RasG12mut (A-549, VL-4, and VL-2).

 
Constitutively Activated K-Ras Overrides the Antagonizing Function of Spry2 on Cell Migration
Next, we tested the influence of Spry2 on cell migration, a known RTK-mediated process, by using scratch assays. Velocity of migration was measured in selected NSCLC cell lines harboring K-Raswt or K-RasG12mut. The migration study with VL-1 was impossible because this cell line underwent apoptosis in response to cell density. As shown in Fig. 4 , Spry2 expression had a pronounced effect on cell migration in K-Raswt WI38 and VL-8 cells (Fig. 4A). In both cell lines, ectopic Spry2 expression potently (2- to 3- fold) prolonged the time to close the gap compared with uninfected cells (data not shown) or cells infected with a control virus (Fig. 4A and B). In comparison, in the three cell lines expressing K-RasG12mut (Fig. 4A), the Spry2-induced reduction in migration velocity was very modest (Fig. 4B). On average, Spry2 decelerated the migration velocity in all three cell lines harboring K-RasG12mut just 1.2- to 1.3 fold, arguing again for the dependency of Spry2 function on a switchable K-Raswt protein.


Figure 4
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  		FIGURE 4. Influence of Spry2 expression on cell migration. A. Cells homozygous for K-Raswt (WI38 and VL-8) were compared with cell lines expressing K-RasG12mut (A-549, VL-4, and VL-2) in a scratch assay (see Materials and Methods). Mean migration velocities are indicated. B. Summary of the data from three to five experiments. Columns, means of the different migration velocities; bars, SD. *, P < 0.05; **, P ≤ 0.01 (Mann-Whitney U test).

 
Spry2 Expression Inhibits Cell Proliferation In vivo and In vitro also in Cells Expressing a Constitutively Active K-Ras
Cell proliferation is another biological process induced by RTK activation. Therefore, we investigated the influence of Spry2 on cell proliferation by generating growth curves and performing clonogenic assays using cell lines homozygous for K-Raswt (Fig. 5A ) or harboring a K-RasG12mut (Fig. 5B). Although infection with control virus had no effect (data of uninfected cells are not shown), ectopic hSpry2 expression significantly reduced NSCLC cell proliferation. This growth-inhibitory function of Spry2 was, in contrast to its ability to fine tune ERK activation and to interfere with cell migration, not restricted to cell lines homozygous for K-Raswt (Fig. 5A).


Figure 5
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  		FIGURE 5. Effect of Spry2 on cell proliferation in vitro and in vivo. Proliferation of cell lines expressing either K-Raswt (A) or K-RasG12mut (B) was determined using growth curve analyses (top) and clonogenic assays (bottom). All growth curves were done at least thrice. For clonogenic assays, representative plates are shown. Columns, means of at least five experiments; bars, SD. *, P < 0.01 (Mann-Whitney U test). Note that VL-2 is less susceptible to the adenoviruses (compare Fig. 3A); thus, after few cell divisions, Spry2 overexpression is lost. Therefore, growth inhibition is obvious within the first 96 h, but afterwards, cell doubling time is comparable. C. Tumor formation of A-549 cells infected with control or Spry2 adenoviruses was analyzed in immunocompromised SCID/BALB/c mice (left). The calculated tumor weight represents a mean value of six mice from two independent experiments (right). Figure 5, control virus; Figure 5, Spry2 virus.

 
Because cell proliferation in all tested cell lines was inhibited by Spry2 expression, we next examined the ability of Spry2 to interfere with tumor formation in vivo (Fig. 5C). In former experiments, A-549 formed reproducibly tumors of comparable size (6 of 6 mice). In addition, A-549 is a well-characterized cell line known to express K-RasG12mut and was therefore chosen for s.c. injection into severe combined immunodeficient mice. Each mouse was injected with 106 cells infected either with Spry2-expressing (right flank) or control virus (left flank). A-549 cells infected with the control virus rapidly formed tumors, whereas cells infected with the Spry2-expressing virus completely failed to do so (Fig. 5C), which was also verified after the mice were sacrificed at day 32 after injection. These data confirm that cell proliferation is inhibited by Spry2 also in cells with constantly active K-Ras and show that Spry2 interferes with tumor formation in vivo.

