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Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

Dzieran J, Fabian J, Feng T, Coulouarn C, Ilkavets I, Kyselova A, Breuhahn K, Dooley S, Meindl-Beinker NM - PLoS ONE (2013)

Bottom Line: TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling.Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients.RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7.

View Article: PubMed Central - PubMed

Affiliation: Molecular Hepatology - Alcohol Associated Diseases, Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

ABSTRACT
Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

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While Smad2 phosphorylation duration correlates to TGF-β and Smad7 expression, cytostatic responsiveness relates to CAGA-reporter-activation.HCC cell lines were treated with 5 ng/ml TGF-β for 0, 1 (3),, 7, 24 and 48 h. (A) Left side: Western blot analysis of Smad2 phosphorylation and GAPDH expression. Right side: To evaluate TGF-β dependent transcriptional activity of Smad2, cells were transfected with plasmids carrying a luciferase gene under control of the activin response element (ARE) and additionally with a FAST-1 expression construct. Luciferase activity of cells treated with 5 ng/ml TGF-β for 9 h was correlated to untreated control samples. (B) Left side: Western blot analysis of Smad1/3 phosphorylation (1: upper/3: lower band); loading control GAPDH. Right side: TGF-β dependent transcriptional activity of Smad3 was evaluated 9 h after TGF-β treatment in cells infected with an adenovirus carrying a luciferase gene controlled by a CAGA response element. Treated samples were correlated to the corresponding untreated control. In the table, cell lines marked as black, show TGF-β induced cell death and growth inhibition whereas cells marked as grey mainly react with the latter one. The gradient from white to black displays increasing basal Smad7 expression levels. (C) Immunofluorescent detection of linker phosphorylated Smad3 in uninduced HCC-T cells. Red fluorescence indicates nuclear localization of pSmad3L. Blue staining indicates nuclei stained with DRAQ5. Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).
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pone-0072252-g005: While Smad2 phosphorylation duration correlates to TGF-β and Smad7 expression, cytostatic responsiveness relates to CAGA-reporter-activation.HCC cell lines were treated with 5 ng/ml TGF-β for 0, 1 (3),, 7, 24 and 48 h. (A) Left side: Western blot analysis of Smad2 phosphorylation and GAPDH expression. Right side: To evaluate TGF-β dependent transcriptional activity of Smad2, cells were transfected with plasmids carrying a luciferase gene under control of the activin response element (ARE) and additionally with a FAST-1 expression construct. Luciferase activity of cells treated with 5 ng/ml TGF-β for 9 h was correlated to untreated control samples. (B) Left side: Western blot analysis of Smad1/3 phosphorylation (1: upper/3: lower band); loading control GAPDH. Right side: TGF-β dependent transcriptional activity of Smad3 was evaluated 9 h after TGF-β treatment in cells infected with an adenovirus carrying a luciferase gene controlled by a CAGA response element. Treated samples were correlated to the corresponding untreated control. In the table, cell lines marked as black, show TGF-β induced cell death and growth inhibition whereas cells marked as grey mainly react with the latter one. The gradient from white to black displays increasing basal Smad7 expression levels. (C) Immunofluorescent detection of linker phosphorylated Smad3 in uninduced HCC-T cells. Red fluorescence indicates nuclear localization of pSmad3L. Blue staining indicates nuclei stained with DRAQ5. Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).

Mentions: As altered expression levels of signaling components may not necessarily reflect activated signal transduction, we investigated the phosphorylation i.e. activation of Smad2 (Figure 5A, Figure S5). Some cell lines (HCC-M, HCC-T, PLC, Hep3B, HuH7) exhibited a prolonged pSmad2 signal after continuous stimulation with TGF-β up to 48h, whereas others (HepG2, HLE, HLF) faded out after 1-7h of TGF-β treatment. In FLC-4 und HuH6 cells pSmad2 signal peaks after 1 hour and then stabilizes on a lower level. This let us sort the cell lines into transiently and long term activated responders with regard to Smad2 phosphorylation.


Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

Dzieran J, Fabian J, Feng T, Coulouarn C, Ilkavets I, Kyselova A, Breuhahn K, Dooley S, Meindl-Beinker NM - PLoS ONE (2013)

While Smad2 phosphorylation duration correlates to TGF-β and Smad7 expression, cytostatic responsiveness relates to CAGA-reporter-activation.HCC cell lines were treated with 5 ng/ml TGF-β for 0, 1 (3),, 7, 24 and 48 h. (A) Left side: Western blot analysis of Smad2 phosphorylation and GAPDH expression. Right side: To evaluate TGF-β dependent transcriptional activity of Smad2, cells were transfected with plasmids carrying a luciferase gene under control of the activin response element (ARE) and additionally with a FAST-1 expression construct. Luciferase activity of cells treated with 5 ng/ml TGF-β for 9 h was correlated to untreated control samples. (B) Left side: Western blot analysis of Smad1/3 phosphorylation (1: upper/3: lower band); loading control GAPDH. Right side: TGF-β dependent transcriptional activity of Smad3 was evaluated 9 h after TGF-β treatment in cells infected with an adenovirus carrying a luciferase gene controlled by a CAGA response element. Treated samples were correlated to the corresponding untreated control. In the table, cell lines marked as black, show TGF-β induced cell death and growth inhibition whereas cells marked as grey mainly react with the latter one. The gradient from white to black displays increasing basal Smad7 expression levels. (C) Immunofluorescent detection of linker phosphorylated Smad3 in uninduced HCC-T cells. Red fluorescence indicates nuclear localization of pSmad3L. Blue staining indicates nuclei stained with DRAQ5. Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).
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pone-0072252-g005: While Smad2 phosphorylation duration correlates to TGF-β and Smad7 expression, cytostatic responsiveness relates to CAGA-reporter-activation.HCC cell lines were treated with 5 ng/ml TGF-β for 0, 1 (3),, 7, 24 and 48 h. (A) Left side: Western blot analysis of Smad2 phosphorylation and GAPDH expression. Right side: To evaluate TGF-β dependent transcriptional activity of Smad2, cells were transfected with plasmids carrying a luciferase gene under control of the activin response element (ARE) and additionally with a FAST-1 expression construct. Luciferase activity of cells treated with 5 ng/ml TGF-β for 9 h was correlated to untreated control samples. (B) Left side: Western blot analysis of Smad1/3 phosphorylation (1: upper/3: lower band); loading control GAPDH. Right side: TGF-β dependent transcriptional activity of Smad3 was evaluated 9 h after TGF-β treatment in cells infected with an adenovirus carrying a luciferase gene controlled by a CAGA response element. Treated samples were correlated to the corresponding untreated control. In the table, cell lines marked as black, show TGF-β induced cell death and growth inhibition whereas cells marked as grey mainly react with the latter one. The gradient from white to black displays increasing basal Smad7 expression levels. (C) Immunofluorescent detection of linker phosphorylated Smad3 in uninduced HCC-T cells. Red fluorescence indicates nuclear localization of pSmad3L. Blue staining indicates nuclei stained with DRAQ5. Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).
Mentions: As altered expression levels of signaling components may not necessarily reflect activated signal transduction, we investigated the phosphorylation i.e. activation of Smad2 (Figure 5A, Figure S5). Some cell lines (HCC-M, HCC-T, PLC, Hep3B, HuH7) exhibited a prolonged pSmad2 signal after continuous stimulation with TGF-β up to 48h, whereas others (HepG2, HLE, HLF) faded out after 1-7h of TGF-β treatment. In FLC-4 und HuH6 cells pSmad2 signal peaks after 1 hour and then stabilizes on a lower level. This let us sort the cell lines into transiently and long term activated responders with regard to Smad2 phosphorylation.

Bottom Line: TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling.Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients.RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7.

View Article: PubMed Central - PubMed

Affiliation: Molecular Hepatology - Alcohol Associated Diseases, Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

ABSTRACT
Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

Show MeSH
Related in: MedlinePlus