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

Anumanthan G, Halder SK, Osada H, Takahashi T, Massion PP, Carbone DP, Datta PK - Br. J. Cancer (2005)

Bottom Line: We also determined the effect of TbetaRII expression in lung adenocarcinoma cell line (VMRC-LCD) that is not responsive to TGF-beta due to lack of TbetaRII expression.Stable expression of TbetaRII in these cells restored TGF-beta-mediated effects including Smad2/3 and Smad4 complex formation, TGF-beta-responsive reporter gene activation, inhibition of cell proliferation and increased apoptosis.Clones expressing TbetaRII showed reduced colony formation in soft-agarose assay and significantly reduced tumorigenicity in athymic nude mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer Biology, Division of Surgical Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

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

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Stable VMRC-LCD cells expressing TβRII. (A) The VMRC-LCD cells were transfected with TβRII-pcDNA3 or empty pcDNA3 vector (Invitrogen) and selected with G418 for 2 weeks to establish the stable clones. In all, 20 ng of total RNA was used for TβRII stable VMRC-LCD clones, vector clones and parental cells. Quantitative real-time RT–PCR was performed to determine the relative mRNA expression level of each TβRII stable clones. Dilutions of RNA isolated from A549 cells was used as standards for comparison. The stable expression of TβRII mRNA in VMRC-LCD clones were compared with the endogenous TβRII expression in A549 cells. (B) Cell lysates from parental, vector clone, and stable TβRII clones were subjected to immunoblotting with anti-TβRII antibody. Expression of TβRII protein in individual clones is shown. Equal amount of protein loading was verified by immunoblotting the membrane with anti-β-actin antibody.
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fig2: Stable VMRC-LCD cells expressing TβRII. (A) The VMRC-LCD cells were transfected with TβRII-pcDNA3 or empty pcDNA3 vector (Invitrogen) and selected with G418 for 2 weeks to establish the stable clones. In all, 20 ng of total RNA was used for TβRII stable VMRC-LCD clones, vector clones and parental cells. Quantitative real-time RT–PCR was performed to determine the relative mRNA expression level of each TβRII stable clones. Dilutions of RNA isolated from A549 cells was used as standards for comparison. The stable expression of TβRII mRNA in VMRC-LCD clones were compared with the endogenous TβRII expression in A549 cells. (B) Cell lysates from parental, vector clone, and stable TβRII clones were subjected to immunoblotting with anti-TβRII antibody. Expression of TβRII protein in individual clones is shown. Equal amount of protein loading was verified by immunoblotting the membrane with anti-β-actin antibody.

Mentions: In order to express TβRII in VMRC-LCD lung adenocarcinoma cells lacking its expression, wild-type TβRII 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 TβRII mRNA in stable VMRC-LCD cells and in lung adenocarcinoma cells (A549) (Figure 2A). These data suggest the physiological level of expression of TβRII in VMRC-LCD cells. We also tested expression of the protein by Western blot analyses (Figure 2B). Three clones that expressed high levels of TβRII (TβRII #10, TβRII #13 and TβRII #17) were selected for further experiments. To test whether overexpressed TβRII is functional, we first analysed the phosphorylation of endogenous-positive regulatory Smads, Smad2 and Smad3. Parental cells, two vector control clones, and three TβRII clones were treated with TGF-β 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 TβRII stable clones although the expressions of these proteins were unchanged. To test whether stable expression of TβRII can restore the complex formation between Smad2/Smad3 and Smad4 in vivo, we performed immunoprecipitation experiments after treating TβRII clones, vector clones, and parental cells with TGF-β 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-β-induced heteromeric complex formation between Smad2/Smad3 and Smad4 was increased in TβRII-expressing clones as compared to parental and vector control clones (Figure 3B). To confirm the TGF-β downstream signalling is intact in VMRC-LCD cells, we performed transcriptional assays using TGF-β-responsive reporter (CAGA)9 MLP-Luc. We observed an increase in transcriptional activity by transfection of TβRII and a further strong induction in response to TGF-β. Transfection of constitutively active TGF-β type I receptor (T204D) (act-TβRI) alone induced the reporter activity in VMRC-LCD cells (Figure 3C), because act-TβRI does not require TGF-β and TβRII for downstream signalling. These results suggest that VMRC-LCD cells lack TβRII expression and the downstream signalling cascade is intact. Together, stable expression of TβRII restores TGF-β/Smad signalling in VMRC-LCD cells.


