Epithelial-to-mesenchymal transition in FHC-silenced cells: The role of CXCR4/CXCL12 axis

I. Aversa, F. Zolea, C. Ieranò, S. Bulotta, A. M. Trotta, M. C. Faniello, C. De Marco, D. Malanga, F. Biamonte, G. Viglietto, G. Cuda, S. Scala, F. Costanzo

Research output: Contribution to journalArticle

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Abstract

Background: Ferritin plays a central role in the intracellular iron metabolism; the molecule is a nanocage of 24 subunits of the heavy and light types. The heavy subunit (FHC) is provided of a ferroxidase activity and thus performs the key transformation of iron in a non-toxic form. Recently, it has been shown that FHC is also involved in additional not iron-related critical pathways including, among the others, p53 regulation, modulation of oncomiRNAs expression and chemokine signalling. Epithelial to mesenchymal transition (EMT) is a cellular mechanism by which the cell acquires a fibroblast-like phenotype along with a decreased adhesion and augmented motility. In this work we have focused our attention on the role of the FHC on EMT induction in the human cell lines MCF-7 and H460 to elucidate the underlying molecular mechanisms. Methods: Targeted silencing of the FHC was performed by lentiviral-driven shRNA strategy. Reconstitution of the FHC gene product was obtained by full length FHC cDNA transfection with Lipofectamine 2000. MTT and cell count assays were used to evaluate cell viability and proliferation; cell migration capability was assayed by the wound-healing assay and transwell strategy. Quantification of the CXCR4 surface expression was performed by flow cytometry. Results: Experimental data indicated that FHC-silenced MCF-7 and H460 cells (MCF-7shFHC, H460shFHC) acquire a mesenchymal phenotype, accompanied by a significant enhancement of their migratory and proliferative capacity. This shift is coupled to an increase in ROS production and by an activation of the CXCR4/CXCL12 signalling pathway. We present experimental data indicating that the cytosolic increase in ROS levels is responsible for the enhanced proliferation of FHC-silenced cells, while the higher migration rate is attributable to a dysregulation of the CXCR4/CXCL12 axis. Conclusions: Our findings indicate that induction of EMT, increased migration and survival depend, in MCF-7 and H460 cells, on the release of FHC control on two pathways, namely the iron/ROS metabolism and CXCR4/CXCL12 axis. Besides constituting a further confirmation of the multifunctional nature of FHC, this data also suggest that the analysis of FHC amount/function might be an important additional tool to predict tumor aggressiveness.

Original languageEnglish
Article number104
JournalJournal of Experimental and Clinical Cancer Research
Volume36
Issue number1
DOIs
Publication statusPublished - Aug 3 2017

Fingerprint

Epithelial-Mesenchymal Transition
Iron
MCF-7 Cells
Phenotype
Ceruloplasmin
Critical Pathways
Ferritins
Chemokines
Wound Healing
Small Interfering RNA
Cell Movement
Transfection
Cell Survival
Flow Cytometry
Complementary DNA
Fibroblasts
Cell Count
Cell Proliferation
Light
Cell Line

Keywords

  • Cancer
  • CXCL12
  • CXCR4
  • EMT
  • Ferritin heavy chain
  • ROS

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Epithelial-to-mesenchymal transition in FHC-silenced cells : The role of CXCR4/CXCL12 axis. / Aversa, I.; Zolea, F.; Ieranò, C.; Bulotta, S.; Trotta, A. M.; Faniello, M. C.; De Marco, C.; Malanga, D.; Biamonte, F.; Viglietto, G.; Cuda, G.; Scala, S.; Costanzo, F.

In: Journal of Experimental and Clinical Cancer Research, Vol. 36, No. 1, 104, 03.08.2017.

Research output: Contribution to journalArticle

Aversa, I, Zolea, F, Ieranò, C, Bulotta, S, Trotta, AM, Faniello, MC, De Marco, C, Malanga, D, Biamonte, F, Viglietto, G, Cuda, G, Scala, S & Costanzo, F 2017, 'Epithelial-to-mesenchymal transition in FHC-silenced cells: The role of CXCR4/CXCL12 axis', Journal of Experimental and Clinical Cancer Research, vol. 36, no. 1, 104. https://doi.org/10.1186/s13046-017-0571-8
Aversa, I. ; Zolea, F. ; Ieranò, C. ; Bulotta, S. ; Trotta, A. M. ; Faniello, M. C. ; De Marco, C. ; Malanga, D. ; Biamonte, F. ; Viglietto, G. ; Cuda, G. ; Scala, S. ; Costanzo, F. / Epithelial-to-mesenchymal transition in FHC-silenced cells : The role of CXCR4/CXCL12 axis. In: Journal of Experimental and Clinical Cancer Research. 2017 ; Vol. 36, No. 1.
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T2 - The role of CXCR4/CXCL12 axis

AU - Aversa, I.

