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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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LX2 cells are activated by conditioned medium (CM) from HepG2 and Hep3B cells and promote tumor growth in mice.A, Representative western blot showing α-SMA activation, using β-actin levels as a control, of human LX2 cells previously exposed to conditioned medium from HepG2 and Hep3B. B and C, mRNA levels of genes associated to HSC activation measured in LX2 cells exposed to conditioned medium from HepG2 and Hep3B. D and E, Evolution of tumor growth in a murine subcutaneous model of Hep3B and HepG2 cells injected alone or combined with LX2 cells. (n = 3). *, p ≤ 0.05, Hep3B/LX2 and HepG2/LX2 vs. Hep3B and HepG2, respectively.
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f2: LX2 cells are activated by conditioned medium (CM) from HepG2 and Hep3B cells and promote tumor growth in mice.A, Representative western blot showing α-SMA activation, using β-actin levels as a control, of human LX2 cells previously exposed to conditioned medium from HepG2 and Hep3B. B and C, mRNA levels of genes associated to HSC activation measured in LX2 cells exposed to conditioned medium from HepG2 and Hep3B. D and E, Evolution of tumor growth in a murine subcutaneous model of Hep3B and HepG2 cells injected alone or combined with LX2 cells. (n = 3). *, p ≤ 0.05, Hep3B/LX2 and HepG2/LX2 vs. Hep3B and HepG2, respectively.

Mentions: To verify if hepatoma-derived medium could also potentiate human HSC cell activation, LX2 cells were exposed to conditioned medium from HepG2 and Hep3B. Although LX2 is a clonal myofibroblastic HSC cell line, frequently used as a surrogate of human activated HSCs, both cell lines further increased LX2 activation monitored by α-SMA (Fig. 2A), supporting the pro-fibrogenic capacity of HCC secretome. Moreover, either HepG2 (Fig. 2B) or Hep3B (Fig. 2C) enhanced the mRNA expression of genes induced during HSC transformation, such as TGFβ, COL1A1 or α-SMA. Of note, CM from HepG2 cells induced a more potent stimulation, maybe indicating a major content of HSC-activating molecules.


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)

LX2 cells are activated by conditioned medium (CM) from HepG2 and Hep3B cells and promote tumor growth in mice.A, Representative western blot showing α-SMA activation, using β-actin levels as a control, of human LX2 cells previously exposed to conditioned medium from HepG2 and Hep3B. B and C, mRNA levels of genes associated to HSC activation measured in LX2 cells exposed to conditioned medium from HepG2 and Hep3B. D and E, Evolution of tumor growth in a murine subcutaneous model of Hep3B and HepG2 cells injected alone or combined with LX2 cells. (n = 3). *, p ≤ 0.05, Hep3B/LX2 and HepG2/LX2 vs. Hep3B and HepG2, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: LX2 cells are activated by conditioned medium (CM) from HepG2 and Hep3B cells and promote tumor growth in mice.A, Representative western blot showing α-SMA activation, using β-actin levels as a control, of human LX2 cells previously exposed to conditioned medium from HepG2 and Hep3B. B and C, mRNA levels of genes associated to HSC activation measured in LX2 cells exposed to conditioned medium from HepG2 and Hep3B. D and E, Evolution of tumor growth in a murine subcutaneous model of Hep3B and HepG2 cells injected alone or combined with LX2 cells. (n = 3). *, p ≤ 0.05, Hep3B/LX2 and HepG2/LX2 vs. Hep3B and HepG2, respectively.
Mentions: To verify if hepatoma-derived medium could also potentiate human HSC cell activation, LX2 cells were exposed to conditioned medium from HepG2 and Hep3B. Although LX2 is a clonal myofibroblastic HSC cell line, frequently used as a surrogate of human activated HSCs, both cell lines further increased LX2 activation monitored by α-SMA (Fig. 2A), supporting the pro-fibrogenic capacity of HCC secretome. Moreover, either HepG2 (Fig. 2B) or Hep3B (Fig. 2C) enhanced the mRNA expression of genes induced during HSC transformation, such as TGFβ, COL1A1 or α-SMA. Of note, CM from HepG2 cells induced a more potent stimulation, maybe indicating a major content of HSC-activating molecules.

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