Expression of a Spry2 Mutant Defective in Antagonizing ERK Activity Inhibits Cell Proliferation Less Potently but Still Significantly
To inhibit Ras/MAPK activation, Spry2 has to be activated by phosphorylation of Tyr55 (Y55; ref. 27). Hence, a mutation of Spry2 protein changing this tyrosine to a phenylalanine (Spry2Y55F) was shown to be defective in inhibiting RTK-mediated ERK phosphorylation. To confirm these data in lung cells, cells harboring either K-Raswt (WI38, Fig. 6A ) or K-RasG12mut (A-549, Fig. 6B) were infected with a control virus or adenoviruses expressing Spry2Y55F or Spry2wt protein. As shown in Fig. 6A, we confirmed that Spry2 mutated at Tyr55 had lost the ability to reduce ERK phosphorylation. Phosphorylation of AKT (pAKT) and ribosomal protein S6, which are signal molecules within the phosphatidylinositol 3-kinase pathway, are neither reduced by Spry2wt protein nor by the mutant Spry2Y55F protein (Fig. 6A and B). As expected for a mutant defective in influencing MAPK activation, Spry2Y55F protein was less effective in inhibiting proliferation of normal lung fibroblasts (Fig. 6C) than the Spry2wt protein. The average doubling time in WI38 cells expressing Spry2Y55F was about 130 h compared with almost 240 h in WI38 cells infected with virus expressing Spry2wt. Nonetheless, overexpression of mutated Spry2Y55F protein clearly reduced cell proliferation in WI38 when compared with cells infected with the control virus (minimal doubling time around 50 h), enforcing the former data that Spry2 inhibits cell proliferation not only by interfering with MAPK activation (compare Fig. 5). In addition, cells with K-RasG12mut (A-549) doubled slower when infected with either Spry2wt or Spry2Y55F (Fig. 6D). Furthermore, we observed that inhibition of cell proliferation in A-549 cells expressing either Spry2Y55F or Spry2wt protein was equal effective during the first 72 h. At later time points, the growth reduction was, like in WI38, more pronounced with Spry2wt protein, suggesting that activation of Spry2 by phosphorylation at Y55 induces an additional inhibitory function also in cell lines expressing K-RasG12mut (Fig. 6D).


Figure 6
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  		FIGURE 6. Effects of mutant Spry2Y55F compared with Spry2wt. Logarithmically growing WI38 cells (A) and A-549 (B) cells were infected with the indicated adenoviruses and analyzed by immunoblotting with the indicated antibodies. The indicated ratios of pERK, pAKT, and pS6 to the respective total proteins (ERK, AKT, and S6) were calculated after densitometric analysis of the respective pairs using Image Quant 5.0. Proliferation of WI38 (C) and A-549 (D) cells infected with control, Spry2wt, and Spry2Y55F adenoviruses was measured by clonogenic assays and growth curve analyses (compare Fig. 5). The clonogenic assays (top) were done six times. Representative plates are shown. Columns, mean numbers of clones formed from 500 cells plated; bars, SD. Colony formation between cells infected with the respective adenoviruses differed significantly in both cell lines. *, P < 0.01 (Mann-Whitney U test). Growth curve analyses (bottom) were done thrice. Points, means; bars, SD.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Autonomous cell growth independent of external signals is a characteristic feature of human cancers. Therefore, changes affecting genes involved in signal transduction are among the most frequent alterations in tumors. In this study, we report that expression of the MAPK inhibitory protein Spry2 is down-regulated in NSCLC, whereas another Spry family member, Spry1, tends to be up-regulated in this tumor type. In accordance with these data, also in hepatoma, Spry2 but not Spry1 expression was shown to be down-regulated in the malignant cells (40). In breast cancer, both Spry1 and Spry2 expression levels are frequently repressed (41), and in prostate cancer, decreases in Spry1 and Spry4 levels contribute to malignant transformation (42, 43). These data indicate that Spry proteins are commonly down-regulated in cancer, but dependent on tumor types, different members of the family are affected.

