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Pleiotropic modes of action in tumor cells of RNASET2, an evolutionary highly conserved extracellular RNase.

Lualdi M, Pedrini E, Rea K, Monti L, Scaldaferri D, Gariboldi M, Camporeale A, Ghia P, Monti E, Tomassetti A, Acquati F, Taramelli R - Oncotarget (2015)

Bottom Line: Indeed, RNASET2 expression levels were consistently found to increase following stress induction.Of note, a remarkable rearrangement of the actin cytoskeleton organization, together with changes in cell adhesion and motility, emerged as putative mechanisms by which such cell-autonomous role could occur.Altogether, these biological features allow to put forward the hypothesis that the RNASET2 protein can act as a molecular barrier for limiting the damages and tissue remodeling events occurring during the earlier step of cell transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Theoretical and Applied Sciences, Università degli Studi dell'Insubria, Varese, Italy.

ABSTRACT
As widely recognized, tumor growth entails a close and complex cross-talk among cancer cells and the surrounding tumor microenvironment. We recently described the human RNASET2 gene as one key player of such microenvironmental cross-talk. Indeed, the protein encoded by this gene is an extracellular RNase which is able to control cancer growth in a non-cell autonomous mode by inducing a sustained recruitment of immune-competent cells belonging to the monocyte/macrophage lineage within a growing tumor mass. Here, we asked whether this oncosuppressor gene is sensitive to stress challenges and whether it can trigger cell-intrinsic processes as well. Indeed, RNASET2 expression levels were consistently found to increase following stress induction. Moreover, changes in RNASET2 expression levels turned out to affect several cancer-related parameters in vitro in an ovarian cancer cell line model. Of note, a remarkable rearrangement of the actin cytoskeleton organization, together with changes in cell adhesion and motility, emerged as putative mechanisms by which such cell-autonomous role could occur. Altogether, these biological features allow to put forward the hypothesis that the RNASET2 protein can act as a molecular barrier for limiting the damages and tissue remodeling events occurring during the earlier step of cell transformation.

No MeSH data available.


Related in: MedlinePlus

RNASET2 silencing affects both cell adhesion and FA dynamics in OVCAR3 cellsA) A colorimetric in vitro cell-adhesion assay was performed on five different ECM components. Significant differences in cell-adhesion on laminin, collagen type I and collagen type IV were observed when RNASET2-silenced OVCAR3 cell clones were compared to control clones. Such differences turned out to be weakened when RNASET2-silenced cells were exposed to recombinant RNASET2 protein. Triplicate experiments were performed with four RNASET2-silenced and four control OVCAR3 clones. Statistical analysis was performed using two-tailed Student's t-test. * p<0,05; ** p<0,01. B) Evaluation of phoshoryated paxillin on total cell lysates upon adhesion on fibronectin (FN) or collagen type I (Col I). Upper panel: immunoblotting for the expression of phospho-paxillin. Lower panel: densitometric analysis reporting the levels of phosphorylated paxillin with respect to α-tubulin expression of cells grown on ECM matrices. Statistical analysis of data from triplicate experiments was performed using Student's t-test. *** p< 0.001. C) Confocal IIF performed on migrating OVCAR3 cell pools. Starved cells were induced to migrate through a wound upon stimulation with FCS for 24 hr, and then fixed with paraformaldehyde. FAs were stained with anti-phoshphorylated paxillin (green); actin was stained with phalloidin (red). Nuclei were counterstained with DAPI. The white arrow indicates dorsal stress fibers present in RNaseT2-silenced cells. Scal bar: 50 μm. D) Control vs. RNASET2-silenced OVCAR3 cells were grown in reduced growth factor-matrigel 3D culture and pictures were taken with a light microscope.
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Figure 7: RNASET2 silencing affects both cell adhesion and FA dynamics in OVCAR3 cellsA) A colorimetric in vitro cell-adhesion assay was performed on five different ECM components. Significant differences in cell-adhesion on laminin, collagen type I and collagen type IV were observed when RNASET2-silenced OVCAR3 cell clones were compared to control clones. Such differences turned out to be weakened when RNASET2-silenced cells were exposed to recombinant RNASET2 protein. Triplicate experiments were performed with four RNASET2-silenced and four control OVCAR3 clones. Statistical analysis was performed using two-tailed Student's t-test. * p<0,05; ** p<0,01. B) Evaluation of phoshoryated paxillin on total cell lysates upon adhesion on fibronectin (FN) or collagen type I (Col I). Upper panel: immunoblotting for the expression of phospho-paxillin. Lower panel: densitometric analysis reporting the levels of phosphorylated paxillin with respect to α-tubulin expression of cells grown on ECM matrices. Statistical analysis of data from triplicate experiments was performed using Student's t-test. *** p< 0.001. C) Confocal IIF performed on migrating OVCAR3 cell pools. Starved cells were induced to migrate through a wound upon stimulation with FCS for 24 hr, and then fixed with paraformaldehyde. FAs were stained with anti-phoshphorylated paxillin (green); actin was stained with phalloidin (red). Nuclei were counterstained with DAPI. The white arrow indicates dorsal stress fibers present in RNaseT2-silenced cells. Scal bar: 50 μm. D) Control vs. RNASET2-silenced OVCAR3 cells were grown in reduced growth factor-matrigel 3D culture and pictures were taken with a light microscope.

