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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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Induction of Smad7 expression by TGF-β correlates with cytostatic responsiveness.(A) Left side: Cells were treated with or without TGF-β for 2 h. Smad7 expression and 18S rRNA levels as reference gene were detected by real time PCR. Right side: For evaluation of transcriptional activity, HCC cell lines were transfected with a construct containing a luciferase gene under control of the Smad7-promotor and treated with TGF-β for 6 h. Treated samples were correlated to untreated controls. (B) Changes in expression levels of Smad3 target genes Bim and PAI-1 were detected after 2 h (PAI-1) or 24 h (Bim) using real time PCR analysis with rS18 as reference gene. The tables highlight cell lines with highest CAGA activity (black fields). 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-g004: Induction of Smad7 expression by TGF-β correlates with cytostatic responsiveness.(A) Left side: Cells were treated with or without TGF-β for 2 h. Smad7 expression and 18S rRNA levels as reference gene were detected by real time PCR. Right side: For evaluation of transcriptional activity, HCC cell lines were transfected with a construct containing a luciferase gene under control of the Smad7-promotor and treated with TGF-β for 6 h. Treated samples were correlated to untreated controls. (B) Changes in expression levels of Smad3 target genes Bim and PAI-1 were detected after 2 h (PAI-1) or 24 h (Bim) using real time PCR analysis with rS18 as reference gene. The tables highlight cell lines with highest CAGA activity (black fields). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).

Mentions: In order to mimic the response of hepatocytes to TGF-β secreted by other cell types, we investigated the impact of TGF-β stimulation on expression of TGF-β signaling components. Smad2 and Smad4 (Figure 3A, 3B, Figure S4) levels did not vary upon 24h TGF-β treatment, whereas Smad3 (Figure 3A, 3B, Figure S4) and Smad7 (Figure 4A) expression was significantly induced mostly in cytostasis responsive cell lines (including HuH6), 24h and 2h after TGF-β treatment. TGF-β-induced expression of Smad7 (Figure 4A) was inversely correlated with intrinsic Smad7 expression, excluding HCC-M and HCC-T (Figure 2.A).


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)

Induction of Smad7 expression by TGF-β correlates with cytostatic responsiveness.(A) Left side: Cells were treated with or without TGF-β for 2 h. Smad7 expression and 18S rRNA levels as reference gene were detected by real time PCR. Right side: For evaluation of transcriptional activity, HCC cell lines were transfected with a construct containing a luciferase gene under control of the Smad7-promotor and treated with TGF-β for 6 h. Treated samples were correlated to untreated controls. (B) Changes in expression levels of Smad3 target genes Bim and PAI-1 were detected after 2 h (PAI-1) or 24 h (Bim) using real time PCR analysis with rS18 as reference gene. The tables highlight cell lines with highest CAGA activity (black fields). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750029&req=5

pone-0072252-g004: Induction of Smad7 expression by TGF-β correlates with cytostatic responsiveness.(A) Left side: Cells were treated with or without TGF-β for 2 h. Smad7 expression and 18S rRNA levels as reference gene were detected by real time PCR. Right side: For evaluation of transcriptional activity, HCC cell lines were transfected with a construct containing a luciferase gene under control of the Smad7-promotor and treated with TGF-β for 6 h. Treated samples were correlated to untreated controls. (B) Changes in expression levels of Smad3 target genes Bim and PAI-1 were detected after 2 h (PAI-1) or 24 h (Bim) using real time PCR analysis with rS18 as reference gene. The tables highlight cell lines with highest CAGA activity (black fields). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).
Mentions: In order to mimic the response of hepatocytes to TGF-β secreted by other cell types, we investigated the impact of TGF-β stimulation on expression of TGF-β signaling components. Smad2 and Smad4 (Figure 3A, 3B, Figure S4) levels did not vary upon 24h TGF-β treatment, whereas Smad3 (Figure 3A, 3B, Figure S4) and Smad7 (Figure 4A) expression was significantly induced mostly in cytostasis responsive cell lines (including HuH6), 24h and 2h after TGF-β treatment. TGF-β-induced expression of Smad7 (Figure 4A) was inversely correlated with intrinsic Smad7 expression, excluding HCC-M and HCC-T (Figure 2.A).

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