Limits...
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.

Show MeSH

Related in: MedlinePlus

Recombinant angiogenin activates HSCs and angiogenin depletion from conditioned medium reduces HSC activation.A, Primary murine HSCs were exposure to recombinant angiogenin (rANG, 1 μg/ml) for several days (0 to 3) and phenotypic transformation was detected by changes in α-SMA, PCNA, CtsB or MMP9 expression at day 7. B, Angiogenin protein (up) and mRNA (down) levels of HepG2 cells stably transfected with shRNA control (shCTRL) or against angiogenin (shANG). C, α-SMA protein expression in HSCs after 4 or 5 days of exposure to control medium (-) or conditioned medium from HepG2 cells with shCTRL or shANG transfection. D, α−SMA protein expression after HSC treatment (day 7) with control medium (−), CM-HepG2 (CM) and CM-HepG2 where angiogenin content was previously depleted by immunoprecipitation (CM+Ab), as denoted by angiogenin detection in agarose beads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4300499&req=5

f4: Recombinant angiogenin activates HSCs and angiogenin depletion from conditioned medium reduces HSC activation.A, Primary murine HSCs were exposure to recombinant angiogenin (rANG, 1 μg/ml) for several days (0 to 3) and phenotypic transformation was detected by changes in α-SMA, PCNA, CtsB or MMP9 expression at day 7. B, Angiogenin protein (up) and mRNA (down) levels of HepG2 cells stably transfected with shRNA control (shCTRL) or against angiogenin (shANG). C, α-SMA protein expression in HSCs after 4 or 5 days of exposure to control medium (-) or conditioned medium from HepG2 cells with shCTRL or shANG transfection. D, α−SMA protein expression after HSC treatment (day 7) with control medium (−), CM-HepG2 (CM) and CM-HepG2 where angiogenin content was previously depleted by immunoprecipitation (CM+Ab), as denoted by angiogenin detection in agarose beads.

Mentions: Increased serum angiogenin levels have been detected in patients with different types of cancer, and in particular, poor HCC prognosis correlates with angiogenin expression14. However, no evidence of angiogenin effect on HSC pathophysiology has been previously reported. To address whether angiogenin may reproduce the effect of HCC-derived CM on HSC activation, primary HSCs were exposed to recombinant angiogenin (rANG) to monitor fibrogenic markers. Similar to CM-HepG2-treated HSC cells, angiogenin-treated HSCs displayed higher α-SMA proteins levels than untreated cells (Fig. 4A). Moreover, other proteins that participate in ECM remodeling, such as cathepsin B (CtsB) or MMP9, or indicative of HSC proliferation, such as PCNA, were also elevated by rANG administration (Fig. 4A).


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)

Recombinant angiogenin activates HSCs and angiogenin depletion from conditioned medium reduces HSC activation.A, Primary murine HSCs were exposure to recombinant angiogenin (rANG, 1 μg/ml) for several days (0 to 3) and phenotypic transformation was detected by changes in α-SMA, PCNA, CtsB or MMP9 expression at day 7. B, Angiogenin protein (up) and mRNA (down) levels of HepG2 cells stably transfected with shRNA control (shCTRL) or against angiogenin (shANG). C, α-SMA protein expression in HSCs after 4 or 5 days of exposure to control medium (-) or conditioned medium from HepG2 cells with shCTRL or shANG transfection. D, α−SMA protein expression after HSC treatment (day 7) with control medium (−), CM-HepG2 (CM) and CM-HepG2 where angiogenin content was previously depleted by immunoprecipitation (CM+Ab), as denoted by angiogenin detection in agarose beads.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Recombinant angiogenin activates HSCs and angiogenin depletion from conditioned medium reduces HSC activation.A, Primary murine HSCs were exposure to recombinant angiogenin (rANG, 1 μg/ml) for several days (0 to 3) and phenotypic transformation was detected by changes in α-SMA, PCNA, CtsB or MMP9 expression at day 7. B, Angiogenin protein (up) and mRNA (down) levels of HepG2 cells stably transfected with shRNA control (shCTRL) or against angiogenin (shANG). C, α-SMA protein expression in HSCs after 4 or 5 days of exposure to control medium (-) or conditioned medium from HepG2 cells with shCTRL or shANG transfection. D, α−SMA protein expression after HSC treatment (day 7) with control medium (−), CM-HepG2 (CM) and CM-HepG2 where angiogenin content was previously depleted by immunoprecipitation (CM+Ab), as denoted by angiogenin detection in agarose beads.
Mentions: Increased serum angiogenin levels have been detected in patients with different types of cancer, and in particular, poor HCC prognosis correlates with angiogenin expression14. However, no evidence of angiogenin effect on HSC pathophysiology has been previously reported. To address whether angiogenin may reproduce the effect of HCC-derived CM on HSC activation, primary HSCs were exposed to recombinant angiogenin (rANG) to monitor fibrogenic markers. Similar to CM-HepG2-treated HSC cells, angiogenin-treated HSCs displayed higher α-SMA proteins levels than untreated cells (Fig. 4A). Moreover, other proteins that participate in ECM remodeling, such as cathepsin B (CtsB) or MMP9, or indicative of HSC proliferation, such as PCNA, were also elevated by rANG administration (Fig. 4A).

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