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

Anumanthan G, Halder SK, Osada H, Takahashi T, Massion PP, Carbone DP, Datta PK - Br. J. Cancer (2005)

Stable VMRC-LCD cells expressing TβRII. (A) The VMRC-LCD cells were transfected with TβRII-pcDNA3 or empty pcDNA3 vector (Invitrogen) and selected with G418 for 2 weeks to establish the stable clones. In all, 20 ng of total RNA was used for TβRII stable VMRC-LCD clones, vector clones and parental cells. Quantitative real-time RT–PCR was performed to determine the relative mRNA expression level of each TβRII stable clones. Dilutions of RNA isolated from A549 cells was used as standards for comparison. The stable expression of TβRII mRNA in VMRC-LCD clones were compared with the endogenous TβRII expression in A549 cells. (B) Cell lysates from parental, vector clone, and stable TβRII clones were subjected to immunoblotting with anti-TβRII antibody. Expression of TβRII protein in individual clones is shown. Equal amount of protein loading was verified by immunoblotting the membrane with anti-β-actin antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361493&req=5

fig2: Stable VMRC-LCD cells expressing TβRII. (A) The VMRC-LCD cells were transfected with TβRII-pcDNA3 or empty pcDNA3 vector (Invitrogen) and selected with G418 for 2 weeks to establish the stable clones. In all, 20 ng of total RNA was used for TβRII stable VMRC-LCD clones, vector clones and parental cells. Quantitative real-time RT–PCR was performed to determine the relative mRNA expression level of each TβRII stable clones. Dilutions of RNA isolated from A549 cells was used as standards for comparison. The stable expression of TβRII mRNA in VMRC-LCD clones were compared with the endogenous TβRII expression in A549 cells. (B) Cell lysates from parental, vector clone, and stable TβRII clones were subjected to immunoblotting with anti-TβRII antibody. Expression of TβRII protein in individual clones is shown. Equal amount of protein loading was verified by immunoblotting the membrane with anti-β-actin antibody.
Mentions: In order to express TβRII in VMRC-LCD lung adenocarcinoma cells lacking its expression, wild-type TβRII 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 TβRII mRNA in stable VMRC-LCD cells and in lung adenocarcinoma cells (A549) (Figure 2A). These data suggest the physiological level of expression of TβRII in VMRC-LCD cells. We also tested expression of the protein by Western blot analyses (Figure 2B). Three clones that expressed high levels of TβRII (TβRII #10, TβRII #13 and TβRII #17) were selected for further experiments. To test whether overexpressed TβRII is functional, we first analysed the phosphorylation of endogenous-positive regulatory Smads, Smad2 and Smad3. Parental cells, two vector control clones, and three TβRII clones were treated with TGF-β 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 TβRII stable clones although the expressions of these proteins were unchanged. To test whether stable expression of TβRII can restore the complex formation between Smad2/Smad3 and Smad4 in vivo, we performed immunoprecipitation experiments after treating TβRII clones, vector clones, and parental cells with TGF-β 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-β-induced heteromeric complex formation between Smad2/Smad3 and Smad4 was increased in TβRII-expressing clones as compared to parental and vector control clones (Figure 3B). To confirm the TGF-β downstream signalling is intact in VMRC-LCD cells, we performed transcriptional assays using TGF-β-responsive reporter (CAGA)9 MLP-Luc. We observed an increase in transcriptional activity by transfection of TβRII and a further strong induction in response to TGF-β. Transfection of constitutively active TGF-β type I receptor (T204D) (act-TβRI) alone induced the reporter activity in VMRC-LCD cells (Figure 3C), because act-TβRI does not require TGF-β and TβRII for downstream signalling. These results suggest that VMRC-LCD cells lack TβRII expression and the downstream signalling cascade is intact. Together, stable expression of TβRII restores TGF-β/Smad signalling in VMRC-LCD cells.

Bottom Line: We also determined the effect of TbetaRII expression in lung adenocarcinoma cell line (VMRC-LCD) that is not responsive to TGF-beta due to lack of TbetaRII expression.Stable expression of TbetaRII in these cells restored TGF-beta-mediated effects including Smad2/3 and Smad4 complex formation, TGF-beta-responsive reporter gene activation, inhibition of cell proliferation and increased apoptosis.Clones expressing TbetaRII showed reduced colony formation in soft-agarose assay and significantly reduced tumorigenicity in athymic nude mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer Biology, Division of Surgical Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

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

Show MeSH
Related in: MedlinePlus