Restoration of TGF-β signalling reduces tumorigenicity in human lung cancer cells

Members of the transforming growth factor-β (TGF-β) family regulate a wide range of biological processes including cell proliferation, migration, differentiation, apoptosis, and extracellular matrix deposition. Resistance to TGF-β-mediated tumour suppressor function in human lung cancer may occur through the loss of type II receptor (TβRII) expression. In this study, we investigated the expression pattern of TβRII in human lung cancer tissues by RT–PCR and Western blot analyses. We observed downregulation of TβRII in 30 out of 46 NSCLC samples (65%) by semiquantitative RT–PCR. Western blot analyses with tumour lysates showed reduced expression of TβRII in 77% cases. We also determined the effect of TβRII expression in lung adenocarcinoma cell line (VMRC-LCD) that is not responsive to TGF-β due to lack of TβRII expression. Stable expression of TβRII in these cells restored TGF-β-mediated effects including Smad2/3 and Smad4 complex formation, TGF-β-responsive reporter gene activation, inhibition of cell proliferation and increased apoptosis. Clones expressing TβRII showed reduced colony formation in soft-agarose assay and significantly reduced tumorigenicity in athymic nude mice. Therefore, these results suggest that reestablishment of TGF-β signalling in TβRII null cells by stable expression of TβRII can reverse malignant behaviour of cells and loss of TβRII expression may be involved in lung tumour progression.

