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[Cancer Research 65, 7660-7665, September 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Angiotensin II Type 2 Receptor Gene Deficiency Attenuates Susceptibility to Tobacco-Specific Nitrosamine-Induced Lung Tumorigenesis: Involvement of Transforming Growth Factor-ß-Dependent Cell Growth Attenuation

Tsutomu Kanehira, Tatsuo Tani, Tetsuo Takagi, Yuichirou Nakano, Eric F. Howard and Masaaki Tamura

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee

Requests for reprints: Masaaki Tamura, Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, 210 Coles Hall, Manhattan, KS 66506. Phone: 785-532-4825; Fax: 785-532-4557; E-mail: mtamura{at}vet.KSU.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To clarify an involvement of angiotensin II signaling in lung neoplasia, we have examined the effect of angiotensin II receptor deficiency on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)–induced lung tumorigenesis. Male angiotensin II type 2 receptor (AT2)-null mice with an SWR/J genetic background and control wild-type mice were treated with NNK (100 mg/kg, i.p.) or saline vehicle. NNK treatment caused the development of lung tumors in all wild-type control mice (100 % tumor prevalence), but only 85% of AT2-null mice developed tumors. The tumor multiplicity in AT2-null mice (1.9 ± 0.3) was significantly smaller than that in wild-type mice (4.1 ± 0.9). Primary cultured lung fibroblasts prepared from both AT2-null and wild-type mice markedly increased the colony counts of A549 lung cancer cells in soft agar, but a consistently higher colony count was observed with the wild-type fibroblasts (fold increase in colony number, 5.6 ± 0.5) than with the AT2-null fibroblasts (3.5 ± 0.8). The underlying mechanism by which angiotensin II regulates cancer cell growth is due to the regulation of active transforming growth factor-ß (TGF-ß) production. Although the total level of TGF-ß was significantly stimulated when A549 cells were cocultured with either type of fibroblasts, the level of active TGF-ß in the conditioned medium was consistently higher with AT2-null fibroblasts than with wild-type fibroblasts. These results imply that the AT2 receptor negatively regulates the level of active TGF-ß and thus increases NNK-induced lung tumorigenesis. The AT2 receptor function in lung stromal fibroblasts may be a potential modulator of tumor susceptibility in chemical carcinogen-induced lung tumorigenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It has been suggested that angiotensin-converting enzyme (ACE) inhibitors that are commonly employed in the treatment of human clinical hypertension also attenuate human cancer cell growth in experimental animals (1–5) and may reduce the risk of several human cancers (6). ACE inhibitors block the formation of angiotensin II, an octapeptide that exerts the many diverse effects of the renin-angiotensin system through its receptors (7). The angiotensin II type 1 (AT1) receptor blocker also reduces lung metastasis in mice (8, 9). This suggests that angiotensin II may have a modulating role in lung neoplasia.

The renin-angiotensin system is one of the phylogenetically oldest hormone systems that has been conserved throughout evolution and plays a key role in the regulation of cardiovascular homeostasis (10). The peptide hormone angiotensin II is the active compound of the renin-angiotensin system, which maintains arterial blood pressure and fluid and electrolyte homeostasis (11). There are two well-defined receptors of angiotensin II (AT1 and AT2; ref. 12). The major isoform AT1 receptor is expressed in a wide variety of tissues (12). The AT1 receptor–mediated signal affects a variety of pathophysiologic reactions, including constriction of blood vessels; secretion of mineralocorticoids; expression of proto-oncogenes such as c-fos, c-myc, and c-jun; and promotion of cell proliferation (13, 14). It also stimulates neovascularization (15, 16) and production of growth factors and cytokines such as transforming growth factor-ß (TGF-ß; ref. 17), vascular endothelial growth factor (15–18), and fibroblast growth factor-2 (FGF-2; ref. 19), all of which are tightly associated with tumor growth. The AT2 receptor, the second major isoform of the angiotensin II receptor, is expressed in a smaller quantity but is inducible and apparently functional under pathophysiologic conditions (20–23). The AT2 receptor frequently mediates signals that counteract the AT1 receptor–mediated biological actions (24, 25). This receptor has been cloned and sequenced (26, 27), but its definitive physiologic functions have yet to be assigned.

