Limits...
Gamma-smooth muscle actin expression is associated with epithelial-mesenchymal transition and stem-like properties in hepatocellular carcinoma.

Benzoubir N, Mussini C, Lejamtel C, Dos Santos A, Guillaume C, Desterke C, Samuel D, Bréchot C, Bourgeade MF, Guettier C - PLoS ONE (2015)

Bottom Line: Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells.This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.

View Article: PubMed Central - PubMed

Affiliation: Inserm, Unité 785, Villejuif, F-94800, France; Univ Paris-Sud, UMR-S 785, Villejuif, F-94800, France; DHU Hepatinov, Villejuif, France.

ABSTRACT

Background and aims: The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process.

Methods: The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series.

Results: We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.

Conclusion: Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.

No MeSH data available.


Related in: MedlinePlus

γSMA is expressed in TGF-β-induced EMT.(A) HuH7 cells and primary HCC tumour cells were treated with TGF-β for 48h and the expression of γSMA and αSMA was determined by immunofluorescence analysis. The images represent the merging of αSMA and γSMA staining. LX-2 stellate cells activated by TGF-β for 48h were used as a positive control for αSMA expression; one out of three representative experiments is shown. (B) HuH7 cells were treated with TGF-β for 48h and cell extracts were analysed by Western Blotting for the expression of αSMA and γSMA. TGF-β-treated LX-2 cells were used as positive controls for αSMA expression and p38 antibody was used as the control loading. (C) Densitometric analyses of the SMA/p38 ratio represent the mean +/- SD of three independent experiments. (D) HuH7 cells were transfected with a V5-tagged vector coding for γSMA. Cells were treated with TGF-β for 48h, and the expression of V5, γSMA and αSMA was determined by immunofluorescence analysis. Expression levels of V5 and γSMA or V5 and αSMA were determined by measuring fluorescence intensity on a linear section of the cell. One representative experiment is shown. (E) HuH7 cells were transfected or not with a V5-tagged vector coding for γSMA and cell extracts were analysed for the expression of V5, αSMA and γSMA.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482489&req=5

pone.0130559.g001: γSMA is expressed in TGF-β-induced EMT.(A) HuH7 cells and primary HCC tumour cells were treated with TGF-β for 48h and the expression of γSMA and αSMA was determined by immunofluorescence analysis. The images represent the merging of αSMA and γSMA staining. LX-2 stellate cells activated by TGF-β for 48h were used as a positive control for αSMA expression; one out of three representative experiments is shown. (B) HuH7 cells were treated with TGF-β for 48h and cell extracts were analysed by Western Blotting for the expression of αSMA and γSMA. TGF-β-treated LX-2 cells were used as positive controls for αSMA expression and p38 antibody was used as the control loading. (C) Densitometric analyses of the SMA/p38 ratio represent the mean +/- SD of three independent experiments. (D) HuH7 cells were transfected with a V5-tagged vector coding for γSMA. Cells were treated with TGF-β for 48h, and the expression of V5, γSMA and αSMA was determined by immunofluorescence analysis. Expression levels of V5 and γSMA or V5 and αSMA were determined by measuring fluorescence intensity on a linear section of the cell. One representative experiment is shown. (E) HuH7 cells were transfected or not with a V5-tagged vector coding for γSMA and cell extracts were analysed for the expression of V5, αSMA and γSMA.

Mentions: Using an anti-SMA antibody, we had previously reported that SMA was up-regulated in HuH7 cells undergoing EMT after treatment with TGF-β [16]. Using specific antibodies directed against alpha or gamma SMA, the present study focused on the expression and polymerization of these two actin isoforms in hepatic cells undergoing EMT following treatment with TGF-β. As shown in Fig 1A, immunofluorescence staining revealed a strong polymerization of γSMA in TGF-β-treated HuH7 cells in conditions where αSMA was not detectable. The same results were obtained when primary HCC cells were treated with TGF-β; suggesting that the gamma isoform of SMA might represent a mesenchymal marker of liver tumour cells. Hepatic LX-2 stellate cells were used as positive controls for αSMA expression, and interestingly the expression of both SMAs was observed in these cells with strong labelling after activation by TGF-β. Furthermore, Western blot analysis demonstrated that αSMA was not detected in hepatic cells, whether they were or were not treated with TGF-β, while as expected, αSMA expression was easily detectable in LX-2 stellate cells after activation by TGF-β (Fig 1B and 1C).