Transforming growth factor-b (TGF-b) belongs to a superfamily of structurally related polypeptides that are involved in various biological processes, including cell growth, differentiation, angiogenesis, apoptosis, and extracellular matrix remodelling (Massague, 1998). The multifunctional effects of TGF-b in cellular actions occur by binding to its receptors with intrinsic serine/ threonine kinase activity. Sequence comparisons indicate that these receptors fall into two subgroups, designated as type I (TbRI) and type II (TbRII) serine -threonine kinase receptors. Transforming growth factor-b initiate signals by binding to TbRII and stabilises the heteromeric complex with TbRI, and as a result, TbRI is transphosphorylated within a glycine -serine-rich domain (GS-domain). The activated TbRI then propagates the signals to intracellular signal mediators including Smads (Engel et al, 1998;Massague and Wotton, 2000).
Smad proteins are classified according to their structure and function in signalling by TGF-b family members (Attisano and Wrana, 1998). They are characterised by homologous regions at their N-and C-termini known as Mad homology (MH-1 and MH-2) domains, respectively. A divergent linker region separates these domains. Both structural and functional differences provide the basis for a division of the Smads into three groups: receptor regulated (R-Smad), common-mediator, and inhibitory Smads. R-Smads including Smad1, 2, 3, 5, 8, and 9, act as direct substrates of specific type I receptors and are activated by phosphorylation of serine residues at the carboxyl terminus. Thus, Smad2 and Smad3 mediate signalling by TGF-b and activin, while Smad1 and presumably Smad5, Smad8, and Smad9 are similarly modified through bone morphogenetic protein (BMP) exposure (Engel et al, 1998). Transforming growth factor-b/activin receptor-phosphorylated Smads (R-Smads) oligomerise with the common mediator Smad4 (Co-Smad), and after nuclear import, they regulate gene expression positively or negatively by binding to DNA or by interacting with transcription factors (Shi and Massague, 2003). A distinct class of distantly related Smads, including Smad6 and Smad7, has been identified as inhibitors of these signalling pathways. Smad7 forms stable associations with activated type I receptors, thereby preventing R-Smads from binding to and being phosphorylated by these receptors (Hayashi et al, 1997;Nakao et al, 1997). Thus, Smad7 inhibits TGF-b, activin, and BMP signalling. Recently, we have cloned a novel WD40 domaincontaining protein called STRAP that associates with both TbRI and TbRII and is involved in the negative regulation of TGF-b signalling . STRAP synergises with Smad7, but not with Smad6, in the inhibition of TGF-b-induced transcriptional responses. STRAP associates with Smad7, recruits it from the cytosol to the activated TbRI, and stabilises the heteromeric complex, thereby assisting Smad7 in preventing Smad2 and Smad3 activation by the receptor complex (Datta and Moses, 2000). A distinct mechanism of inhibition for Smad6 and its primary role in regulating BMP signals have been proposed (Hata et al, 1998;Ishisaki et al, 1999). The expression of Smad6 and Smad7 is increased in response to BMP, activin, and TGF-b, suggesting a negative feedback of these pathways (Nakao et al, 1997;Ishisaki et al, 1999).
Perhaps one of the most important biological effects of TGF-b is its ability to inhibit proliferation of many cell types, including most epithelial cells . As a result, the mechanism(s) of TGF-b growth inhibition has been the subject of intense investigation over the past decade. Transforming growth factor-b inhibits progression of cells from G1 into the S phase of the cell cycle Howe et al, 1991). Cell cycle progression is mostly governed by Cdks, which are activated by Cyclins binding and inhibited by the Cdk inhibitors. Although there is not much data linking the known TGF-b-signalling pathways with the regulators of cell cycle progression implicated in TGF-b-induced growth arrest. Several lines of evidence suggest that Smad signalling is functionally connected directly or indirectly in suppressing the growth of epithelial cells. The primary event that initiates the TGF-b-induced growth arrest may be associated with increased expression of p15 INK4B , p21 Cip1 , and suppression of c-Myc expression. Deregulation of Cdk inhibitors may contribute to TGF-b resistance in cancer. Loss of p21 Cip1 has been observed in advanced breast cancers in association with a poor patient prognosis (Jiang et al, 1997). In epithelial cells from the skin, lung, and breast, TGF-b rapidly elevates expression of the Cdk4/6 inhibitor p15 INK4B (Hannon and Beach, 1994). In keratinocytes, colon and ovarian epithelial cells, TGF-b elevates the expression of p21 Cip1 (Elbendary et al, 1994;Datto et al, 1995). Smad signalling is required for TGF-b-mediated induction of p15 INK4B (Feng et al, 2000), and p21 Cip1 (Pardali et al, 2000). Transforming growth factor-b stimulation of epithelial cells induces the formation of a Smad complex that specifically recognises a TGF-b-inhibitory element in the c-Myc promoter, and this response may be critical for TGF-b-induced c-Myc downregulation and growth arrest (Chen et al, 2001). Dominant-negative Smad3 (Liu et al, 1997) or the inhibitory Smad, Smad7 (Itoh et al, 1998) blocks TGF-b-induced growth inhibition in epithelial cells.
Cell growth is modulated by positive and negative signals, and loss of negative growth constraints may contribute to oncogenic processes. Such perturbations may occur as a consequence of the loss of the tumour suppressor functions of TGF-b. One of the physiological roles of the TGF-b-signalling pathway is to provide protection against malignant transformation. Loss of TGF-b receptor expression has been reported in several tumours, including colon cancer (Markowitz et al, 1995), breast cancer (Sun et al, 1994), prostrate cancer (Guo and Kyprianou, 1998), and TGF-b expression is elevated in these tumours. Resistance to TGFb in lung cancer may occur through several mechanisms that involve functional alteration of signalling molecules. A recent study has demonstrated that most lung cancer cell lines have lost the growth-inhibitory response to TGF-b signal (Osada et al, 2001). It has been suggested that TGF-b resistance stemming from inactivation of TbRII could be a multiple process involving both genetic and epigenetic events. Moreover, extensive analyses found that mutations within the coding sequence of the TbRII gene are rare in NSCLC. Replication error phenotype, a high incidence of mutation in poly-adenosine (poly-A) tract of the TbRII gene was found leading to loss of TbRII in colon and gastric cancer (Markowitz et al, 1995). Therefore, mutation in the poly-A tract or truncation in the kinase domain of TbRII is infrequent in lung cancer. Mutations of Smad2 and Smad4 genes have been found in a limited fraction of lung cancers (5 -10%) Uchida et al, 1996). Two previous reports have shown (Hougaard et al, 1999;Osada et al, 2001) that four out of 33 lung cancer cell lines are responsive to TGF-b-induced growth inhibition and 29 cell lines are unresponsive to TGF-b. However, among these unresponsive cell lines, 21 cell lines show either no expression or weak expression of TbRII. Therefore, downregulation of TbRII can explain TGF-b unresponsiveness in certain fraction of lung cancers. A recent study has suggested that impairment of TGF-b signalling may contribute to tumour progression by cell proliferation rather than by modulation of angiogenesis (Park et al, 2002). These findings suggest that cancer cells could result in escape from autocrine growth inhibitory effect of TGF-b due to the loss of TbRII. A correlation between diminished expression of TbRII in breast cancer cells and enhanced in vivo malignant behaviour has been observed in studies based on patient specimens and an established cell line (Gobbi et al, 2000), suggesting that attenuation of the growth inhibitory TGF-b autocrine loop in breast cancers worsens clinical outcome. However, little is known about the TbRII status in lung cancer and whether restoring TGF-b signalling by the introduction of functional TGF-b type II receptor alters tumorigenicity in TGF-b unresponsive lung cancer cell lines that do not express the type II receptor. In the present study, we have carried out RT -PCR and Western blot analyses with human lung tumour tissues to analyse the expression pattern of TbRII for the first time. Out of 46 lung tumour samples analysed by RT -PCR, squamous cell carcinoma (80%), adenocarcinoma (42%), and large cell carcinoma (72%) show downregulation or loss of TbRII, and Western blot analyses suggest reduced expression of TbRII in 77% of lung tumour samples. We have determined the effects of stable expression of TbRII in TGF-b-unresponsive human lung tumour-derived VMRC-LCD cells lacking TbRII expression. TbRII expression restores TGF-b downstream signalling as determined by the induction of Smad2/3 and Smad4 complex formation, TGFb-responsive reporter gene activation, inhibition of cell proliferation and induction of apoptosis. We also show that clones expressing TbRII reduce colony formation in soft-agarose assay and tumorigenicity in athymic nude mice. Therefore, our data support the notion that impairment of TGF-b tumour suppressor function may contribute to lung tumour progression.