We have previously reported that the AT2 receptor possesses an oncogenic function through the regulation of the initiation of azoxymethane-induced colon tumorigenesis in mice (28). Because the lung is a major site of angiotensin II generation and also a target tissue for angiotensin II, it is of interest to examine whether angiotensin II signaling is also involved in carcinogen-induced lung tumorigenesis.

In the present study, we examined the effect of AT2 receptor deficiency (AT2-null) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)–induced lung tumorigenesis in mice. In addition to the in vivo study, we also studied the effect of lung stromal fibroblasts, which were prepared from either AT2-null or control wild-type mice, on human cancer cell growth in vitro. These studies revealed that angiotensin II AT2 receptor signaling plays an important role in NNK-induced lung tumorigenesis through attenuation of TGF-ß activation in lung stromal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Angiotensin II and [Sar1, Ile8] angiotensin II were purchased from Peninsula Laboratories (Belmont, CA). The AT2 receptor blocker PD123,319 and the AT2 receptor agonist CGP42112A were from Sigma Chemical Co. (St. Louis, MO). [125I]Na and [3H]thymidine were from New England Nuclear (Boston, MA). Losartan was a gift from DuPont (Wilmington, DE). Collagenase A and DNase 1 were from Boehringer Mannheim (Indianapolis, IN). Trypsin was from ICN Biomedical, Inc. (Costa Mesa CA). Mink lung epithelial cells (MLEC) stably transfected with the human PAI-1 gene fused to the firefly luciferase reporter gene were prepared by Dr. D. Rifkin (Department of Cell Biology, New York University; ref. 29) and were generously provided as a gift. Culture media and primers for PCR were from the DNA Synthesis and Reagent Supply Core facility in the Vanderbilt University Diabetes Center. All other chemicals were of analytic grade.

Animals and genotyping. The original male hemizygote AT2-null mutant (Agtr2–/y) mice were the offspring of Agtr2 deletion mutants produced by homologous recombination in embryonic stem cells derived from strain 129/Ola (30). Female offspring from chimeric males and C57BL/6J females were backcrossed with SWR/J males for 10 generations such that the genetic background of the mice is susceptible to NNK-induced lung tumorigenesis. Wild-type male littermates served as controls. Southern blot analysis of tail DNA was used to evaluate for the Agtr2 genotype as previously described (30). In addition to Southern blot analysis, PCR-based Agtr2 genotyping was also done as a quick genotyping procedure. Results from both procedures showed 100% matches. In brief, published sequences (26, 30) were used to synthesize primers for the AT2 receptor (forward 5'-CACCAGCAGAAACATCAC-3' and reverse 5'-CCAAACAAGGGGAACTAC-3') and the neomysin resistant (Neo-r) gene product (forward 5'-AGCCAACGCTATGTCCTGAT-3' and reverse 5'-AGACAATCGGCTGCTCTGAT-3'). Extracted tail DNA (10-20 ng) was amplified (35 cycles) at 95°C for 1 minute (denaturation), at 58°C for 1 minute (annealing), and at 72°C for 1 minute (elongation) with 0.5 µmol/L of each primer, 1.25 units DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN), and 0.2 mmol/L deoxynucleotide triphosphates in PCR buffer (Roche Molecular Biochemicals). PCR products of the AT2 receptor (478 bp) and Neo-r gene product (593 bp) were visualized by 1% agarose gel electrophoresis. AT2 (+) and Neo-r (–), AT2 (+) and Neo-r (+), and AT2 (–) and Neo-r (+) were assigned as wild type, heterozygote, and AT2-null, respectively. All animals were maintained in a humidity- and temperature-controlled room on 12-hour light/dark cycles. All procedures for handling animals were approved by the Institutional Committee for Animal Care and Use of Vanderbilt University.

Experimental protocol for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone administration in vivo. All NNK-handling procedures were approved by the office of Safety and Environmental Health of Vanderbilt University. Five-week-old male wild-type and AT2-null mice (minimum six mice per group) received regular mouse chow (#5015, Purina Mills, Inc., Indianapolis, IN). Mice were treated with bolus i.p. administrations of NNK (100 mg/kg). The control group for the NNK treatment received saline. Mice were sacrificed 20 weeks after the NNK treatment. After macroscopic examination, the whole lung was stained by injecting India Black ink solution through the trachea and fixed with Fekete's solution (31). The tumor burdens were evaluated by counting tumor number in the lung under a dissection microscope. A single observer throughout the study carried out the measurements blindly. All of the tumor-bearing lung tissues were fixed with 10% formalin, sectioned, and stained with H&E for histologic examination.