Several mechanisms may account for the reduced Spry2 expression in NSCLC cells. In agreement with published data describing Spry2 expression in rodents (17, 36), in human nonmalignant adult lungs, Spry2 was primarily expressed in the bronchial epithelium. During malignant progression, many epithelial tumors are supposed to undergo a transition endowing the cancer cell with typical mesenchymal properties to facilitate invasion and metastasis. Spry2 down-regulation in tumors might thus be connected to this dedifferentiation process. Accordingly, cell lines derived from NSCLC metastases tended to be low in Spry2 expression. In addition, in prostate cancer, an inverse correlation between Spry expression and tumor grade was reported (44). In this tissue, low Spry expressions correlated with enhanced methylation of the Spry2 and Spry4 promoters, respectively (43, 44), whereas in NSCLC, only 3 of 12 cell lines exhibited slightly enhanced Spry2 expression following treatment with demethylating agents. Accordingly, in breast cancer and hepatocellular carcinoma, methylation of the Spry2 promoter was unchanged (40, 41). Besides epigenetic silencing, additionally, selection for lower gene doses might be involved in Spry2 repression. In prostate, McKie et al. observed frequently loss of heterozygosity in microsatellite markers flanking the Spry2 gene locus. (44), whereas in hepatocellular carcinoma, no loss of heterozygosity was observed in this region (40). Using comparative genomic hybridization, Luk et al. showed that in 21% of NSCLC cases, the chromosomal region 13q31 harboring the Spry2 gene is underrepresented (45). Some of the NSCLC-derived cell lines investigated in this study have 13q31 underrepresented.3 For example, A-549, VL-8, and VL-10 are among the cell lines that have a lowered 13q31 gene dosage and express low levels of Spry2 (data not shown). Furthermore, Spry expression is suggested to be regulated as part of an autoregulatory feedback loop (27). In agreement with this hypothesis, we observed that only NSCLC cell lines harboring mutated K-Ras express high levels of Spry2. This may explain why, with respect to histologic subtypes, Spry2 levels tend to be higher in adenocarcinoma-derived than in SCC-derived cell lines. K-Ras mutations are more frequent in adenocarcinoma compared with SCC (3). Nevertheless, the mechanisms responsible for down-regulation of Spry2 in NSCLC cancer are not completely elucidated, and ongoing studies in our laboratory will focus on this issue.

To investigate the contribution of Spry2 down-regulation to the malignant phenotype of NSCLC cells, we expressed the protein by an adenoviral approach. Ectopic expression of Spry2 caused inhibition of ERK phosphorylation and decelerated tumor formation, proliferation, and cell migration. However, Spry2-mediated repression of ERK activity was exclusively observed in cells containing only K-Raswt. In agreement with these data, additionally, Gross et al. showed that Spry2 inhibits Raf binding activity of H-Raswt but not of a H-RasR12 mutant in NIH3T3 cells (20). These observations indicate that constitutively active Ras can circumvent Spry2 function in the MAPK pathway regulation. Because initial attempts to immunoprecipitate Spry2 with K-Ras have been unsuccessful (data not shown), we speculate that Spry2 interferes with ERK phosphorylation upstream of K-Ras.

Accordingly, attenuation of cell migration by Spry2 was significantly more potent in NSCLC cell lines expressing only K-Raswt compared with cell lines harboring K-RasG12mut. Previous reports show that Spry and Spry-like Spred proteins inhibit cell migration by interfering with Rac-1 activation or by direct interaction with RhoA, respectively (21, 46, 47). Because both RhoA (through GAP120 and GAP190 interaction) and Rac-1 (via phosphatidylinositol 3-kinase) were shown to be activated by Ras (48), it is possible that Spry2 inhibits cell migration via the same mechanism responsible for inhibition of ERK phosphorylation. Nonetheless, we cannot exclude that Spry2 interferes with mechanisms directly regulating Rac1 and/or RhoA, which could be circumvented by activated Ras.