Mentions: To evaluate whether the observed effects of RNASET2 on cell morphology and migration could be modulated by a differential activation of extracellular matrix/integrin signaling, we also carried out an in vitro cell adhesion assay on control and RNASET2-silenced OVCAR3 cells grown on different ECM-derived components. As shown in Figure 7A, we could not detect any significant difference in cell adhesion on fibronectin or vitronectin. However, cell adhesion to laminin and collagens type I and IV was markedly increased in RNASET2-silenced OVCAR3 cells. Moreover, recombinant RNASET2 was able to partially restore a reduced adhesion in the latter cells, particularly with respect to collagen I (Figure 7A).


Pleiotropic modes of action in tumor cells of RNASET2, an evolutionary highly conserved extracellular RNase.

Lualdi M, Pedrini E, Rea K, Monti L, Scaldaferri D, Gariboldi M, Camporeale A, Ghia P, Monti E, Tomassetti A, Acquati F, Taramelli R - Oncotarget (2015)

RNASET2 silencing affects both cell adhesion and FA dynamics in OVCAR3 cellsA) A colorimetric in vitro cell-adhesion assay was performed on five different ECM components. Significant differences in cell-adhesion on laminin, collagen type I and collagen type IV were observed when RNASET2-silenced OVCAR3 cell clones were compared to control clones. Such differences turned out to be weakened when RNASET2-silenced cells were exposed to recombinant RNASET2 protein. Triplicate experiments were performed with four RNASET2-silenced and four control OVCAR3 clones. Statistical analysis was performed using two-tailed Student's t-test. * p<0,05; ** p<0,01. B) Evaluation of phoshoryated paxillin on total cell lysates upon adhesion on fibronectin (FN) or collagen type I (Col I). Upper panel: immunoblotting for the expression of phospho-paxillin. Lower panel: densitometric analysis reporting the levels of phosphorylated paxillin with respect to α-tubulin expression of cells grown on ECM matrices. Statistical analysis of data from triplicate experiments was performed using Student's t-test. *** p< 0.001. C) Confocal IIF performed on migrating OVCAR3 cell pools. Starved cells were induced to migrate through a wound upon stimulation with FCS for 24 hr, and then fixed with paraformaldehyde. FAs were stained with anti-phoshphorylated paxillin (green); actin was stained with phalloidin (red). Nuclei were counterstained with DAPI. The white arrow indicates dorsal stress fibers present in RNaseT2-silenced cells. Scal bar: 50 μm. D) Control vs. RNASET2-silenced OVCAR3 cells were grown in reduced growth factor-matrigel 3D culture and pictures were taken with a light microscope.
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Related In: Results  -  Collection