Cells. Mouse lung fibroblasts (MLF) were isolated from 4-week-old AT2-null and wild-type mice according to the method of Shannon et al. (32), with slight modifications. In brief, MLF were prepared by 0.2% collagenase and 0.05% trypsin digestion and were cultured in DMEM/Ham's F-12 medium (1:1) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C with 5% CO2. MLF between passage numbers one to three were used for the study. A549 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in Ham's F-12 medium with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin and incubated at 37°C. A549 cells between passages 3 and 10 were used for the study.

Coculture in soft agar. MLF were grown in a 35-mm tissue culture dish. Once MLF were grown to ~30% confluency, 1 mL 0.8% agar in Ham's F-12 medium was poured into the dish (bottom layer). A549 cells (1 x 105 cells per dish) were suspended in 1 mL of Ham's F-12 medium containing 0.4% agar and plated on top of the bottom agar layer. The cells were incubated at 37°C with 5% CO2 for 8 days for growth of colonies. Colonies of >50 µm were counted by an automated colony counter (Biologics, Inc., Gainesville, VA).

Coculture in plates. MLFs were grown in the bottom wells of 6-well transwell plates. Once MLFs attained 70% to 80% confluency, the medium was replaced with 2.5 mL of fresh Ham's F-12 medium. A549 cells (1 x 105 cells per dish) were seeded in the cell culture inserts with a pore size of 3 µm and a pore density of 8.0 x 105 pores/cm2 (Becton Dickinson, Franklin Lakes, NJ), allowing the bidirectional diffusion of molecules but not the migration of cells and incubated at 37°C with 5% CO2. Culture medium was collected after 48 hours of coculturing and stored at –75°C until use.

Luciferase assay for transforming growth factor-ß. The luciferase assay for determination of TGF-ß levels was done as described previously (29). MLECs stably transfected with the firefly luciferase reporter gene-fused PAI-1 promoter (1.6 x 104 cells per well in 96-well plate) were used for the determination of active TGF-ß. Total levels of secreted TGF-ß in the conditioned media were determined by activating latent TGF-ß by heating media at 80°C for 10 minutes. The luciferase activity in MLEC lysates was analyzed for TGF-ß content using a Monolight 3010 luminometer (PharMingen, San Diego, CA). Porcine TGF-ß1 (R&D Systems, Minneapolis, MN) was used as a standard.

[3H]Thymidine incorporation assay. The [3H]thymidine incorporation assay in the presence or absence of serum was done as described previously (33).

Radioligand receptor–binding assay. The radioligand receptor–binding assay was done by using intact cultured cells and either [125I][Sar1, Ile8] angiotensin II in the presence of PD123,319 for the angiotensin II AT1 receptor or [125I]CGP42112A for the angiotensin II AT2 receptor (34). The [125I]-labeled peptides were separately prepared from [Sar1, Ile8] angiotensin II or CGP42112A and [125I]Na by the lactoperoxidase method (34). Specific binding was normalized by the protein quantity.

Statistical analysis. Data obtained from the in vivo study, colony counting, luciferase reporter assay, and [3H]thymidine incorporation assay were averaged and are presented as means ± SE. Significant differences between groups were evaluated by one-way ANOVA with the Student-Newman-Keuls test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of AT2 receptor deficiency on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone–induced lung tumorigenesis. To evaluate whether the AT2 receptor function is associated with chemical carcinogen-induced tumorigenesis in the lung, AT2-null mutant and wild-type control mouse groups were treated with either NNK (100 mg/kg, i.p.) or the saline vehicle. NNK caused the development of multiple lung tumors in all wild-type mice and in 84.6% of the AT2-null mice 20 weeks after the NNK injection (Table 1). The tumor multiplicity in the null mice was significantly smaller than in the wild-type mice (Table 1). The macroscopic tumors were observed throughout the lung. There was no characteristic developmental site of the tumors. The size of the tumors was relatively uniform (1-2 mm diameter). Histologic analysis revealed that tumor types in the wild-type and the AT2-null mice were identical. The majority of the tumors were histologically seen as adenoma. Saline controls in both mouse groups did not develop any tumors. These results indicate that the disruption of the AT2 receptor significantly attenuates NNK-induced tumorigenesis in the lung. It is suggested that the AT2 receptor–mediated signal is associated with NNK-induced lung tumorigenesis in this mouse strain.