In contrast to cell migration, cell proliferation was reduced by Spry2 in all tested NSCLC cell lines. These data argue for a Spry2 function in cell proliferation independent of Ras/ERK activation. In agreement with this conclusion, a Spry2 mutant (Spry2Y55F) that was reported to be diminished with respect to MAPK inhibition (28, 32) also significantly attenuated proliferation. Nevertheless, the effect of Spry2Y55F was lower compared with the Spry2wt protein, indicating a cumulating contribution of MAPK-mediated signals. In A-549, harboring K-RasG12mut, Spry2Y55F was also less effective compared with the Spry2wt protein. Because in the K-RasG12mut background, the difference between Spry2wt and Spry2Y55F became visible only from 72 h onwards (Fig. 6D), we hypothesize that activated Ras, with a reported half-life of around 30 h (49), is lost due to protein degradation, and that Spry2wt but not Spry2Y55F might interfere with the activation also of newly synthesized, dominant-active K-Ras. The mechanisms as to how Spry2 achieves inhibition of cell proliferation without affecting ERK phosphorylation are to be clarified. A recent study suggests that Spry2 inhibits proliferation via a mechanism connected to the coinciding elevation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression (50). However, neither AKT nor S6 phosphorylation were reduced by ectopic Spry2. In contrast, pAKT was rather elevated in response to Spry2 overexpression. Similar results were reported by de Alvaro et al. (51). Therefore, we conclude, in accordance with several other reports (8), that also in NSCLC cells Spry2 expression does not inhibit the phosphatidylinositol 3-kinase pathway. The most prominent Ras-independent signal cascades induced through RTK signaling involve generation of the second messengers phosphoinositol triphosphate and diacylglycerol by phospholipase-C (2). Accordingly, studies in Xenopus clearly showed that Spry exerts parts of its inhibitory functions via influencing Ca2+ efflux (52), known to be activated by phospholipase-C (2). In addition, it was shown that phospholipase-C can activate Spry2 expression, suggesting the presence of an alternative autoregulatory feedback loop (53). Studies investigating the influence of Spry2 on Ras-independent signaling are initiated in our laboratory.

In summary, our data show that Spry2 expression is almost generally repressed in NSCLC, and that this alteration contributes to the malignant phenotype by enhancing RTK-mediated processes like cell migration and proliferation. In addition, we show that Spry2 inhibits RTK-mediated signals both upstream of Ras but also by mechanisms independent of the Ras/MAPK signaling axis. Our observations implicate that Spry2 represents a tumor suppressor in NSCLC and suggest Spry2 re-expression as a promising therapeutic strategy against lung tumors even with mutated K-Ras.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Patient Material and Cell Lines
For reverse transcription-PCR analysis, a panel of cDNA samples synthesized from four NSCLC and one SCLC (patient 3) and the corresponding normal tissue from individual patients (BD Biosciences) was used. Concerning the histologic subtypes, patient 1 was diagnosed with LCC, and patients 2, 4, and 5 represent SCC.

Immunohistochemistry was done using tissue sections derived from 25 lung cancer patients who underwent surgical resection due to proven NSCLC at the Otto Wagner Hospital. The pathologic stage of each tumor was classified according to the WHO classifications. All patients had given informed consent. Eleven of 25 tumor samples were derived from clinical stage I patients; 4 of 25 samples were from stage II patients; 6 of 25 samples were from stage IIIa patients; and 3 of 25 samples were from stage IIIb patients. One patient could not be classified. Eleven tumors each were identified as adenocarcinoma and SCC, whereas three tumors were LCC. Surgical specimens of four patients with pneumothorax were used as nonmalignant controls.