Gamma-smooth muscle actin expression is associated with epithelial-mesenchymal transition and stem-like properties in hepatocellular carcinoma.

Benzoubir N, Mussini C, Lejamtel C, Dos Santos A, Guillaume C, Desterke C, Samuel D, Bréchot C, Bourgeade MF, Guettier C - PLoS ONE (2015)

γSMA is expressed in TGF-β-induced EMT.(A) HuH7 cells and primary HCC tumour cells were treated with TGF-β for 48h and the expression of γSMA and αSMA was determined by immunofluorescence analysis. The images represent the merging of αSMA and γSMA staining. LX-2 stellate cells activated by TGF-β for 48h were used as a positive control for αSMA expression; one out of three representative experiments is shown. (B) HuH7 cells were treated with TGF-β for 48h and cell extracts were analysed by Western Blotting for the expression of αSMA and γSMA. TGF-β-treated LX-2 cells were used as positive controls for αSMA expression and p38 antibody was used as the control loading. (C) Densitometric analyses of the SMA/p38 ratio represent the mean +/- SD of three independent experiments. (D) HuH7 cells were transfected with a V5-tagged vector coding for γSMA. Cells were treated with TGF-β for 48h, and the expression of V5, γSMA and αSMA was determined by immunofluorescence analysis. Expression levels of V5 and γSMA or V5 and αSMA were determined by measuring fluorescence intensity on a linear section of the cell. One representative experiment is shown. (E) HuH7 cells were transfected or not with a V5-tagged vector coding for γSMA and cell extracts were analysed for the expression of V5, αSMA and γSMA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482489&req=5

pone.0130559.g001: γSMA is expressed in TGF-β-induced EMT.(A) HuH7 cells and primary HCC tumour cells were treated with TGF-β for 48h and the expression of γSMA and αSMA was determined by immunofluorescence analysis. The images represent the merging of αSMA and γSMA staining. LX-2 stellate cells activated by TGF-β for 48h were used as a positive control for αSMA expression; one out of three representative experiments is shown. (B) HuH7 cells were treated with TGF-β for 48h and cell extracts were analysed by Western Blotting for the expression of αSMA and γSMA. TGF-β-treated LX-2 cells were used as positive controls for αSMA expression and p38 antibody was used as the control loading. (C) Densitometric analyses of the SMA/p38 ratio represent the mean +/- SD of three independent experiments. (D) HuH7 cells were transfected with a V5-tagged vector coding for γSMA. Cells were treated with TGF-β for 48h, and the expression of V5, γSMA and αSMA was determined by immunofluorescence analysis. Expression levels of V5 and γSMA or V5 and αSMA were determined by measuring fluorescence intensity on a linear section of the cell. One representative experiment is shown. (E) HuH7 cells were transfected or not with a V5-tagged vector coding for γSMA and cell extracts were analysed for the expression of V5, αSMA and γSMA.
Mentions: Using an anti-SMA antibody, we had previously reported that SMA was up-regulated in HuH7 cells undergoing EMT after treatment with TGF-β [16]. Using specific antibodies directed against alpha or gamma SMA, the present study focused on the expression and polymerization of these two actin isoforms in hepatic cells undergoing EMT following treatment with TGF-β. As shown in Fig 1A, immunofluorescence staining revealed a strong polymerization of γSMA in TGF-β-treated HuH7 cells in conditions where αSMA was not detectable. The same results were obtained when primary HCC cells were treated with TGF-β; suggesting that the gamma isoform of SMA might represent a mesenchymal marker of liver tumour cells. Hepatic LX-2 stellate cells were used as positive controls for αSMA expression, and interestingly the expression of both SMAs was observed in these cells with strong labelling after activation by TGF-β. Furthermore, Western blot analysis demonstrated that αSMA was not detected in hepatic cells, whether they were or were not treated with TGF-β, while as expected, αSMA expression was easily detectable in LX-2 stellate cells after activation by TGF-β (Fig 1B and 1C).

Bottom Line: Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells.This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.

View Article: PubMed Central - PubMed

Affiliation: Inserm, Unité 785, Villejuif, F-94800, France; Univ Paris-Sud, UMR-S 785, Villejuif, F-94800, France; DHU Hepatinov, Villejuif, France.

ABSTRACT

Background and aims: The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process.

Methods: The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series.

Results: We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.

Conclusion: Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.

No MeSH data available.


Related in: MedlinePlus