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  		Table 1. Summary of the number of lung tumors in male AT2-null and wild-type mice

 
AT2 receptor expression in wild-type mouse lung and cultured human lung cancer cells. Although the in vivo study suggests that there is an involvement of angiotensin II signaling in NNK-induced lung tumorigenesis, the angiotensin II action site is unclear. Accordingly, angiotensin II receptor expression in lung tissue, typical human lung cancer cell lines (H23, H520, H529, H647, H1793, H2429, and A549), a murine lung cancer cell line (Lewis lung carcinoma cells), and primary cultured lung fibroblasts was examined by the receptor-ligand binding assay using [125I][Sar1, Ile8] angiotensin II and [125I]CGP42112. The expression levels of the AT1 and AT2 receptors in the lung plasma membranes of wild-type mice were 20.4 ± 5.3 and 9.8 ± 3.5 fmol/mg protein, respectively. All of the tested cancer cell lines exhibited negligible receptor-specific binding of [125I]-labeled ligand (<1.0 fmol/mg protein). Primary cultured fibroblasts at passage one showed both AT1 and AT2 receptor expression (n = 4) at levels of 27.5 and 42.1 fmol/mg protein, respectively. These results may suggest that angiotensin II receptor–dependent regulation of tumorigenesis and/or tumor growth is mediated mainly through the host stromal cells.

Effect of AT2 receptor deficiency in lung fibroblasts on colony growth of A549 cells in soft agar. Stromal cells often play a critical role in tumorigenesis, and they are important factors in the determination of neoplastic outgrowth of microtumors (35, 36). Because fibroblasts are a primary component of stromal tissues in the lung and lung fibroblasts prepared from weaning age mice express the angiotensin II receptors in a good quantity, we used them to examine the effect of AT2 receptor expression in lung fibroblasts on the growth of human lung cancer cells. The A549 cell line was selected as typical human lung cancer cells for this study because this cell line is derived from human adenocarcinoma and because NNK induces adenocarcinoma in mouse lung. Cocultured MLF from both wild-type and AT2-null mice markedly increased the colony counts of A549 human lung cancer cells (Fig. 1). Both the colony number and size of cancer cells cocultured with AT2-null MLF [fold increase in colony number (n = 4), 3.5 ± 0.8] were consistently and significantly smaller than those cocultured with wild-type MLF (5.6 ± 0.5). These results indicate that stromal fibroblasts derived from weaning age mice are capable of stimulating cancer cell growth. This growth stimulation effect of MLF is perhaps due to growth factors secreted from stromal fibroblasts because the fibroblasts and cancer cells do not contact each other. These results also suggest that the AT2 receptor–mediated signal(s) may stimulate fibroblast-dependent growth of cancer cells.



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  		Figure 1. Effect of AT2 receptor expression in fibroblasts on the colony growth of A549 cells in soft agar. Fibroblasts (2.5 x 104 cells) prepared from either wild type or AT2-null mouse lung were cultured for 3 days, and then human cancer cells (A549) were placed in the upper agar layer and cultured in serum-free medium at 37°C in 5% CO2 for 8 days. A, the colony number (>50 µm diameter) was counted by an automated colony counter. The experiment was repeated three times with triplicate determinations and the data are shown as a fold increase in comparison with the A549 colony number cultured without fibroblasts (control). A549 without fibroblasts (B) and co-cultured with fibroblasts from wild-type (C) or AT2-null (D) mouse lung. Both colony number and size of cancer cells in the sample cocultured with AT2-null mouse fibroblasts were significantly smaller than those cocultured with wild-type fibroblasts.