Ten of 15 NSCLC cell lines analyzed were established at our institute as described (5): surgical specimens from one histologic confirmed adenocarcinoma (VL-1), seven SSC (VL-3 and VL-5 to VL-10), and two LCC (VL-2 and VL-4) were used at passage numbers between 15 and 30. Six of 10 cell lines (VL-1 to VL-4, VL-9, and VL-10) were derived from primary tumors, and four (VL-5 to VL-8) were derived from lymph node metastases. Additionally, five of the adenocarcinoma cell lines (A-427, A-549, Calu-3, Calu-6, and SK-LU-1) and the normal embryonic lung fibroblasts (WI38 at passage 16) were purchased from the American Type Culture Collection.

Antibodies
Polyclonal rabbit serum to Spry2 was raised against glutathione S-transferase–tagged NH2-terminal 150 amino acids of human Spry2. Rabbit sera against Spry1 were generated using glutathione S-transferase–tagged NH2-terminal 200 amino acids of human Spry1. According to the manufacturer's protocol, purified glutathione S-transferase, NH2-terminal 150 amino acids of human Spry2, and NH2-terminal 200 amino acids of human Spry1 were coupled to CNBr-activated Sepharose (GE Healthcare). The antibodies were affinity purified from rabbit serum by incubation with a glutathione S-transferase affinity column, followed by Spry2 and Spry1 protein affinity columns, respectively. To also deplete hSpry1-specific antibodies, the crude Spry2 antiserum was cycled twice over hSpry1 protein coupled to beads before affinity purification against hSpry2. Antibodies against pERK, pAKT, AKT, pS6, and S6 were purchased from Cell Signaling; Spry and total ERK1/2 were from Upstate; and ß-actin was from Santa Cruz Biotechnology.

Immunohistochemistry
Immunohistochemistry was done as described previously (54) using Fast Red as a chromogen (Ventana) and hemalaun as counterstain. The intensity of immunohistochemical staining was assessed semiquantitatively by two authors (H.S. and C.E.M.). In all cases, both malignant and normal epithelial cells stained positive, the latter consistently showing strong staining intensity. We therefore scored overall staining intensities of malignant cells with respect to the ones observed in corresponding normal epithelial cells: 3, strong positive staining resembling or slightly below that of the normal epithelium; 2, clearly below the normal epithelium but still with distinct immunoreactions; 1, weak to very weak staining.

Reverse Transcription-PCR
Expression levels of Spry2 transcripts in tumor tissues were determined by semiquantitative reverse transcription-PCR procedure as described previously (55), using ß-actin as housekeeping gene. Dynamics of PCR amplification was evaluated at different PCR cycle numbers. For data presentation, 30 cycles for Spry1 and Spry2 and 25 cycles for ß-actin were used. Several controls were included in each experiment. Oligonucleotides are as follows: Spry2, 5'-ATGGAGGCCAGAGCTCAGAGTG-3' and 5'-GTTCAGAGGAGCTGCTGCTGG-3'; Spry1, 5'-GAGAGCATGGTGGAATATGG-3' and 5'-GAGTTAGACCTTGGCAACAG-3'; ß-actin, 5'-GTGGGGCGCAGGCACCA-3' and 5'CTCCTTAATGTCACGCACGATTTC-3'.

RFLP Analysis of K-Ras Mutations in Codon 12
The artificial RFLP was done as described by Nishikawa et al. (38). Briefly, genomic DNA was extracted using QiaAMPBlood kit (Qiagen GmbH), and exon 1 of K-Ras was amplified by PCR. By using the forward primer 5'-ACTGAATATAAACTTGTGGTCCATGGAGCT-3' and the reverse primer 5'-TTTACCATATTGTTGGATCATATTC-3', an artificial BstXI restriction enzyme site was introduced. In the case of K-Raswt, a second BstXI site, including the first two nucleotides (GG) of codon 12, is generated. After restriction digest with BstXI (Roche), the fragment size was analyzed using PAGE gel electrophoresis and staining with ethidium bromide.

