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p63 promotes cell survival through fatty acid synthase.

Sabbisetti V, Di Napoli A, Seeley A, Amato AM, O'Regan E, Ghebremichael M, Loda M, Signoretti S - PLoS ONE (2009)

Bottom Line: Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN.FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival.Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

ABSTRACT
There is increasing evidence that p63, and specifically DeltaNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN), a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT) was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

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Silencing of either Total or ΔN specific p63 isoforms induces apoptosis with no significant changes in cell proliferation.A–C. Flow cyometric analysis of SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (A), SCC9-Tp63 and SCC9-Dp63 cells with or without treatment tetracycline (B) and iPrEC-Tp63 and iPrEC-Dp63 cells with or without treatment tetracycline (C). Left panels show quantification (percentage) of the sub G1 population measured by PI staining. Right panels show the fractions of cells in the various phases of the cell cycle, as determined by BrdU incorporation. Data are shown as means +/− standard error and are representative of three independent experiments. D. Immunoblot analysis of apoptosis-related proteins in SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (left panel), in SCC9-Tp63 cells with or without treatment with tetracycline (middle panel) and in iPrEC-Tp63 cells with or without treatment with tetracycline (right panel).
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pone-0005877-g002: Silencing of either Total or ΔN specific p63 isoforms induces apoptosis with no significant changes in cell proliferation.A–C. Flow cyometric analysis of SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (A), SCC9-Tp63 and SCC9-Dp63 cells with or without treatment tetracycline (B) and iPrEC-Tp63 and iPrEC-Dp63 cells with or without treatment tetracycline (C). Left panels show quantification (percentage) of the sub G1 population measured by PI staining. Right panels show the fractions of cells in the various phases of the cell cycle, as determined by BrdU incorporation. Data are shown as means +/− standard error and are representative of three independent experiments. D. Immunoblot analysis of apoptosis-related proteins in SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (left panel), in SCC9-Tp63 cells with or without treatment with tetracycline (middle panel) and in iPrEC-Tp63 cells with or without treatment with tetracycline (right panel).

Mentions: We next assessed whether the decrease in cell viability observed upon p63 downregulation was a result of apoptosis, cell cycle arrest, or both. We addressed this question by performing flow cytometric analysis in both SCC9 and iPrEC cell lines after p63 silencing. We found that p63 knockdown of either all p63 isoforms or ΔNp63 isoforms caused a significant increase in the sub G1 population of cells when compared to the control (P<0.05 at 48, 72, and 96 h, two-tailed t-test) (Figure 2A–2C, left panels). In line with these data, knockdown of p63 in both SCC9 and iPrEC cells resulted in an increase in the cleaved caspase 3 levels and a concomitant decrease in the expression total caspase 3. Accordingly, cleaved poly (ADP-ribose) polymerase (PARP) protein levels were increased after p63 knockdown (Figure 2D). These data provide evidence that p63 plays a role in the survival of both transformed and immortalized epithelial cells. The observation that Tp63 and Dp63 siRNAs produced similar results suggests that p63 anti-apoptotic function is mostly mediated by ΔNp63 isoforms.


p63 promotes cell survival through fatty acid synthase.

Sabbisetti V, Di Napoli A, Seeley A, Amato AM, O'Regan E, Ghebremichael M, Loda M, Signoretti S - PLoS ONE (2009)

Silencing of either Total or ΔN specific p63 isoforms induces apoptosis with no significant changes in cell proliferation.A–C. Flow cyometric analysis of SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (A), SCC9-Tp63 and SCC9-Dp63 cells with or without treatment tetracycline (B) and iPrEC-Tp63 and iPrEC-Dp63 cells with or without treatment tetracycline (C). Left panels show quantification (percentage) of the sub G1 population measured by PI staining. Right panels show the fractions of cells in the various phases of the cell cycle, as determined by BrdU incorporation. Data are shown as means +/− standard error and are representative of three independent experiments. D. Immunoblot analysis of apoptosis-related proteins in SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (left panel), in SCC9-Tp63 cells with or without treatment with tetracycline (middle panel) and in iPrEC-Tp63 cells with or without treatment with tetracycline (right panel).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691576&req=5

pone-0005877-g002: Silencing of either Total or ΔN specific p63 isoforms induces apoptosis with no significant changes in cell proliferation.A–C. Flow cyometric analysis of SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (A), SCC9-Tp63 and SCC9-Dp63 cells with or without treatment tetracycline (B) and iPrEC-Tp63 and iPrEC-Dp63 cells with or without treatment tetracycline (C). Left panels show quantification (percentage) of the sub G1 population measured by PI staining. Right panels show the fractions of cells in the various phases of the cell cycle, as determined by BrdU incorporation. Data are shown as means +/− standard error and are representative of three independent experiments. D. Immunoblot analysis of apoptosis-related proteins in SCC9 cells transfected with Tp63, Dp63 or Scr siRNAs (left panel), in SCC9-Tp63 cells with or without treatment with tetracycline (middle panel) and in iPrEC-Tp63 cells with or without treatment with tetracycline (right panel).
Mentions: We next assessed whether the decrease in cell viability observed upon p63 downregulation was a result of apoptosis, cell cycle arrest, or both. We addressed this question by performing flow cytometric analysis in both SCC9 and iPrEC cell lines after p63 silencing. We found that p63 knockdown of either all p63 isoforms or ΔNp63 isoforms caused a significant increase in the sub G1 population of cells when compared to the control (P<0.05 at 48, 72, and 96 h, two-tailed t-test) (Figure 2A–2C, left panels). In line with these data, knockdown of p63 in both SCC9 and iPrEC cells resulted in an increase in the cleaved caspase 3 levels and a concomitant decrease in the expression total caspase 3. Accordingly, cleaved poly (ADP-ribose) polymerase (PARP) protein levels were increased after p63 knockdown (Figure 2D). These data provide evidence that p63 plays a role in the survival of both transformed and immortalized epithelial cells. The observation that Tp63 and Dp63 siRNAs produced similar results suggests that p63 anti-apoptotic function is mostly mediated by ΔNp63 isoforms.

Bottom Line: Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN.FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival.Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

ABSTRACT
There is increasing evidence that p63, and specifically DeltaNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN), a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT) was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

Show MeSH
Related in: MedlinePlus