 
Effect of an agonist and antagonists of angiotensin II receptors on wild-type mouse lung fibroblast–induced colony growth of A549 cells. Because MLF are capable of stimulating the colony growth of cancer cells and because this stimulation is suggested to be due to a paracrine mechanism of growth factors, the effect of angiotensin II on cancer cell growth was examined in the same soft agar culture system. The addition of angiotensin II dose-dependently attenuated the wild-type MLF-induced colony growth stimulation (Fig. 2A), and this attenuation by angiotensin II was completely blocked by the AT1 receptor antagonist losartan but not by the AT2 receptor antagonist PD123,319 (Fig. 2B). Wild-type MLF-induced colony growth stimulation was attenuated to approximately half by the AT2 receptor blocker alone (1 µmol/L), but the AT1 receptor blocker alone had no significant effect (Fig. 2B). The colony growth of cancer cells alone was not affected by exogenous angiotensin II or its receptor blockers (data not shown). These results suggest that MLF secrete angiotensin II in this culture system and that the AT1 receptor-mediated signal attenuates cancer cell growth, but the AT2 receptor–mediated signal stimulates cancer cell growth.



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  		Figure 2. Effect of angiotensin II (Ang II, A), angiotensin II receptor antagonists (B), and an ACE inhibitor (C) on wild-type MLF-induced colony growth of A549 cells. Colony growth was determined as illustrated in Fig. 2. All chemicals were added to the culture when coculture was initiated. Angiotensin II dose-dependently attenuated MLF-induced colony growth (A) and this attenuation was blocked by losartan (B). The ACE inhibitor lisinopril dose-dependently increased MLF-induced colony growth (C). Columns, averages of two to three experiments with triplicate determinations; bars, SE. B, concentrations of chemicals: angiotensin II, 10 nmol/L; losartan, 1 µmol/L; PD123,319, 1 µmol/L. *, P < 0.05 compared with number of A549 colonies in sample cocultured with fibroblasts alone.

 
To evaluate angiotensin II production in this culture system, the effect of an ACE inhibitor on MLF-induced colony growth was examined (Fig. 2C). The ACE inhibitor lisinopril dose-dependently increased MLF-induced cancer cell growth. This result supports the above hypothesis that angiotensin II is secreted in the culture and plays a significant role in MLF-induced cancer cell growth.

Effect of AT2 receptor expression in fibroblasts on transforming growth factor-ß production. TGF-ß often plays a critical role in growth of cancer cells (37–39). Because angiotensin II has been shown to regulate TGF-ß production in a variety of cells (19, 40), the effect of AT2 receptor expression in MLF on TGF-ß production was evaluated in a transwell coculture system of MLF and A549 cells. The orientation of the cell culture was designed the same way as in the soft agar culture (MLF on the bottom layer and cancer cells in the top insert). Levels of both active and latent form of TGF-ß in the culture medium derived from wild-type and AT2-null MLF were similar (Fig. 3). Although coculture of A549 cells and either type of fibroblast markedly augmented the total TGF-ß (active + latent form) to a similar level, the level of active TGF-ß was significantly higher in the AT2-null MLF coculture than in the wild-type MLF coculture. These results may suggest that AT2 receptor–mediated signaling attenuates active TGF-ß production.



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  		Figure 3. Effect of AT2 receptor expression in lung fibroblasts on TGF-ß production. The lung fibroblasts prepared from wild-type or AT2-null mice were cocultured with or without A549 cells. Active TGF-ß secreted into the medium was determined by the PAI-1/Luciferase reporter assay. Total levels of secreted TGF-ß were measured by heat activation of the latent TGF-ß in conditioned media (80°C for 10 minutes). Content of the latent form of the TGF-ß was estimated by subtracting active TGF-ß from the amount of total TGF-ß. The experiment was repeated twice with triplicate determinations. Columns, average level of active (solid columns) and inactive (hashed columns) TGF-ß; bars, SE. *, P < 0.05 compared with TGF-ß level with A549 cells alone. #, P < 0.05 compared with active TGF-ß level in the conditioned medium from wild-type MLF cocultured with A549 cells.

 
Effect of transforming growth factor-ß1 on cancer cell growth. We obtained intriguing results suggesting that the AT2 receptor function suppresses active TGF-ß production thereby AT2-null MLF-dependent growth promotion of cancer cells is less than that by wild-type MLF. However, TGF-ß does not always regulate cancer cell growth because some cancer cells escape TGF-ß-dependent growth regulation (41, 42). To evaluate whether TGF-ß indeed regulates the growth of cancer cells in our culture system, cells were treated with a low dose of TGF-ß1 and DNA synthesis was determined by measuring [3H]thymidine uptake. TGF-ß1 (0.1-1 ng/mL) induced a dose-dependent inhibition of [3H]thymidine uptake in A549 cells with a maximal inhibition to ~30% of the corresponding control (Fig. 4). This result indicates that an increase in active TGF-ß is the potential mechanism by which AT2-null MLF stimulate cancer cell growth less than wild-type MLF.



