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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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TGF-β induced cell death is Smad3 dependent.RNA interference technology was used to downregulate Smad2 (S2) and Smad3 (S3) in PLC/PRF/5 (upper panel), Hep3B (middle panel) and HuH7 (lower panel) cells. An unspecific siRNA sequence (co) was used as control. Knockdown was allowed to take place for 48 h. Afterwards, each setup was treated with or without 5 ng/ml TGF-β for 1 (Immunoblot) or 3 days (cell death and proliferation) (Left). Western blot analysis against phosphorylated and total Smad2 and Smad1/3 was performed to confirm successful knockdown. GAPDH was used as loading control (Middle). After 3 days with or without TGF-β, cell death rates were evaluated using an LDH assay. Untreated cells for each siRNA were defined as 0 (grey line). Filled bars show TGF-β treated samples (Right). LDH content of viable (adherent) cells was used to determine proliferation rates. TGF-β treated samples (filled bars) were related to untreated siRNA samples (grey line).
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pone-0072252-g008: TGF-β induced cell death is Smad3 dependent.RNA interference technology was used to downregulate Smad2 (S2) and Smad3 (S3) in PLC/PRF/5 (upper panel), Hep3B (middle panel) and HuH7 (lower panel) cells. An unspecific siRNA sequence (co) was used as control. Knockdown was allowed to take place for 48 h. Afterwards, each setup was treated with or without 5 ng/ml TGF-β for 1 (Immunoblot) or 3 days (cell death and proliferation) (Left). Western blot analysis against phosphorylated and total Smad2 and Smad1/3 was performed to confirm successful knockdown. GAPDH was used as loading control (Middle). After 3 days with or without TGF-β, cell death rates were evaluated using an LDH assay. Untreated cells for each siRNA were defined as 0 (grey line). Filled bars show TGF-β treated samples (Right). LDH content of viable (adherent) cells was used to determine proliferation rates. TGF-β treated samples (filled bars) were related to untreated siRNA samples (grey line).

Mentions: Taken together, the clusters demonstrate that disrupted Smad3 downstream signaling is required for loss of cytostatic TGF-β effects in liver cancer. Additionally, TGF-β strongly enhanced Smad3 expression and its transcriptional activity in cell lines with retained TGF-β mediated cytostasis. For functional proof of the critical role of Smad3 in TGF-β mediated cytostasis, we knocked down Smad3 or Smad2 in Hep3B, HuH7 and PLC, and investigated the resulting TGF-β effects on apoptosis and proliferation inhibition (Figure 8). In line with our hypothesis, we find that Smad3 knock down diminishes TGF-β induced cytostasis, while the effect of Smad2 knock down is comparably little. The fact that siRNA against Smad2 also reduces Smad3 expression to some extent may even direct the observed Smad2 knock down effects towards Smad3 function.


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)

TGF-β induced cell death is Smad3 dependent.RNA interference technology was used to downregulate Smad2 (S2) and Smad3 (S3) in PLC/PRF/5 (upper panel), Hep3B (middle panel) and HuH7 (lower panel) cells. An unspecific siRNA sequence (co) was used as control. Knockdown was allowed to take place for 48 h. Afterwards, each setup was treated with or without 5 ng/ml TGF-β for 1 (Immunoblot) or 3 days (cell death and proliferation) (Left). Western blot analysis against phosphorylated and total Smad2 and Smad1/3 was performed to confirm successful knockdown. GAPDH was used as loading control (Middle). After 3 days with or without TGF-β, cell death rates were evaluated using an LDH assay. Untreated cells for each siRNA were defined as 0 (grey line). Filled bars show TGF-β treated samples (Right). LDH content of viable (adherent) cells was used to determine proliferation rates. TGF-β treated samples (filled bars) were related to untreated siRNA samples (grey line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750029&req=5

pone-0072252-g008: TGF-β induced cell death is Smad3 dependent.RNA interference technology was used to downregulate Smad2 (S2) and Smad3 (S3) in PLC/PRF/5 (upper panel), Hep3B (middle panel) and HuH7 (lower panel) cells. An unspecific siRNA sequence (co) was used as control. Knockdown was allowed to take place for 48 h. Afterwards, each setup was treated with or without 5 ng/ml TGF-β for 1 (Immunoblot) or 3 days (cell death and proliferation) (Left). Western blot analysis against phosphorylated and total Smad2 and Smad1/3 was performed to confirm successful knockdown. GAPDH was used as loading control (Middle). After 3 days with or without TGF-β, cell death rates were evaluated using an LDH assay. Untreated cells for each siRNA were defined as 0 (grey line). Filled bars show TGF-β treated samples (Right). LDH content of viable (adherent) cells was used to determine proliferation rates. TGF-β treated samples (filled bars) were related to untreated siRNA samples (grey line).
Mentions: Taken together, the clusters demonstrate that disrupted Smad3 downstream signaling is required for loss of cytostatic TGF-β effects in liver cancer. Additionally, TGF-β strongly enhanced Smad3 expression and its transcriptional activity in cell lines with retained TGF-β mediated cytostasis. For functional proof of the critical role of Smad3 in TGF-β mediated cytostasis, we knocked down Smad3 or Smad2 in Hep3B, HuH7 and PLC, and investigated the resulting TGF-β effects on apoptosis and proliferation inhibition (Figure 8). In line with our hypothesis, we find that Smad3 knock down diminishes TGF-β induced cytostasis, while the effect of Smad2 knock down is comparably little. The fact that siRNA against Smad2 also reduces Smad3 expression to some extent may even direct the observed Smad2 knock down effects towards Smad3 function.

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