Cell culture and biological reagents
Fresh lung tumour and the corresponding normal tissue specimens were collected from 46 patients. These patients had received neither radiotherapy nor chemotherapy before surgery. They underwent pulmonary resection for primary NSCLC at Vanderbilt University School of Medicine Hospital. Informed consent was received and the project was approved by the local Institutional Review Board. VMRC-LCD parental cell line (human lung adenocarcinoma derived) was maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS). Vector control and TbRII stable clones were maintained in Geneticin (G418 sulfate; Life Technologies Inc., Carlsbad, CA) selection at 400 mg ml À1 . All cell lines were maintained at 371C in presence of 5% CO 2 in a humidified incubator. Transient transfections were performed using lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's specifications. Anti-Smad2, anti-Smad3 (Zymed Laboratories Inc., Sanfrancisco, CA), anti-p21 Cip1 , anti-Smad4 antibody (Santa Cruz Biotechnology Inc., Santacruz, CA), antiphospo-Smad2 antibody (Cell Signalling, Beverly, MA) and mouse anti-b-actin antibody (Sigma Biochemicals, St Louis, MO) were used in this study. Antiphospho-Smad3 antibody was a generous gift from Dr Edward B. Leof (Mayo Clinic, Rochester, MN). The TGF-b receptor kinase inhibitor, SB-431542 was kindly provided by Dr. Nicholas J. Laping from the Glaxo SmithKline, King of Prussia, PA, USA. SB-431542 was also purchased from Tocris Cookson Inc., Ellisville, MO, USA.

RNA extraction and reverse transcription (RT)-PCR analysis
Total RNA from the tissue specimens was isolated by using Tripure RNA Purification Kit (Roche Applied Science, Indianapolis, IN). RNA quality and concentration were estimated spectrophotometrically at 260 nm. RT -PCR was performed using SUPERSCRIPT II Kit RETROscript TM (Ambion Inc., Austin, TX). The cDNA was synthesised from total RNA (1 mg) using oligo(dT) 15 primer and reverse transcriptase. The TbRII (493 bp) gene fragment was amplified by using the following primers; 5 0 -gcacgttcagaagtcggtta-3 0 (forward) and 5 0 -gcggtagcagtagaagatga-3 0 (reverse). The primer sequences for GAPDH fragment (600 bp) were 5 0 -ccacccatgg caaattccatggca-3 0 (forward) and 5 0 -tctagacggcaggtcaggtccacc-3 0 (reverse). PCR amplification was carried out using 2 ml of the above RT reaction mixture and 1 Â PCR buffer, 1.5 mM MgCl 2 , 2 U of Taq DNA polymerase, and 10 nm of each Primer. After initial denaturation at 941C for 3 min, amplification conditions were as follows: 30 cycles of 941C for 30 s, 551C for 30 s, and 721C for 1 min; followed by a final 5 min extension at 721C. The RT -PCR products were separated on a 1.5% agarose gel.

Transcriptional response assay
VMRC-LCD cells were transiently transfected with CMV-b-gal, and p3TP-Lux, or (CAGA) 9 MLP-Luc or p21 Cip1 -Luc reporter plasmids. Transfected cells were incubated in 0.2% FBS in presence or absence of 2 or 5 ng ml À1 of TGF-b1 for 22 h. Cell lysates were used to measure both luciferase and b-gal activities, and the normalised luciferase activity was presented.

Immunoprecipitation and Western blot analyses
VMRC-LCD cells were serum starved for 2 h and treated in the presence or absence of 5 ng ml À1 of TGF-b for 90 min. Cells were lysed and equal amount of each protein lysate was incubated with both anti-Smad2 and anti-Smad3 polyclonal antibodies for 2 h at 41C, followed by incubation with 20 ml of protein G-Sepharose beads (Sigma Biochemicals, St Louis, MO) for 1 h. The immune complexes were analysed by Western blotting with mouse anti-Smad4 antibody. For other Western blots, extracts were prepared from VMRC-LCD cells treated in presence or absence of TGF-b at different time intervals indicated in figure legends.
[ 3 H]-Thymidine incorporation assay VMRC-LCD cells were treated in the presence or absence of TGF-b1 for 36 h in 10% FBS medium. 4 mCi well À1 [ 3 H]-thymidine (NEN, Boston, MA) was added in each well for an additional 4 h. Cells were then fixed in 10% cold trichloroacetic acid (TCA), washed, and lysed in 0.2 N NaOH. Radioactivity incorporated into TCA-insoluble [ 3 H]-thymidine was measured by scintillation counting and presented.