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  		Figure 4. Effect of TGF-ß on cancer cell growth. After A549 cells were grown to a confluent condition, the cells were treated for 22 hours with the indicated concentrations of TGF-ß1 in the presence (hashed columns) or absence of serum (solid columns). The cells were pulse-labeled for an additional 2 hours with [3H]-thymidine, and thymidine incorporation was quantified as described in Materials and Methods. The experiment was repeated three times with triplicate determinations. Columns, average incorporation; bars, SE. *, P < 0.05 compared with thymidine incorporation in A549 cells incubated without TGF-ß1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Increasing evidence suggests that angiotensin II signaling may play important roles in tumorigenesis in several tissues (1–3, 5). However, the specific roles of the angiotensin II receptors in tumorigenesis have not been elucidated. We have previously shown a pro-oncogenic role of the AT2 receptor in chemical carcinogen azoxymethane-induced colon tumorigenesis (28). In the present study, we used the well-characterized in vivo model of NNK-induced rodent lung tumorigenesis and an in vitro model of colony growth of cancer cells in soft agar to examine the effect of AT2 receptor gene disruption in lung tumorigenesis. Tobacco-specific nitrosamine NNK-induced lung cancer in rodents is a well-studied model system for human lung cancer, and its developmental mechanism is similar to human lung adenocarcinoma in its morphology, proliferation characteristics, and gene mutation involvement (43, 44). Anchorage-independent cancer cell growth is one of the characteristic features of cancer cells. Elucidation of the role of AT2 receptors in lung tumorigenesis and in the growth mechanism of human cancer cells is important. Therefore, the outcome from these in vivo and in vitro studies should be most valuable and significant.

First, we examined the role of the AT2 receptor in NNK-induced tumorigenesis using AT2 receptor-deficient mice with an SWR/J genetic background. Although a large number of mouse studies concerning NNK-induced lung carcinogenesis have been conducted with A/J mice (43), SWR mice have been shown to be the most susceptible strain to a variety of lung-specific chemical carcinogens (45, 46). Indeed, the present study showed that this mouse strain is also susceptible to NNK-induced lung tumorigenesis. Accordingly, this study with the SWR mouse strain seems comparable with those carried out previously with A/J mice. The results clearly show that the presence of the AT2 receptor is very important in NNK-induced lung tumorigenesis, because NNK treatment induced multiple tumors in all of the wild-type mice but in only 86% of the AT2-null mice (Table 1). The tumor multiplicity in the null mice was also significantly smaller than in the control wild-type mice. Therefore, this in vivo study shows that an intact AT2 receptor is favorable for NNK-induced lung tumorigenesis.

Although these observations clearly indicate an involvement of the AT2 receptor in NNK-induced lung tumorigenesis, the types of cells and the mechanism by which the AT2 receptor modulates tumorigenesis were not clarified. However, because most human and murine lung cancer cell lines do not express angiotensin II receptors but stromal fibroblasts do (results of the present study), it is reasonable to speculate that the action site of the AT2 receptor may be within the lung stromal cells. In support of this speculation, the significance of the microenvironment for tumor development has been well documented in experimental in vivo systems (47, 48). Interaction between tumor cells and microenvironment is mediated through either tumor cell– and/or stromal cell–produced growth factors and various cytokines (35, 36). A recent report also showed that disruption of TGF-ß type II receptor expression in the fibroblasts promoted neoplastic conversion of the epithelial cells in the forestomach and prostate (49). In the present study, the effect of AT2 receptor expression in fibroblasts on the growth of human lung cancer cells (A549) was examined. Although cocultured lung fibroblasts from both wild-type and AT2-null mouse strains significantly increased the number of A549 cell colonies, consistently higher colony counts were observed with the wild-type mouse lung fibroblasts than with the AT2-null mouse lung fibroblasts (Fig. 1). These results indicate that the AT2-null mouse fibroblasts are less supportive of cancer cell growth than those from wild-type mice. These results are essentially consistent with those from our in vivo studies. The angiotensin II AT1 receptor–mediated signaling attenuates fibroblast-dependent cancer cell growth, whereas the angiotensin II AT2 receptor–mediated signaling seems positively involved in the stromal fibroblast–dependent growth of cancer cells (Fig. 2A and B). Additionally, the present study indicates that lung fibroblasts are capable of producing functional angiotensin II because the angiotensin II–converting enzyme inhibitor dose-dependently increased the growth of cancer cells (Fig. 2C). The AT2 receptor antagonist-dependent attenuation of cancer cell growth (Fig. 2B) also supports de novo synthesis of angiotensin II. Taken together, these results suggest that locally generated angiotensin II acts as a local growth regulator of lung cancer cells. Therefore, stromal fibroblast activities seem an important determinant of susceptibility to carcinogen-induced lung tumorigenesis. In support of this conclusion, angiotensin II has been shown to be involved in the regulation of fibroblast-dependent production of growth factors and cytokines such as FGF-2 (19, 40, 50). However, because lung epithelial cells express angiotensin II receptors (51, 52), an involvement of angiotensin II signaling in carcinogen-induced lung epithelial cell transformation remains to be clarified.