AU - Zolea, F.

AU - Ieranò, C.

AU - Bulotta, S.

AU - Trotta, A. M.

AU - Faniello, M. C.

AU - De Marco, C.

AU - Malanga, D.

AU - Biamonte, F.

AU - Viglietto, G.

AU - Cuda, G.

AU - Scala, S.

AU - Costanzo, F.

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Y1 - 2017/8/3

N2 - Background: Ferritin plays a central role in the intracellular iron metabolism; the molecule is a nanocage of 24 subunits of the heavy and light types. The heavy subunit (FHC) is provided of a ferroxidase activity and thus performs the key transformation of iron in a non-toxic form. Recently, it has been shown that FHC is also involved in additional not iron-related critical pathways including, among the others, p53 regulation, modulation of oncomiRNAs expression and chemokine signalling. Epithelial to mesenchymal transition (EMT) is a cellular mechanism by which the cell acquires a fibroblast-like phenotype along with a decreased adhesion and augmented motility. In this work we have focused our attention on the role of the FHC on EMT induction in the human cell lines MCF-7 and H460 to elucidate the underlying molecular mechanisms. Methods: Targeted silencing of the FHC was performed by lentiviral-driven shRNA strategy. Reconstitution of the FHC gene product was obtained by full length FHC cDNA transfection with Lipofectamine 2000. MTT and cell count assays were used to evaluate cell viability and proliferation; cell migration capability was assayed by the wound-healing assay and transwell strategy. Quantification of the CXCR4 surface expression was performed by flow cytometry. Results: Experimental data indicated that FHC-silenced MCF-7 and H460 cells (MCF-7shFHC, H460shFHC) acquire a mesenchymal phenotype, accompanied by a significant enhancement of their migratory and proliferative capacity. This shift is coupled to an increase in ROS production and by an activation of the CXCR4/CXCL12 signalling pathway. We present experimental data indicating that the cytosolic increase in ROS levels is responsible for the enhanced proliferation of FHC-silenced cells, while the higher migration rate is attributable to a dysregulation of the CXCR4/CXCL12 axis. Conclusions: Our findings indicate that induction of EMT, increased migration and survival depend, in MCF-7 and H460 cells, on the release of FHC control on two pathways, namely the iron/ROS metabolism and CXCR4/CXCL12 axis. Besides constituting a further confirmation of the multifunctional nature of FHC, this data also suggest that the analysis of FHC amount/function might be an important additional tool to predict tumor aggressiveness.

AB - Background: Ferritin plays a central role in the intracellular iron metabolism; the molecule is a nanocage of 24 subunits of the heavy and light types. The heavy subunit (FHC) is provided of a ferroxidase activity and thus performs the key transformation of iron in a non-toxic form. Recently, it has been shown that FHC is also involved in additional not iron-related critical pathways including, among the others, p53 regulation, modulation of oncomiRNAs expression and chemokine signalling. Epithelial to mesenchymal transition (EMT) is a cellular mechanism by which the cell acquires a fibroblast-like phenotype along with a decreased adhesion and augmented motility. In this work we have focused our attention on the role of the FHC on EMT induction in the human cell lines MCF-7 and H460 to elucidate the underlying molecular mechanisms. Methods: Targeted silencing of the FHC was performed by lentiviral-driven shRNA strategy. Reconstitution of the FHC gene product was obtained by full length FHC cDNA transfection with Lipofectamine 2000. MTT and cell count assays were used to evaluate cell viability and proliferation; cell migration capability was assayed by the wound-healing assay and transwell strategy. Quantification of the CXCR4 surface expression was performed by flow cytometry. Results: Experimental data indicated that FHC-silenced MCF-7 and H460 cells (MCF-7shFHC, H460shFHC) acquire a mesenchymal phenotype, accompanied by a significant enhancement of their migratory and proliferative capacity. This shift is coupled to an increase in ROS production and by an activation of the CXCR4/CXCL12 signalling pathway. We present experimental data indicating that the cytosolic increase in ROS levels is responsible for the enhanced proliferation of FHC-silenced cells, while the higher migration rate is attributable to a dysregulation of the CXCR4/CXCL12 axis. Conclusions: Our findings indicate that induction of EMT, increased migration and survival depend, in MCF-7 and H460 cells, on the release of FHC control on two pathways, namely the iron/ROS metabolism and CXCR4/CXCL12 axis. Besides constituting a further confirmation of the multifunctional nature of FHC, this data also suggest that the analysis of FHC amount/function might be an important additional tool to predict tumor aggressiveness.

KW - Cancer

KW - CXCL12

KW - CXCR4

KW - EMT

KW - Ferritin heavy chain

KW - ROS

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