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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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Differential profile of angiogenesis-related proteins in the secretome of hepatoma (HepG2 and Hep3B) and human HSC activated (LX2) cells.A and B, Representative merged images of antibody microarrays for protein detection in conditioned medium (CM) from HepG2 or Hep3B (green) and compared to CM-LX2 (red) protein pattern. B and D, protein quantification was measured after calibration with internal standards and background controls. (n = 2). Indicated in red the proteins more expressed in LX2 respect to hepatoma cells, and in green the proteins more expressed in Hep3B/HepG2 compared to LX2.
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f3: Differential profile of angiogenesis-related proteins in the secretome of hepatoma (HepG2 and Hep3B) and human HSC activated (LX2) cells.A and B, Representative merged images of antibody microarrays for protein detection in conditioned medium (CM) from HepG2 or Hep3B (green) and compared to CM-LX2 (red) protein pattern. B and D, protein quantification was measured after calibration with internal standards and background controls. (n = 2). Indicated in red the proteins more expressed in LX2 respect to hepatoma cells, and in green the proteins more expressed in Hep3B/HepG2 compared to LX2.

Mentions: Since HepG2 and Hep3B cell lines were able to activate HSCs, while another human liver cell line such as LX2 did not, we analyzed a panel of cytokines, chemokines, growth factors and angiogenic factors in an attempt to identify activating molecules in the differential protein profile secreted by HCC and LX2 cells using commercial protein microarrays (Suppl. Fig. 1). In comparing the protein expression of HepG2 and Hep3B with respect to LX2 cells (Fig. 3), we observed that out of the 43 target proteins, angiogenin exhibited a marked increased expression (up to 10–20 fold) in HCC cells, being particularly high in HepG2 secretome (Fig 2B). Other proteins, such as vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), previously described as HSC activators, were clearly enhanced (Fig. 2B), while moderate increases expression of endostatin and I-TAC (CXCL11), in Hep3B secretome, were also detected (Fig. 2D). On the other hand, enhanced extracellular expression of MCP-1, IL-6 (Fig 1B), uPAR, MMP1 and MMP9 (Fig. 2D) were observed in LX2 cells compared to hepatoma cell lines, reflecting the endogenous activation of LX2 cells. Since the role of angiogenin in HSC biology has not been previously addressed, we next focused on the impact and mechanisms underlying the HSC activation by angiogenin.


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)

Differential profile of angiogenesis-related proteins in the secretome of hepatoma (HepG2 and Hep3B) and human HSC activated (LX2) cells.A and B, Representative merged images of antibody microarrays for protein detection in conditioned medium (CM) from HepG2 or Hep3B (green) and compared to CM-LX2 (red) protein pattern. B and D, protein quantification was measured after calibration with internal standards and background controls. (n = 2). Indicated in red the proteins more expressed in LX2 respect to hepatoma cells, and in green the proteins more expressed in Hep3B/HepG2 compared to LX2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Differential profile of angiogenesis-related proteins in the secretome of hepatoma (HepG2 and Hep3B) and human HSC activated (LX2) cells.A and B, Representative merged images of antibody microarrays for protein detection in conditioned medium (CM) from HepG2 or Hep3B (green) and compared to CM-LX2 (red) protein pattern. B and D, protein quantification was measured after calibration with internal standards and background controls. (n = 2). Indicated in red the proteins more expressed in LX2 respect to hepatoma cells, and in green the proteins more expressed in Hep3B/HepG2 compared to LX2.
Mentions: Since HepG2 and Hep3B cell lines were able to activate HSCs, while another human liver cell line such as LX2 did not, we analyzed a panel of cytokines, chemokines, growth factors and angiogenic factors in an attempt to identify activating molecules in the differential protein profile secreted by HCC and LX2 cells using commercial protein microarrays (Suppl. Fig. 1). In comparing the protein expression of HepG2 and Hep3B with respect to LX2 cells (Fig. 3), we observed that out of the 43 target proteins, angiogenin exhibited a marked increased expression (up to 10–20 fold) in HCC cells, being particularly high in HepG2 secretome (Fig 2B). Other proteins, such as vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), previously described as HSC activators, were clearly enhanced (Fig. 2B), while moderate increases expression of endostatin and I-TAC (CXCL11), in Hep3B secretome, were also detected (Fig. 2D). On the other hand, enhanced extracellular expression of MCP-1, IL-6 (Fig 1B), uPAR, MMP1 and MMP9 (Fig. 2D) were observed in LX2 cells compared to hepatoma cell lines, reflecting the endogenous activation of LX2 cells. Since the role of angiogenin in HSC biology has not been previously addressed, we next focused on the impact and mechanisms underlying the HSC activation by angiogenin.

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