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Angiogenin secretion from hepatoma cells activates hepatic stellate cells to amplify a self-sustained cycle promoting liver cancer.

Bárcena C, Stefanovic M, Tutusaus A, Martinez-Nieto GA, Martinez L, García-Ruiz C, de Mingo A, Caballeria J, Fernandez-Checa JC, Marí M, Morales A - Sci Rep (2015)

Bottom Line: Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules.Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin.These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Death and Proliferation, IIBB-CSIC, IDIBAPS, Barcelona, Spain.

ABSTRACT
Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

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Angiogenin nuclear deposition and HSC activation is reduced by neomycin administration.A, Angiogenin subcellular location (green) was visualized by confocal immunofluorescence in HSCs treated with rANG (1 μg/ml) and/or neomycin preincubation (100 μM) by nuclear (red) co-staining with Hoechst 33258. Representatives images of α-SMA protein expression: B, analyzed after HSC treatment with different doses of angiogenin (0.1 and 1 μg/ml) and/or neomycin (100 μM) for 3 days; and C, after HSC treatment with control medium (−), or CM-HepG2 (CM) and/or neomycin (N) for 5 days.
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f5: Angiogenin nuclear deposition and HSC activation is reduced by neomycin administration.A, Angiogenin subcellular location (green) was visualized by confocal immunofluorescence in HSCs treated with rANG (1 μg/ml) and/or neomycin preincubation (100 μM) by nuclear (red) co-staining with Hoechst 33258. Representatives images of α-SMA protein expression: B, analyzed after HSC treatment with different doses of angiogenin (0.1 and 1 μg/ml) and/or neomycin (100 μM) for 3 days; and C, after HSC treatment with control medium (−), or CM-HepG2 (CM) and/or neomycin (N) for 5 days.

Mentions: Previous studies have reported that the uptake of angiogenin and its nuclear translocation are required for translational changes and ribosomal biogenesis. Moreover, angiogenin-induced reprogramming may be antagonized by genetic knockdown or by interfering with its nuclear location by the aminoglycoside antibiotic neomycin121718. To verify if angiogenin undergoes a distribution change in activated HSCs, and to validate the potential use of neomycin as an inhibitor of angiogenin, we followed angiogenin distribution in LX2 cells by confocal microscopy (Fig. 5A). After administration of rANG, both nuclear and perinuclear distribution of angiogenin was detected in LX2 cells. Interestingly, neomycin incubation antagonized angiogenin nuclear trafficking, resulting in increased perinuclear location (Fig. 5A), blocked nuclear deposition that precluded interaction of DNA with angiogenin, as indicated by the lack of colocalization with the DNA-binding dye Hoechst 33258. To evaluate if neomycin blockage of angiogenin signaling in HSC is translated in a direct effect on proliferative and profibrotic markers we analyzed neomycin effect on HSC-treated with conditioned medium and with rANG. In accordance with a role of angiogenin in HSC transformation, neomycin reduced the phenotypic changes induced by CM-HepG2 in primary HSCs as indicated by α-SMA, MMP9 and PCNA expression (Fig. 5B). Of note, neomycin decreased HSC activation by rANG, measured by α-SMA protein level (Fig. 5C).


Angiogenin secretion from hepatoma cells activates hepatic stellate cells to amplify a self-sustained cycle promoting liver cancer.

Bárcena C, Stefanovic M, Tutusaus A, Martinez-Nieto GA, Martinez L, García-Ruiz C, de Mingo A, Caballeria J, Fernandez-Checa JC, Marí M, Morales A - Sci Rep (2015)

Angiogenin nuclear deposition and HSC activation is reduced by neomycin administration.A, Angiogenin subcellular location (green) was visualized by confocal immunofluorescence in HSCs treated with rANG (1 μg/ml) and/or neomycin preincubation (100 μM) by nuclear (red) co-staining with Hoechst 33258. Representatives images of α-SMA protein expression: B, analyzed after HSC treatment with different doses of angiogenin (0.1 and 1 μg/ml) and/or neomycin (100 μM) for 3 days; and C, after HSC treatment with control medium (−), or CM-HepG2 (CM) and/or neomycin (N) for 5 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300499&req=5

f5: Angiogenin nuclear deposition and HSC activation is reduced by neomycin administration.A, Angiogenin subcellular location (green) was visualized by confocal immunofluorescence in HSCs treated with rANG (1 μg/ml) and/or neomycin preincubation (100 μM) by nuclear (red) co-staining with Hoechst 33258. Representatives images of α-SMA protein expression: B, analyzed after HSC treatment with different doses of angiogenin (0.1 and 1 μg/ml) and/or neomycin (100 μM) for 3 days; and C, after HSC treatment with control medium (−), or CM-HepG2 (CM) and/or neomycin (N) for 5 days.
Mentions: Previous studies have reported that the uptake of angiogenin and its nuclear translocation are required for translational changes and ribosomal biogenesis. Moreover, angiogenin-induced reprogramming may be antagonized by genetic knockdown or by interfering with its nuclear location by the aminoglycoside antibiotic neomycin121718. To verify if angiogenin undergoes a distribution change in activated HSCs, and to validate the potential use of neomycin as an inhibitor of angiogenin, we followed angiogenin distribution in LX2 cells by confocal microscopy (Fig. 5A). After administration of rANG, both nuclear and perinuclear distribution of angiogenin was detected in LX2 cells. Interestingly, neomycin incubation antagonized angiogenin nuclear trafficking, resulting in increased perinuclear location (Fig. 5A), blocked nuclear deposition that precluded interaction of DNA with angiogenin, as indicated by the lack of colocalization with the DNA-binding dye Hoechst 33258. To evaluate if neomycin blockage of angiogenin signaling in HSC is translated in a direct effect on proliferative and profibrotic markers we analyzed neomycin effect on HSC-treated with conditioned medium and with rANG. In accordance with a role of angiogenin in HSC transformation, neomycin reduced the phenotypic changes induced by CM-HepG2 in primary HSCs as indicated by α-SMA, MMP9 and PCNA expression (Fig. 5B). Of note, neomycin decreased HSC activation by rANG, measured by α-SMA protein level (Fig. 5C).

Bottom Line: Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules.Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin.These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Death and Proliferation, IIBB-CSIC, IDIBAPS, Barcelona, Spain.

ABSTRACT
Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

Show MeSH
Related in: MedlinePlus