Cell proliferation assay
VMRC-LCD cells were seeded into 12-well plates. Cells were then treated in the presence or absence of TGF-b (0.5 or 5 ng ml À1 ) for a total of 5 days. Transforming growth factor-b-containing media was replaced every other day. Cells were counted after 5 days and the average cell numbers from triplicate measurements were plotted.

Cell death ELISA
VMRC-LCD cells (2 Â 10 4 cells well À1 ) were seeded into 12-well plates and allowed to attach for 20 h. Cells were serum starved for 20 h and then treated in the presence or absence of 5 or 10 ng ml À1 of TGF-b1 for 24 h. Cells (floating and adherent) were lysed in 200 ml of lysis buffer. Apoptosis in these cells was quantified by using a cell-death detection ELISA kit (Roche Molecular Biochemicals, Laval, Quebec, Canada) according to the manufacturer's instructions. This quantitative sandwich enzyme immunoassay specifically measures the histone region (H1, H2A, H2B, H3, and H4) of mono-and oligonucleosomes that are released during apoptosis. Photometric development was monitored kinetically by reading the plate at 405 nm at 5-min intervals by using a THERMOmax microplate reader (Molecular Devices Corp., Menlo Park, CA, USA). All data points were assessed in triplicate.

Soft-agarose assay
Soft-agarose assays were performed to compare the clonogenic potential of control and TbRII-transfected cells. In all, 5 Â 10 4 cells from each pool were suspended in 1 ml of 0.4% sea plaque agarose containing 10% FBS medium and then plated on the top of 1 ml of semisolidified 0.8% agarose in the same medium in 35 mm plates. For each parental, vector control, and TbRII clones, triplicate wells were plated. Plates were incubated for 2 weeks at 371C in the presence of 5% CO 2 in a humidified incubator. Colonies grown on soft agarose were counted by automated colony counter.

Tumorigenicity study
Cells from exponential cultures of VMRC-LCD (5 Â 10 6 cells) were inoculated subcutaneously behind the anterior fore limb of 6-week-old athymic nude mice. Mice were maintained in a pathogen-free facility and tumours were measured two times in a week. Growth curves for xenografts were determined by externally measuring tumours in two dimensions using a slide caliper. Tumour volume was determined from the equation: V ¼ (L Â W 2 ) Â 0.5, where L is length and W is width of the tumour. Growth curves for tumours were plotted from the mean volume7s.d. of tumours from six mice. The use of these mice in research was approved by Vanderbilt University Institutional Animal Care and Use Committee.

Downregulation of TbRII in non-small-cell lung cancers (NSCLC)
Reduced expression of TGF-b receptors is known to be associated with unresponsiveness to TGF-b-mediated growth inhibitory function and may be involved in tumour progression. To verify whether TbRII level is downregulated in human lung tumours, we analysed for TbRII expression by RT -PCR using RNA samples from 46 lung tumour specimens (20 squamous cell carcinoma, 19 adeno carcinoma, and seven large cell carcinoma) ( Figure 1A). The TbRII expression was found to be decreased in 80% of squamous cell carcinoma, 42% adenocarcinoma, and 72% large cell carcinoma. Since we observed reduced TbRII mRNA expression in NSCLC, we tested the expression of TbRII protein in lysates made from tumour specimens by Western blot analysis. Out of 22 lung tumour samples (including squamous, adeno, and large cell carcinoma) analysed with corresponding control, 17 tumours (77%) showed reduced TbRII protein level ( Figure 1B). 10 specimens showed almost no expression of TbRII. These results suggest that the expression of TbRII is reduced in majority of NSCLC patients.