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Figure 7: RNASET2 silencing affects both cell adhesion and FA dynamics in OVCAR3 cellsA) A colorimetric in vitro cell-adhesion assay was performed on five different ECM components. Significant differences in cell-adhesion on laminin, collagen type I and collagen type IV were observed when RNASET2-silenced OVCAR3 cell clones were compared to control clones. Such differences turned out to be weakened when RNASET2-silenced cells were exposed to recombinant RNASET2 protein. Triplicate experiments were performed with four RNASET2-silenced and four control OVCAR3 clones. Statistical analysis was performed using two-tailed Student's t-test. * p<0,05; ** p<0,01. B) Evaluation of phoshoryated paxillin on total cell lysates upon adhesion on fibronectin (FN) or collagen type I (Col I). Upper panel: immunoblotting for the expression of phospho-paxillin. Lower panel: densitometric analysis reporting the levels of phosphorylated paxillin with respect to α-tubulin expression of cells grown on ECM matrices. Statistical analysis of data from triplicate experiments was performed using Student's t-test. *** p< 0.001. C) Confocal IIF performed on migrating OVCAR3 cell pools. Starved cells were induced to migrate through a wound upon stimulation with FCS for 24 hr, and then fixed with paraformaldehyde. FAs were stained with anti-phoshphorylated paxillin (green); actin was stained with phalloidin (red). Nuclei were counterstained with DAPI. The white arrow indicates dorsal stress fibers present in RNaseT2-silenced cells. Scal bar: 50 μm. D) Control vs. RNASET2-silenced OVCAR3 cells were grown in reduced growth factor-matrigel 3D culture and pictures were taken with a light microscope.
Mentions: To evaluate whether the observed effects of RNASET2 on cell morphology and migration could be modulated by a differential activation of extracellular matrix/integrin signaling, we also carried out an in vitro cell adhesion assay on control and RNASET2-silenced OVCAR3 cells grown on different ECM-derived components. As shown in Figure 7A, we could not detect any significant difference in cell adhesion on fibronectin or vitronectin. However, cell adhesion to laminin and collagens type I and IV was markedly increased in RNASET2-silenced OVCAR3 cells. Moreover, recombinant RNASET2 was able to partially restore a reduced adhesion in the latter cells, particularly with respect to collagen I (Figure 7A).

Bottom Line: Indeed, RNASET2 expression levels were consistently found to increase following stress induction.Of note, a remarkable rearrangement of the actin cytoskeleton organization, together with changes in cell adhesion and motility, emerged as putative mechanisms by which such cell-autonomous role could occur.Altogether, these biological features allow to put forward the hypothesis that the RNASET2 protein can act as a molecular barrier for limiting the damages and tissue remodeling events occurring during the earlier step of cell transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Theoretical and Applied Sciences, Università degli Studi dell'Insubria, Varese, Italy.

ABSTRACT
As widely recognized, tumor growth entails a close and complex cross-talk among cancer cells and the surrounding tumor microenvironment. We recently described the human RNASET2 gene as one key player of such microenvironmental cross-talk. Indeed, the protein encoded by this gene is an extracellular RNase which is able to control cancer growth in a non-cell autonomous mode by inducing a sustained recruitment of immune-competent cells belonging to the monocyte/macrophage lineage within a growing tumor mass. Here, we asked whether this oncosuppressor gene is sensitive to stress challenges and whether it can trigger cell-intrinsic processes as well. Indeed, RNASET2 expression levels were consistently found to increase following stress induction. Moreover, changes in RNASET2 expression levels turned out to affect several cancer-related parameters in vitro in an ovarian cancer cell line model. Of note, a remarkable rearrangement of the actin cytoskeleton organization, together with changes in cell adhesion and motility, emerged as putative mechanisms by which such cell-autonomous role could occur. Altogether, these biological features allow to put forward the hypothesis that the RNASET2 protein can act as a molecular barrier for limiting the damages and tissue remodeling events occurring during the earlier step of cell transformation.

No MeSH data available.


Related in: MedlinePlus