TGF-ß is a multifunctional growth factor expressed by many cell lines and tissue types (38, 39). Although this growth factor has been shown to regulate immune response, angiogenesis, chondrogenesis, myogenesis, and production of extracellular matrix proteins, it usually acts as a potent inhibitor of growth in most cells, especially those of the epithelial lineage (37–39). During carcinogenesis, the disruption of TFG-ß signaling has been shown to be critical. Although the present study indicates that angiotensin II is a potential mediator for suppressed tumorigenesis in the AT2-null mice, angiotensin II-dependent regulation of TGF-ß signaling might be an underlying mechanism. Accordingly, the effect of AT2 receptor expression in lung fibroblasts on TGF-ß transcription was examined by the luciferase reporter assay. In the present study, the level of bioactive TGF-ß (this method does not distinguish among the isoforms of TGF-ß) in the medium conditioned with AT2-null mouse lung fibroblasts/A549 cells was significantly higher than that in the medium conditioned with wild-type mouse lung fibroblasts/A549 cells (Fig. 3). These results suggest that the angiotensin II AT2 receptor may suppress TGF-ß activation. Increased activation of TGF-ß in the AT2-null fibroblasts thereby effectively attenuates fibroblast-dependent growth of cocultured lung cancer cells. This assumption is supported by the experimental facts that fibroblasts and cancer cells did not contact each other and angiotensin II alone did not show any effect on the colony growth of A549 cells in the absence of cocultured fibroblasts (data not shown). Therefore, TGF-ß activation seems downstream of angiotensin II AT2 receptor signaling. To the best of our knowledge, the present study is the first to show that the angiotensin II AT2 receptor attenuates TGF-ß activation. Although other cytokines and growth factors have been shown to be produced in fibroblasts, their involvement in the downstream signaling of angiotensin II awaits further study.

The present study shows an oncogenic modulator function of the AT2 receptor in NNK-induced lung tumorigenesis in the mouse. Results suggest that the AT2 receptor function in the lung may be a determinant of tumor susceptibility in NNK-induced tumorigenesis. Because pharmacologic control of the AT2 receptor function in vivo is a viable therapeutic technique (53), it is feasible to determine whether AT2 receptor blockers attenuate NNK-induced lung tumorigenesis. This may lead to the development of a potential chemoprevention procedure for human lung cancer. To the best of our knowledge, the present study is the first to show that the angiotensin II AT2 receptor possesses an oncogenic modulator function in NNK-induced lung tumorigenesis in mice.


   Acknowledgments
 
Grant support: NIH grants RO3 CA091428, P50 CA90949, and P30 CA68485.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Tadashi Inagami (Department of Biochemistry, Vanderbilt University) for sharing the original strain of the AT2-null mice; Dr. Brian Law (Department of Cancer Biology, Vanderbilt University) for his practical instruction and assistance in the TGF-ß-related experiments; Dr. Adriana Gonzalez (Department of Pathology, Vanderbilt University) for her assistance in the diagnosis of lung tumor type; Pamela J. Tamura (Department of Chemistry, Vanderbilt University), Dr. Gerald Frank (Department of Biochemistry, Vanderbilt University), and Dr. Erwin J. Landon (Department of Pharmacology, Vanderbilt University) for critical reading and constructive comments during the preparation of the article.

Received 1/26/05. Revised 4/14/05. Accepted 6/15/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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