Stable expression of TbRII in VMRC-LCD cells induces Smad2/Smad3 and Smad4 complex formation
In order to express TbRII in VMRC-LCD lung adenocarcinoma cells lacking its expression, wild-type TbRII was transfected and cells were selected with G418 to generate stable clonal cell lines. Quantitative RT -PCR was performed to test and to compare the expression of TbRII mRNA in stable VMRC-LCD cells and in lung adenocarcinoma cells (A549) (Figure 2A). These data suggest the physiological level of expression of TbRII in VMRC-LCD cells. We also tested expression of the protein by Western blot analyses ( Figure 2B). Three clones that expressed high levels of TbRII (TbRII #10, TbRII #13 and TbRII #17) were selected for further experiments. To test whether overexpressed TbRII is functional, we first analysed the phosphorylation of endogenous-positive regulatory Smads, Smad2 and Smad3. Parental cells, two vector control clones, and three TbRII clones were treated with TGF-b for 90 min and cell lysates were subjected to Western blot analyses by antiphospho-Smad2 and antiphospho-Smad3 antibodies. Phosphorylation of Smad2 and Smad3 ( Figure 3A, first and third panel) was found to be increased in TbRII stable clones although the expressions of these proteins were unchanged. To test whether stable expression of TbRII can restore the complex formation between Smad2/Smad3 and Smad4 in vivo, we performed immunoprecipitation experiments after treating TbRII clones, vector clones, and parental cells with TGF-b for 90 min. Equal amounts of cell lysates were used for immunoprecipitation with both anti-Smad2 and anti-Smad3 antibodies. The immune complexes were analysed by Western blot with anti-Smad4 monoclonal antibody. Transforming growth factor-b-induced heteromeric complex formation between Smad2/Smad3 and Smad4 was increased in TbRII-expressing clones as compared to parental and vector control clones ( Figure 3B). To confirm the TGF-b downstream signalling is intact in VMRC-LCD cells, we performed transcriptional assays using TGF-b-responsive reporter (CAGA) 9 MLP-Luc. We observed an increase in transcriptional activity by transfection of TbRII and a further strong induction in response to TGF-b. Transfection of constitutively active TGF-b type I receptor (T204D) (act-TbRI) alone induced the reporter activity in VMRC-LCD cells ( Figure 3C), because act-TbRI does not require TGF-b and TbRII for downstream signalling. These results suggest that VMRC-LCD cells lack TbRII expression and the downstream signalling cascade is intact. Together, stable expression of TbRII restores TGF-b/Smad signalling in VMRC-LCD cells.

TbRII expression restores TGF-b-induced transcriptional responses and p21 Cip1 expression
To explore the functional significance of TbRII expression in VMRC-LCD cells, we focused our analyses on TGF-b-induced transcriptional responses. We used two TGF-b-responsive reporters. The first reporter (CAGA) 9 MLP-Luc contains multiple Smad3/Smad4-binding CAGA boxes upstream of a minimal adenovirus major late promoter. The second reporter p3TP-Lux containing element from the PAI-1 promoter has both Smad-and N T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16   T33 T34 T35 T36 T37 T38 T39 T40 T41 T42 T43 T44 T45 T46 N   T17 T18 T19 T20 T21 T22 T23 T24 T25 T26 T27 T28 T29 T30 T31 T32  Ap1-binding elements. Stable TbRII clones showed higher reporter activity in response to TGF-b, whereas parental and vector clones did not show any significant response to TGF-b ( Figure 4A). Similarly, (CAGA) 9 MLP-Luc reporter activity was strongly induced in response to TGF-b in TbRII-expressing clones when compared with parental and vector control ( Figure 4B). Interestingly, both reporters were induced significantly in TbRII clones without TGF-b treatment as compared to parental and vector control cells. In an attempt to determine why TbRII expression alone induces transcriptional activation, we have performed experiment to block endogenous and exogenous TGF-b effect by a TGF-b receptor kinase inhibitor (SB-431542) (Halder et al, 2005). VMRC-LCD parental cells were transiently cotransfected with TGF-b-responsive reporter (CAGA) 9 MLP-Luc and TbRII. Cells were treated with TGF-b (5 ng ml À1 ) in the presence or absence of SB-431542 for 22 h. We observed an increase in transcriptional response by TbRII alone. This effect was completely blocked by SB-431542 treatment ( Figure 4C) suggesting that expression of TbRII can restore signalling induced by endogenous TGF-b. VMRC-LCD cell line has been shown to secret significant amount of functional TGF-b (Halder et al, 2005). As expected, this inhibitor also blocks the effect of exogenous TGF-b. To compare how well the restoration of TGF-b signalling has worked, we performed reporter assay using VMRC-LCD cells stably expressing TbRII and TGF-b responsive lung adenocarcinoma cells A549 that express TbRII. We have observed four-and seven-fold induction in TGF-b-induced transcription in VMRC-LCD cells expressing TbRII and in A549 cells ( Figure 4D), respectively. These results coupled with the Figure  Parental cells, vector control and stable TbRII clones were preincubated for 2 h in serum-free medium and then treated with TGF-b1 (5 ng ml À1 ) for 90 min. Cell lysates were subjected to immunoblotting with antiphospho Smad2, anti-Smad2, antiphospho Smad3, anti-Smad3, and anti-Smad4 antibodies. Equal amount of protein loading was tested by immunoblotting the membrane with anti-b-actin antibody. (B) Parental cells, vector control, and stable TbRII clones were treated with TGF-b as above. Equal amount of cell lysates were subjected to immunoprecipitation with anti-Smad2 and anti-Smad3 polyclonal antibodies and the immunoprecipitates were analysed by immunoblotting with anti-Smad4 antibodies. (C) VMRC-LCD parental cells were transiently transfected with (CAGA) 9 MLP-Luc, and CMV-b-gal, TbRII or act-TbRI (T204D) expression plasmids. Cells were treated with 5 ng ml À1 TGF-b for 22 h. Luciferase activity was normalised to b-gal activity, and the relative luciferase activity was expressed as the mean7s.d. of triplicate measurements. These experiments were repeated at least three times. *Po0.001 for all groups in a multiple comparison test with Bonferroni adjustments after rank transforming the data.
of TGF-b signalling has any effect on endogenous protein expression, we analysed the expression of p21 Cip1 protein in parental, vector control, and three stable TbRII clones in response to TGF-b at different time points. The cell lysates were subjected to Western blot analyses using anti-p21 Cip1 antibody ( Figure 4F). Parental and vector clones did not show any induction. However, TGF-b-induced p21 Cip1 protein expression significantly in all three stable TbRII clones, and its expression is maintained up to 16 h after the treatment. These results show that VMRC-LCD cells are unresponsive to TGF-b due to the lack of TbRII expression, and re-expression of TbRII makes these cells responsive to TGF-b.