Recombinant Adenovirus Generation and Cell Infection
The coding sequence of human Spry2 was amplified by PCR using Pfx Polymerase (Invitrogen) with upstream primer 5'-TAGCGAATTCGGATCCATGGAGGCCAGAGCTCAGAG-3' (Spry2-s) and downstream primer 5'-TAGCGAATTCCTCGAGCTATGTTGGTTTTTCAAAGT-3' (Spry2-as) to add appropriate cloning sites. The Spry2Y55F mutation was introduced by PCR using primers 5'-AATGAATTCACAGAGGGGCCT-3' and 5'-TGTGAATTCATTGGTGTTTCG-3' in combination with Spry2-s and Spry2-as primers. The PCR-amplified DNA fragments were cloned via BamHI/EcoRI into a pADlox plasmid to generate pADlox-Spry2wt and pADlox-Spry2Y55F, and correct cloning was confirmed by sequencing. Recombinant viruses were produced as described (56) by cotransfection of adenoviral DNA and pADlox-Spry2wt and pADlox-Spry2Y55F plasmid DNA, respectively. Empty (pADlox) and control (pADlox-LacZ) viruses were generated using the same procedure. For infection, viruses were diluted in serum-free medium. If not indicated otherwise, adenoviruses were generally used at a multiplicity of infection of 50.

RNA Extraction and Northern Blotting
RNA preparation and Northern blotting procedures followed published methods (57). The 948-bp Spry2 coding sequence was used as a probe for labeling with High Prime Labeling kit (Roche).

Immunoblotting
Protein isolation and immunoblotting were done as described (5). Western blot signals were quantified using Image Quant software (Molecular Dynamics).

Scratch Assay
Cells were infected with the respective adenoviruses, and after 24 h, 2 x 105 cells were seeded into a six-well plate and allowed to grow to 90% confluency. An "X"-shaped scratch was set into the cell monolayers with a sterile pipette tip. Wounded monolayers were then washed thrice to remove cell debris, and comparable proportions of three scratches per experimental group were selected and marked. At the indicated times, still images were taken under a Leica TE100 microscope equipped with a CCD camera, and the gap width was measured using Metamorph 6.1 software (Universal Imaging Corp.) to calculate migration velocity (µm/h).

Growth Curves
Twenty-four hours after infection, 104 cells were seeded into Petri dishes and re-incubated in the appropriate medium containing 10% FCS. Two plates each were counted at different time points (dependent on the cell line) using a Bürker-Türk cell-counting chamber. Every experiment was done at least twice in duplicates.

Clonogenic Assay
Twenty-four hours after infection, 500 cells each were seeded into six-well plates in sextuplicates. Two to 3 weeks later, the cells were stained and fixed with GIEMSA solution (Merck), and colonies were counted.

Tumor Formation in Severe Combined Immunodeficient Mice
Two days after infection with control and Spry2-expressing adenoviruses, 1 x 106 A-549 cells were re-suspended in 100 µL PBS and injected s.c. into immunocompromised SCID/BALB/c recipient mice. The tumor size was periodically determined using a Vernier caliper, and the tumor weight was calculated from tumor size according to the formula: diameter x diameter x length / 2. Thirty-two days after cell injection, the respective areas were surgically removed and fixed in 4% formaldehyde solution. All experiments were done according to the Austrian guidelines for animal care and protection.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Grant support: Herzfelder'sche Familienstiftung, Hochschuljubiläumsstiftung der Stadt Wien grant H-1224/2003, Medizinisch-wissenschaftlicher Fonds des Bürgermeisters der Bundeshauptstadt Wien grant 2483, and FWF grant P17630-B12.

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.

Note: H. Sutterlüty and C-E. Mayer contributed equally to the main findings of the study.

3 C. Pirker, personal communication. Back

Received 8/25/06; revised 2/27/07; accepted 3/12/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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