Expression of TbRII in VMRC-LCD cells induces TGF-b-mediated growth inhibition
One of the most important biological effects of TGF-b is its ability to inhibit proliferation of many cell types. To determine the effect of TbRII expression on TGF-b-induced growth inhibition, we first V e c to r # 2 V e c to r # 7 P a r e n ta l T R II # 1 0 T R II # 1 3 T R II # 1 7 V e c to r # 2 V e c to r # 7 P a r e n ta l T R II # 1 0 T R II # 1 3 T R II # 1 7 performed [ 3 H]-thymidine incorporation assay using control cells and TbRII-expressing clones. DNA synthesis in TbRII clones is decreased in response to TGF-b in a dose-dependent manner. However, we did not observe significant change in thymidine incorporation in parental and vector control cells in response to TGF-b ( Figure 5A). To compare the growth inhibitory effect of TGF-b on DNA synthesis between stable TbRII-expressing VMRC-LCD clone and A549 cells with endogenous TbRII expression, we Cells were then fixed, lysed, and the radioactivity incorporated was counted. Radioactivity incorporated without TGF-b treatment is considered as 100%, and the results are expressed as the mean7s.d. for triplicate measurements. (B) Stable TbRII clone and A549 cells were plated as above and treated with TGF-b for 36 h. Cells were processed as above and the radioactivity incorporated was counted. Radioactivity incorporated without TGF-b treatment is considered as 100%, and the results are expressed as the mean7s.d. for triplicate measurements. *Po0.05 for all groups in (A) and (B) were compared by Wilcoxon's test. (C) Cell counting assay. In all, 8 Â 10 3 cells from parental, vector control, and stable TbRII clones were seeded into each well of 12-well plate and then treated with TGF-b (0.5 or 5 ng ml À1 ) in 10% FBS-containing medium. Cells were counted after 5 days and plotted. Each data point is expressed as the mean7s.d. of triplicate measurements. Each experiment was repeated three times with similar results. (D) VMRC-LCD parental, vector control, and TbRII stable clones were treated with 5 ng ml À1 TGFb in the presence or absence of SB-431542 (10 mM) for 5 days. Cells were counted and the cell numbers were plotted. Individual data points are the mean7s.d. of triplicate determinations. *Po0.005 for all groups in (C) and (D) in a linear mixed effect model on the log-transformed data.

TGF-
performed thymidine incorporation assay. Inhibition in thymidine incorporation by TGF-b was 30% in VMRC-LCD stable cells and 25% in A549 cells ( Figure 5B). These results suggest that both cell lines has comparable inhibitory effect on thymidine incorporation in response to TGF-b. We further tested the effect of TbRII expression on cell proliferation by cell counting assays. Parental cells, vector and TbRII clones were treated with TGF-b and the cells were counted after 5 days. Transforming growth factor-b treatment did not affect the growth of both parental and vector control cells, whereas the growth of stable TbRII-expressing cells were significantly inhibited by TGF-b treatment ( Figure 5C). Interestingly, the growth of TbRII clones were inhibited in absence of TGF-b and this could be due to endogenous TGF-b secretion. To confirm the inhibitory role of endogenous TGF-b on cell growth in TbRII stable cells, we treated the VMRC-LCD parental, vector clones and TbRII clones with TRKI (SB-431542) in the presence or absence of TGF-b. Growth of parental cells and vector clones was not affected by either TGF-b or the inhibitor. However, growth inhibition induced by endogenous or exogenous TGF-b was significantly blocked by the inhibitor in stable TbRII clones ( Figure 5D) suggesting the reestablishment of autocrine endogenous TGF-b signalling in these cells. These results suggest that loss of TbRII is important for VMRC-LCD cells to be resistant to TGFb-induced growth suppression.

TGF-b induces apoptosis in VMRC-LCD cells expressing TbRII
TGF-b is known to induce apoptosis depending on cell types that might contribute to TGF-b-mediated tumour suppressor function.
To evaluate the role of stable expression of TbRII in apoptosis in VMRC-LCD cells, we performed a quantitative cell death ELISA assay using VMRC-LCD cells and stable TbRII clones in the presence or absence of TGF-b. TbRII stable clones showed significant increase in apoptosis in a dose-dependent manner ( Figure 6). However, parental and vector control cells did not show any change in apoptosis in response to TGF-b. Sodium butyrate induced apoptosis in both control and TbRII expressing clones in a similar way. Taken together, we can conclude that expression of TbRII in VMRC-LCD cells restore tumour suppressor function of TGF-b.

Stable expression of TbRII decreases tumorigenicity of VMRC-LCD cells
Loss of TGF-b signalling in human tumours is believed to be critical in carcinogenesis. Anchorage-independent growth in semisolid medium and the formation of xenografts in immunocompromised mice are generally considered to be read-outs for assessing the tumorigenicity of human cells. To assess the effect of TbRII expression on the malignant properties of VMRC-LCD cells, we compared the ability of the control and TbRII stable cells to form colonies in soft agarose. Control clones and TbRII stable clones were tested for growth in soft agarose in 35 mm culture plates at 5 Â 10 4 cells well À1 . After 2 weeks of incubation, colonies were counted by automated colony counter. Significant reduction in colony formation (both size and number) was observed in TbRII stable clones when compared with parental and vector cells ( Figure 7A). These data suggest that stable expression of TbRII  decreases the anchorage-independent growth of VMRC-LCD cells. Reduction in cloning efficiency in soft agarose suggested that restoration of TGF-b sensitivity might also render VMRC-LCD cells less tumorigenic. To test this hypothesis, we injected exponentially growing cells from parental, vector control and TbRII stable clones (5 Â l0 6 ) subcutaneously in athymic nude mice and followed the progression of xenograft formation. Clones stably expressing TbRII consistently formed smaller tumours compared to tumours arising from parental and vector cells ( Figure 7B). tumours formed from parental and vector control cells grew substantially faster. Together, these results suggest that overexpression of TbRII in a TGF-b-resistant lung cancer cell line restored TGF-b sensitivity. It is possible that the unresponsiveness to TGF-b response in VMRC-LCD cells is merely due to lack of TbRII expression and restoration of TbRII alone might restore TGF-b tumour suppressor function.

DISCUSSION
The malignant transformation in several types of cancer, including lung cancer, results in a loss of tumour suppressor effects of TGFb. Loss of TGF-b response has been shown to be associated with tumour development and/or tumour progression in a number of cancer cell lines (Masui et al, 1986;Arteaga et al, 1988;Kimchi et al, 1988;Hoosein et al, 1993;Moustakas et al, 1993). However, resistance to TGF-b in cancer may occur through several mechanisms such as reduced expression of TbRI and/or TbRII, mutations or functional inactivation of TbRII, inactivating mutations in Smad2 and Smad4 and overexpression of inhibitory proteins including Smad7 (Kleeff et al, 1999;Halder et al, 2005). In addition, increased production of TGF-b by cancer cells during tumour progression favours tumour growth, angiogenesis, and metastasis. Reduced expression of TbRII has been implicated as a mechanism for TGF-b resistance in both NSCLC and small-cell lung cancer (SCLC). A recent report has suggested that tumours of lung adenocarcinoma from nonsmokers show reduced expression of TbRII (Powell et al, 2003). In the present study, analyses of RNA and protein from lung tumours demonstrate that TbRII expression is reduced or lost in 80% of squamous cell carcinoma, 42% of adenocarcinoma and 72% of large cell carcinoma in comparison to nontumour lesions. We have observed that stable expression of TbRII restores TGF-b-induced transcription, growth inhibition and apoptosis in a lung adenocarcinoma cell line lacking TbRII. As a result these cells become less tumorigenic as determined by softagar assay and tumour xenograft studies. These studies suggest that impairment of TGF-b signalling through the loss of TbRII is involved in lung tumour progression. Cell growth is modulated by positive and negative signals, and loss of negative growth constraints may contribute to oncogenic processes. Such perturbations may occur as a consequence of the loss of the tumour suppressor functions of TGF-b. Reduced expression of TbRII has been reported in poorly differentiated lung adeno, and squamous cell carcinoma by immunohistochemical studies (Kang et al, 2000). In contrast, another study reported that a reduced expression of TGF-b1, TbRI and TbRII correlated with less lung tumour aggressiveness and a better prognosis (Takanami et al, 1997). In these studies the expression levels were determined by immunohistochemistry. However, although we have observed reduced expression of TbRII in squamous, adeno and large cell carcinoma by RT -PCR and Western blot, we do not find any direct correlation between expression level and prognosis or differentiation status. Our study is in agreement with the previous studies with lung tumour-derived cell lines that both NSCLC and SCLC cell lines show either no expression or weak expression of TbRII in 65 -75% cases (Hougaard et al, 1999;Osada et al, 2001).
One of the physiological roles of the TGF-b-signalling pathway is to provide protection against malignant transformation. While TGF-b inhibits proliferation of different normal cell types, most tumour cells including gastric, colon, and lung carcinomas are resistant to TGF-b-induced growth arrest. As mutations or deletions in receptors or Smads are not common in NSCLC or SCLC, downregulation of the expression of TbRII may be a common mechanism for lung tumours to be resistant to TGF-b tumour suppressor functions. To test the effect of the loss of TbRII expression in lung cancer, we have used lung adenocarcinoma cell line VMRC-LCD that is insensitive to growth inhibitory effects of TGF-b and that lacks TbRII expression. Our study shows that expression of physiological level of functional TbRII restores TGFb-mediated transcription by inducing complex formation between Smad2/3 and Smad4, suggesting that TGF-b/Smad-signalling cascade downstream of receptors is intact. A comparable level of TGF-b-induced transcriptional response and growth inhibition has been observed in stable TbRII-expressing VMRC-LCD clones and A549 cells with endogenous TbRII expression. Although these two cell lines are originally derived from lung adenocarcinoma, direct comparison in TGF-b signalling is not possible due to different gene expression level and mutational status. Interestingly, TbRIIexpressing clones are growth inhibited by both endogenous and exogenous TGF-b supporting the fact that lung cancer cells could escape from TGF-b-induced growth inhibition by losing the expression of TbRII. In previous study, we observed that VMRC-LCD cells produce significant amount of TGF-b (Halder et al, 2005) that may inhibit growth of these clones in an autocrine manner after stable expression of TbRII. The effect of endogenous TGF-b on growth inhibition in VMRC-LCD cells is further supported by the fact that a TGF-b receptor kinase inhibitor specifically blocks this effect in TbRII-expressing clones. Although TGF-b is known to be a potent growth inhibitor, it also functions as an inducer of apoptosis. Stable expression of TbRII in these cells restores TGF-b-induced apoptosis. It is possible that loss of TGF-b-mediated apoptosis by reduced expression of TbRII may be involved in the lung tumour progression. The increased expression and activation of TGF-b by tumour cells may induce pro-oncogenic effects that result in the progression of epithelial tumours to the metastatic stage. However, after restoration of TGF-b signalling in VMRC-LCD cells we do not observe any change in the tumour-promoting effects of TGF-b including cell motility, epithelial to mesenchymal transition, migration and invasion (data not shown).
Loss of TGF-b response has been reported to be associated with tumour development and/or progression in a number of cancer cell lines. While some TGF-b-resistant cancer cell lines have been shown to retain intact TGF-b receptors, majority of the cancer cell lines show the loss of TGF-b tumour suppressor function that could be associated with reduced expression of TGF-b receptors. We have observed reduced cloning efficiency in soft-agar assay by TbRII-expressing clones. In addition, stable expression of TbRII in these cells decreases tumorigenicity in vivo. Therefore, loss of TbRII expression leads to the generation of an aggressive phenotype in lung carcinoma cells and restoration of TGF-binduced tumour suppressor function through the expression of TbRII plays an important role in decreasing tumorigenicity.
In conclusion, these data support the notion that TGF-b type II receptor plays a critical role in cell proliferation and lung carcinogenesis. The defective expression of TbRII may provide an important molecular mechanism in explaining unresponsiveness to TGF-b in lung carcinomas. Stable expression of TbRII alone plays an important functional role in reducing tumour growth by restoring TGF-b-mediated tumour suppressor functions in lung tumour cells that lack TbRII. Our study showed that overexpression of TbRII restored TGF-b sensitivity and reduced the tumour growth. As majority of NSCLC and SCLC cell lines, that are not responsive to TGF-b-induced growth inhibition, show weak or no expression of TbRII (Hougaard et al, 1999;Osada et al, 2001), restoration of TGF-b signalling through the